Guidelines support the use of enteral nutrition to improve clinical outcomes in critical illness; however, the optimal calorie and protein intake remains unclear. The purpose of this meta-analysis was to quantitatively analyze randomised controlled trials with regard to clinical outcomes related to varying calorie and protein administration in critically ill adult patients.We searched Medline, EMBASE, and Cochrane databases to identify randomised controlled trials that compared the effects of initially different calorie and protein intake in critical illness. The risk ratio (RR) and weighted mean difference with 95% confidence intervals (CI) were calculated using random-effects models. The primary endpoint was mortality; secondary endpoints included infection, pneumonia, gastrointestinal intolerance, hospital and intensive care unit lengths of stay, and mechanical ventilation days.P = 0.40), infection , or the risk of gastrointestinal intolerance . In subgroup analysis, the low-energy subgroup, fed 33.3 to 66.6% of goal energy, showed a lower mortality than the high-energy group . The improvements in mortality and gastrointestinal intolerance were absent when calorie intake was >66.6% of goal energy in the low-energy group. High-energy intake combined with high-protein intake reduced the infections ; however, when the daily protein intake was similar in both groups, a high-energy intake did not decrease the infections. No statistical differences were observed in other secondary outcomes.In the eight randomised controlled trials that enrolled 1,895 patients there was no statistical difference between the low-energy and high-energy groups in mortality , compared to high energy, may reduce mortality, and a higher protein intake combined with high energy (≥0.85 g/kg per day) may decrease the infection rate. However, the contribution of energy versus protein intake to outcomes remains unknown.The online version of this article (doi:10.1186/s13054-015-0902-0) contains supplementary material, which is available to authorized users. Enteral nutrition (EN) may not only supply nutrition to patients but also protect intestinal epithelial cells, improve intestinal tight junctions, support intestinal structure and function, and prevent bacterial translocation -5. HowevThe European Society for Clinical Nutrition and Metabolism’s guidelines suggest that an exogenous energy supply in excess of 20 to 25 kcal/kg body weight per day may be associated with less favourable outcomes during the acute phase of a critical illness (grade C) . SimilarA cohort study of patients in a respiratory ICU reported that inadequate calorie delivery is associated with higher odds of mortality . SurprisIn this systematic review, we conducted a search of the published literature to identify all relevant clinical trials using the keywords or MeSH headings of ‘trophic feeding’, ‘hypocaloric nutrition’, ‘permissive underfeeding’, ‘gradual enteral nutrition’, ‘standard enteral nutrition’, ‘intensive enteral nutrition’, ‘concentrated enteral nutrition’, ‘hypercaloric nutrition’, ‘normocaloric nutrition’, ‘full feeding’, ‘critical illness’, ‘critically ill’, ‘ICU’, ‘acute lung injury’, ‘respiratory insufficiency’, and ‘intensive care’ in combination with the Boolean operators AND and OR. Two of the authors (Feng Tian and Xinying Wang) independently performed computerised searches in the MEDLINE, EMBASE, and Cochrane Controlled Trials Register databases. We also searched previous review articles for additional original studies. The final search was conducted on 1 November 2014. No language restrictions were included in the searches. Additionally, abstracts from major scientific meetings were included if they were available for data extraction; authors were approached for additional or missing data, if necessary.Two investigators (Feng Tian and Xuejin Gao) independently reviewed all original studies to determine inclusion or exclusion; in the case of disagreement, a third author was consulted. Original studies were selected for inclusion if they met the following criteria: 1) the research design was an RCT or randomized trial or study; 2) the population comprised critically ill adult patients admitted to the ICU (>16 years old); 3) the intervention of the study was designed such that the two groups received significantly different calorie intakes by EN; and 4) the clinical outcome of overall mortality of critically ill patients was reported.Studies were excluded if they met either of the following criteria: 1) the target calorie supply was different between the two groups; and 2) EN was administered for <2 days.Two authors (Feng Tian and Xuejin Gao) independently extracted the following variables where they were published and available: demographics, sample size, critical illness severity, amount of daily calorie and protein intake, body mass index (BMI), intervention provided in both groups, and clinical outcomes.The same two authors independently assessed the study quality and risk of bias using the methods detailed in the Cochrane Handbook for Systematic Reviews of Interventions . DisagreThe primary outcome of this meta-analysis was overall mortality. Hospital mortality in all studies was combined. If hospital mortality was not reported, 60- or 90-day or ICU mortality was used instead. Secondary outcomes included infections (bacteraemia or sepsis was used if the number of infection cases was not reported), pneumonia (ventilator-associated pneumonia and infectious pneumonia), length of stay (LOS) in both hospital (LOS-HOS) and ICU (ICU LOS), and mechanical ventilation days (MVD); these were also pooled for all studies. Despite differences in the definition of gastrointestinal intolerance , we combined the outcomes where diarrhoea was reported.P value (Cochran’s Q statistic), where a P < 0.05 and I2 statistic >50% indicated significant heterogeneity. Subgroup analyses were conducted to find the source of heterogeneity, and a test for heterogeneity interaction was performed to determine whether there was a difference in effect sizes between subgroups. The subgroups were chosen based on their suspected influences on the analyses and previous relevant studies. For instance, the varying calorie intake between studies could influence the aggregate results. Therefore, the eight studies were divided into three subgroups according to the percentage of target energy achieved in the low-energy (LE) group . The cut-offs of 33.3% and 66.6% were based on a previous cohort study and a recent meta-analysis [The risk ratio (RR) and associated 95% confidence interval (CI) were used to summarize binary outcomes including mortality, infections, and pneumonia. The overall weighted mean difference (WMD) with 95% CI was estimated for LOS and MVD. A more conservative random-effects model was used in this meta-analysis because of anticipated heterogeneity. Statistical heterogeneity among trials is expressed as the analysis ,20. The Our detailed search strategy is illustrated in the flow diagram in Figure Randomisation methods were reported in all eight studies. Allocation concealment was used in six trials using sealed envelopes -26, and,2 [The basic characteristics of patients in the included studies are shown in Tables 2 -27; one 2 .Table 1The mean daily delivered caloric and protein levels differed among studies. In the LE group, the mean daily percentage of target calories in two studies was <33.3%; in four studies, it was between 33.3% and 66.6%; and in two studies, it was >66.6%. In the HE group, the percentage of target calories for patients was >70% in seven studies and >90% in two studies, but it was 59.2% in one study. Although the caloric intake was 59.2% for the HE group in the study by Taylor and colleagues , the meaP = 0.40; I2 = 31%; P = 0.18). Subgroup analysis was performed according to the percentage of the goal energy achieved and showed that mortality was significantly different among the three subgroups. Compared with the HE group, mortality was significantly lower in the LE group . In contrast, compared with the HE group, mortality was not different in the LE subgroup that was fed <33.3%  or >66.6% of the goal energy . There was significant heterogeneity among the subgroups    and revealed that mortality was not significantly different for patients in the LE group compared with the HE group .Six of the eight studies reported infections; these studies included 1,683 patients -23,26-28P = 0.02; I2 = 0%; P = 0.41). Interestingly, when the daily protein intake was similar, infectious complications were not different between the LE and HE groups . There was significant heterogeneity among the subgroups  . Subgroup analysis showed that pneumonia was not affected by the percentage of the goal energy achieved  or the daily protein intake  . Subgroup analysis showed that gastrointestinal intolerance in the LE group that was fed 33.3% to 66.6% of the goal energy was significantly lower than that in the HE group . However, this advantage was not obvious in the LE group that was fed >66.6% of the goal energy , compared with the HE group fed >95% of the goal energy  [2 = 0%; P = 0.91) or daily protein intake   -24. SubgP = 0.57; I2 = 0%; P = 0.60) [2 = 0%; P = 0.80) or daily protein intake   -24. SubgP = 0.36; I2 = 46%; P = 0.17) [For the two studies of 440 patients that provided data on MVD, our meta-analysis showed no difference between the LE and HE groups  ,26  as compared to HE may reduce mortality, and a higher protein intake combined with HE (≥0.85 g/kg per day) may decrease the infection rates. However, because of the study limitations, the optimal dose of initial calorie and protein intake is still uncertain, and further large-sample RCTs are needed to confirm our conclusions.Initial moderately low calorie intake could improve the prognosis of critical illness and excessively low or high calorie intake should be avoided in these patients.A relatively high protein intake might benefit critically ill patients, but the daily administration of protein should not exceed the upper limit of the recommended range.The current data are not sufficient to draw conclusions regarding the optimal initial calorie and protein intake to be administered by EN in non-malnourished critically ill adults because of the limited number and heterogeneity of the trials, including the varying calorie and protein intake in the eight included studies.More rigorously designed studies using similar protocols are required to determine the optimal levels of calorie and protein intake.


Hyperadrenocorticism (HAC) is associated with an increased prevalence of hypertension. This study investigated the left ventricular function using two-dimensional speckle-tracking echocardiography (2D-STE) in small breed dogs affected with spontaneous HAC.n = 9), dogs with pituitary-dependent hyperadrenocorticism , and dogs with adrenal-dependent hyperadrenocorticism  were included in this study. Conventional echocardiography, global longitudinal and circumferential strain, and strain rate were assessed.Age-matched healthy controls . Neither LVFWd nor IVSd was correlated with systemic blood pressure . The values of all variables derived from the global strain and strain rate in longitudinal and circumferential directions followed the same pattern: highest in the controls, followed by PDH and then ADH . On multiple regression analyses, global longitudinal strain, global longitudinal strain rate in systole and early diastole, and global circumferential strain all decreased linearly with increased IVSd .On group-wise comparison, left ventricular free wall (LVFWd) and interventricular septal thickness in diastole (IVSd) were thickest in the ADH group, followed by the PDH and controls (Left ventricular hypertrophy (LVH) was more prevalent in the HAC group compared to the control group. Association between hypertension and development of LVH was not identified. Decreased global longitudinal and circumferential strains were associated with increased IVSd. 2D-STE revealed significant decreases in systolic functions that were undetected using conventional echocardiography in the ADH and PDH groups. Hyperadrenocorticism (HAC) is a condition of chronically elevated circulating glucocorticoid concentration. In response to stimulation of the renin-angiotensin system and mineralocorticoid and glucocorticoid receptors of myocytes, development of systemic hypertension, left ventricular hypertrophy (LVH), and myocardial fibrosis are commonly observed in human patients with HAC -3. The lHuman patients with HAC and concomitant hypertension have a two-fold greater risk of detection of cardiac mass abnormality compared with normotensive patients and hypertensive controls . HyperteClient-owned dogs visiting the National Taiwan University Veterinary Hospital (NTUVH) during 2010 and 2012 for routine wellness checkups were included in this study. Thorough medical histories were obtained, and physical examinations, routine blood analyses , chest radiographic examinations, abdominal ultrasonographic examinations, echocardiographic examinations , M-mode, and Doppler) and 2D-STE were performed to exclude the presence of any forms of systemic, cardiac, renal, and adrenal disease. Nine dogs  were deemed clinically healthy and categorized as age-matched healthy controls (controls).Client-owned dogs affected with HAC during the same period of time were included in this group. Inclusion criteria of HAC included evidence of clinical signs consistent with a diagnosis of HAC , results of routine serum biochemical analyses consistent with a diagnosis of HAC , and results of ACTH stimulation tests consistent with a diagnosis of HAC. Dogs with inconclusive results from ACTH stimulation tests were excluded from the study. All included dogs with HAC were required to have received an ACTH stimulation test with intramuscular injection of 0.25 mg of synthetic ACTH  prior to treatment. Blood samples for the detection of serum cortisol concentrations were collected via cephalic venipuncture immediately before and one hour after synthetic ACTH was injected . Serum cSystolic blood pressure was measured using the indirect method of Doppler flow detection with a 9.5 MHz transducer. A Doppler Ultrasonic Flow Detector  with an inflatable cuff width of 1.9 or 2.5 cm (approximately 40% of the circumference of the antebrachium) was used. During systemic blood pressure measurement, the antebrachium was maintained at the level of the heart. A series of five readings (with 10 to 20 seconds between consecutive measurements) was obtained for each dog. To minimize procedural stress, all dogs were allowed to assume a comfortable position with only gentle restraint by their owners. The dogs remained in the same position throughout systemic blood pressure measurement. The final systemic blood pressure value was calculated as the mean of five readings. The heart rate was manually recorded by pulse Doppler ultrasound detection over a period of 20 seconds after the systemic blood pressure value was measured. Dogs were considered to be hypertensive if the average systemic blood pressure exceeded 160 mm Hg .Conventional echocardiography was performed without sedation in gently restrained recumbent positions. Standard echocardiographic views were obtained in right and left lateral recumbency using the 2D-guided M mode with ultrasound units equipped with 2–5 and 5–7.5 MHz transducers . Measurements of left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular free-wall thickness (LVFWd) and interventricular septal thickness (IVSd) at end-diastole, ejection fraction (EF), and fractional shortening (FS) were obtained from the right parasternal long-axis view. The left atrium to aorta ratio (LA/AO) was measured in the right parasternal short-axis view. Values for the inflow of the maximal early (E) and late (A) diastolic mitral and tricuspid flow velocities, E/A ratio, and isovolumic relaxation time (IVRT) were determined by pulsed-wave Doppler from the left apical four- or five-chamber view.Inclusion of LVH was based on conventional echocardiographic examinations. LVH was defined as regional or generalized concentric hypertrophy with a diastolic wall thickness ≧ 7 mm of the LVFWd or ≧ 6 mm of the IVSd in dogs with a body weight of < 5 kg, and with a diastolic wall thickness of ≧ 8 mm of the LVFWd or ≧ 7 mm of the IVSd in dogs with a body weight of 5–11 kg .S), circumferential strain rate in early diastole (CSRE), circumferential strain rate in late diastole (CSRA) and peak circumferential strain (CS) were measured from the right parasternal short-axis view at the level of the papillary muscles. The software automatically divided the left ventricle into six segments based on standard segmentation model used in human patients [S), longitudinal strain rate in early diastole (LSRE), longitudinal strain rate in late diastole (LSRA), and peak longitudinal strain (LS) were measured from the left apical long-axis four-chamber view. The software automatically divided into three segments in each the left ventricular free wall and interventricular septum based on standard segmentation model used in human patients [2D-STE examinations were performed at the end of conventional echocardiography for all dogs. The standard left apical long-axis four-chamber and the right parasternal short-axis views at the level of papillary muscles were obtained using a frame rate above 60 per second. For each dog, three consecutive cardiac cycles were recorded using one single loop, and digitally stored in a hard disk for offline analysis on a workstation . The 2D-STE variables included strain, and strain rate. Circumferential strain rate in peak systole . Descriptive statistical analyses were performed with a with commercial computer statistics software  Version 12.0.1, SPSS Inc. in Chicago, IL, USA.). Continuous data were tested for normality by use of the D’Agostino-Pearson omnibus test and Shapiro-Wilk test. All tests were two-tailed, and a P value for multiple comparisons. The relationships between STE-derived variables and age, systemic blood pressure, body weight, LVFWd, and IVSd were assessed using Pearson’s correlation coefficient.Overall group-wise in continuous data , as well as conventional echocardiographic and STE-derived variables, among the PDH, ADH, and control groups were investigated using analysis of variance and post hoc tests with a Scheffe-adjusted t tests.Least squares multiple linear regression analyses were performed with the measured STE-derived variables as dependent variables and the independent variables of gender, age, heart rate, systemic blood pressure, IVSd, and LVFWd. A Chi-square test was used to assess differences in categorical variables among the groups. Comparison of the systemic blood pressure, conventional echocardiographic variables, and 2D-STE derived variables between LVH dogs and controls was performed using Student’s unpaired two-tailed The intra-observer coefficients of variations (CV) of the conventional echocardiographic and 2D-STE derived variables were calculated by a variance component analysis. The CV was determined by dividing the SD value by the overall mean. Six clinically healthy dogs were scanned on three nonconsecutive occasions within one day by the same examiner (HYC), who was unaware of values from the initial examination.The breeds represented in the controls included one miniature dachshund and two each of Maltese terriers, miniature Schnauzers, mixed breeds, and Yorkshire terriers. The mean age of the control was 10.5 ± 2.8 years (range: 7 to 15 years) and the mean body weight was 6.2 ± 2.7 kg (range: 2.6 to 10.0 kg). Nineteen dogs with HAC were included: 10 dogs with pituitary-dependent hyperadrenocorticism (PDH) and nine dogs with adrenal-dependent hyperadrenocorticism (ADH). The breed distribution of the 19 dogs with HAC was as follows: eight Maltese terriers, five Shih-Tzus, and one each of Chihuahua, miniature Schnauzer, Pomeranian, toy poodle, short-haired dachshund, and Yorkshire terrier. The mean age of the 10 dogs  with PDH was 12.5 ± 2.0 years (range: 9 to 15 years) and the mean body weight of this groups was 4.5 ± 1.4 kg (range: 3.2 to 6.6 kg). The mean age of the nine dogs  with ADH was 12.3 ± 2.0 years (range: 9 to 15 years), and the mean body weight of this group was 5.9 ± 2.7 kg (range: 2.1 to 10.0 kg).P = 0.615).No statistically significant differences in body weight and heart rate among controls, the PDH, and the ADH groups were found Table . The femP = 0.005 versus PDH group and controls, Table P = 0.009 versus PDH group and controls, Table The mean systemic blood pressure was highest in the ADH group . Five dogs  were characterized with symmetric LVH, four dogs  were characterized with focal and asymmetric LVH.In conventional echocardiographic examinations, most indices among controls, the PDH, and the ADH groups were not different, except LVFWd and IVSd, mitral E wave, and IVRT values Table . Both LVP = 0.008, Table P = 0.036 and P = 0.001, respectively, Table r2 = 0.05, P = 0.238 and r2 = 0.09, P = 0.113, respectively). Neither LVFWd nor IVSd was correlated with body weight . Systemic blood pressure was negatively correlated with E/IVRT .IVRT was highest in ADH, followed by PDH and controls (n = 9) and controls . No significant differences in systemic blood pressure was found between HAC dogs with LVH (n = 9) and without LVH . Significant decreases in mitral E/A ratio and significant increases in IVRT were also found in LVH dogs compared to controls .No significant differences in systemic blood pressure, FS, or EF were found between dogs with LVH . No correlation between age, body weight, and 2D-STE indices were found.Systemic blood pressure was negatively correlated with global CRSS , global LSRE , global LS , global CSRS  and global CS . The LVFWd was negatively correlated with global LSRS , global LS  and global CS . The E/IVRT was positively correlated with global CSRS , global CSRE  and global CS .The IVSd was negatively correlated with global LSRS, LSRE, LS, global CSRS, CSRE, and CS, were significantly decreased in dogs with LVH compared to controls ; however, these differences between dogs with LVH (n = 9) and without LVH (n = 10) in the HAC group did not reach statistical significance.Seven of eight global 2D-STE variables, namely LSRS, and LSRE all decreased linearly with IVSd and were affected by gender , IVSd had the highest r2 in the final model.In the multiple regression analyses, global LS, global LSRr2 = 0.223, P = 0.007). Global CSRS decreased linearly with IVSd and increased linearly with heart rate , IVSd had the highest r2 in the final model. Global CSRE increased linearly with heart rate and decreased linearly with systemic blood pressure , systemic blood pressure had the highest r2 in the final model.Global CS decreased linearly with IVSd (adjusted CV (%) of 2D-STE variables were all less than 20% Table .Table 6Chronic excessive cortisol concentrations might cause hypertension. Subsequent left ventricular remodeling, hypertrophy, and altered diastolic function are the most important risk factors of cardiovascular mortality in human patients with HAC . Sixty-eCardiac hypertrophy secondary to hypertension and chronic exposure to excess circulating cortisol may also contribute directly to left ventricular concentric remodeling in human patients with HAC. Subsequently, functional abnormality, such as diastolic dysfunction (reduced E/A ratio and/or E velocity) has also been reported in human patients with HAC ,26. In tDevelopment of LVH is generally believed to be associated with concomitant hypertension in HAC. However in this study, the wall thickness of the left ventricle was not correlated with systemic blood pressure. Significant differences in systemic blood pressure were not found between dogs with LVH and controls, or between HAC dogs with LVH and HAC dogs without LVH. No association between hypertension and development of LVH was identified in this study. The mechanism of development of LVH in dogs with HAC could not be clarified in this study.In 2D-STE analysis, global peak systolic and early diastolic strain rate, as well as global strain in longitudinal and circumferential dynamics exhibited a consistent pattern: profound decreases in the ADH and the PDH groups compared to controls, i.e. significant decreases in systolic and diastolic functions were observed in the ADH and PDH groups when compared to controls. Left ventricular systolic and diastolic dysfunction was also reported in human patients with HAC ,28. DiasIn this study, 2D-STE revealed alterations in left ventricular mechanics despite a preserved systolic function detected by conventional echocardiography in dogs with HAC. The indices of left ventricular systolic function derived from conventional echocardiography, such as FS, EF, LVEDD and LVESD, were not significantly different between the HAC group and controls. FS and EF did not reach significant difference between dogs with LVH and the control. However decreased systolic function was revealed using 2D-STE. Decreased systolic function was most prominent in dogs with LVH associated with ADH, with decreasing prominence observed in the PDH group and finally in controls. This finding suggests that dogs with HAC may have subclinical systolic dysfunction that may remain undetected using conventional echocardiographic examination. Similar findings are also reported in human patients with HAC-associated hypertrophic cardiomyopathy: subclinical left ventricular systolic dysfunction is indicated using STE, although the assessment of FS and EF is usually normal or supernormal ,29.S, global LSRE, global CS, and CSRS decreased while IVSd increased, suggesting that decreased magnitude and rate of myocardial deformation is followed by increased LVH. Based on these findings, increased IVSd could be used as predictor of subclinical LV dysfunction in small breed dogs with HAC. In addition, global CSRE decreased linearly with increased systemic blood pressure. Based on this finding, increased systemic blood pressure could be used as predictor of subclinical LV diastolic dysfunction in small breed dogs with HAC.Global LS, global LSRE/IVRT is an index to predict for left ventricular filling pressure, and increases when LV filling pressure is increased in dogs with congestive heart failure characterized by volume overload . In thisLongitudinal deformation was affected by gender in this study. Although dogs with HAC are more commonly female, no gender predilection has been reported. In this study, 57% of the dogs with HAC were females, a rate similar to that previously reported ; howeverIn this study, the differences of longitudinal 2D-STE variables among groups were prominent when the analyses were based upon averaged global deformation comparing to the analyses across six myocardial regions. This might be due the definition of segments. The definition of segment and post-processing software was based on human nomenclature or anatomy, and it may not be necessarily applied to the dogs . AverageLimitations of this study include low number of dogs. The dexamethasone suppression test and ACTH concentrations were not applied to make a differential diagnosis of ADH and PDH. HAC of iatrogenic origin was predominant in the area where the authors practice. The differential diagnosis of ADH and PDH was based on the results of the ACTH stimulation test and ultrasonographic images of adrenal glands. Many HAC cases in which bilaterally and irregularly enlarged/round adrenals were presented were excluded from the study. No corresponding aldosterone concentrations were included in this study.


G protein-coupled receptor 43/free fatty acid receptor 2 (GPR43/FFAR2) is essential for polymorphonuclear (PMN) recruitment. We investigated the expression of GPR43/FFAR2 in the colon from Crohn’s disease patients and whether dietary fiber in enteral nutrition increases GPR43+ polymorphonuclear infiltration in mucosa. Segments of ascending colon and white blood cells from peripheral blood were obtained from 46 Crohn’s disease patients and 10 colon cancer patients. The Crohn’s disease patients were grouped by the activity of disease (active or remission) and enteral nutrition with or without dietary fiber. Histological feature, expression and location of GPR43/FFAR2 and level of tumor necrosis factor-α (TNF-α), interleukine-6 (IL-6) and myeloperoxidase were assessed. The results of hematoxylin-eosin and immunohistochemistry staining revealed that the infiltration of immune cells, including GPR43+ PMN, was more severe in active Crohn’s disease patients who consumed normal food or enteral nutrition with dietary fiber than in remission patients and colon cancer patients. This finding was supported by the results of GPR43 and myeloperoxidase expression. Active Crohn’s disease (CD) patients who consumed enteral nutrition without dietary fiber exhibited severe immune cell infiltration similar to the other active CD patients, but GPR43+ PMNs were rarely observed. The level of TNF-α mRNA in active Crohn’s disease patients was higher than those of the other patients. In conclusion, the use of dietary fiber in enteral nutrition by active Crohn’s disease patients might increase GPR43+ PMNs infiltration in colon mucosa. This effect was not observed in Crohn’s disease patients in remission. A lack of enteral nutrition, such as occurs during total parenteral nutrition, leads to atrophy and hypofunction throughout the gastrointestinal tract , and theet al.  and Fern [et al.  reported [et al. . SCFAs, 2+ and a decrease in intracellular cyclic adenosine monophosphate (cAMP). GPR43/FFAR2 is expressed in adipose tissue, the gastrointestinal tract, and in inflammatory cells (mainly neutrophils). It is believed that GPR43 is involved in lipid metabolism, gastrointestinal tract function, and inflammatory disease [In 1997, the gene of G-protein coupled receptor 43 (GPR43), along with G-protein coupled receptor 40/41/42 (GPR40/41/42), was found in the course of search for novel subtypes of the unrelated galanin receptor . In 2003 disease ,14.et al. [et al. [Neutrophils  play an important role in the development of Crohn’s disease (CD). It is believed that the intrinsic functions of neutrophils  in CD are intact ,16,17, bet al.  and Mark [et al.  found th [et al. . This re [et al.  and lead [et al. .et al. [et al. [et al. [et al. [SCFA can recruit immune cells, and this process is mediated by GPR43/FFAR2, which is expressed in neutrophil cells. Neutrophils (or PMNs) from GPR43 knock out (GPR43 KO) mice cannot be recruited by SCFA ,23,24. H [et al.  found th [et al.  and Masu [et al.  believe et al. [Karaki et al.  investiget al.  and neutet al. .This was a retrospective study. Ten colon cancer patients (can.) and 46 Crohn’s disease patients were included in our study. Crohn’s disease patients were divided into 5 groups, according to The activity of Crohn’s disease was judged by the Crohn’s disease activity index (CDAI) score before operation. Active Crohn’s disease was defined as CDAI ≥ 150, and Crohn’s disease in remission was defined as CDAI < 150. All of the patients underwent an operation during their hospitalization in our hospital from December 2013 to February 2015. For all of the patients with selective operations in our department, enteral nutrition was routinely consumed at least 10 days prior to surgery. Seven patients who received emergency operations without the use of enteral nutrition before surgery were included as controls (ac-food). For all of these included patients, the ascending colon or part of the ascending colon was removed during the operations.This study was approved by the Ethics Committee of Jinling Hospital (2013NLY-212). All patients signed written consent that their surgical specimen and other information would be used for scientific research.Segments of ascending colon and samples of venous blood were obtained from these 56 patients.From each patient, 3 segments of the ascending colon (approximately 1 cm × 1 cm) were cut from the region of surgical specimen without visible lesions (can.) but very close to the ulcer (patients of Crohn’s disease). Two segments were frozen at −80 °C, and the third segment was placed in a 4% paraformaldehyde solution.During hospitalization, 2 mL venous blood was obtained from each patient and anticoagulated by Ethylenediaminetetraacetic acid (EDTA). White blood cells (WBC) were separated and washed by Hank’s buffer twice and then frozen at −80 °C.The process of paraffin embedding, slicing, and Haematoxylin-eosin (H&E) staining followed the standard procedure.Immunohistochemistry (IHC) staining was used to investigate the location of GPR43 and peptide-YY (PYY). After antigen retrieval (with citrate buffer) and organization background closing (with goat serum), sections of paraffin embedded colon were incubated with primary antibodies  and anti-PYY ) for 1 h. Then, the samples were incubated with a biotin-linked second antibody (goat-anti-rabbit) for 20 min, with streptavidin labeled with horseradish-peroxidase (S-A/HRP) for 20 min and with diaminobenzidine (DAB) for 5 min. The sections were then stained with hematoxylin, and the cover slides were fixed with 50% glycerin.Western blot was used to investigate the amount of ZO-1, GPR43 and Peptide-YY in the mucosa and the amount of GPR43 in the WBC. We chose α-tubulin as the internal control.The mucosae were separated from frozen human colon samples. The method for the preparation of protein solution from mucosa and WBC followed the standard process.Equal amounts of protein (20 µL) were separated by electrophoresis in 15% , 10%  or 5%  SDS-polyacrylamide gels. Separated protein was transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked by skimmed milk, the membranes were incubated with primary antibodies , Boston, USA 1:1000), anti-tubulin , anti-GPR43  or anti-PYY ) at 4 °C overnight. Then, the PVDF membranes were incubated with the corresponding HRP-linked secondary antibody  for 1 h at room temperature. Standard ECL reagent (for α-tubulin and ZO-1) and Immobilon Western HRP Substrate  were used for chemiluminescence. Kodak films were used for the detection of fluorescence from the bands of target proteins. The pixel density of the bands on the film was measured using ImageJ software. The data were presented as target protein expression/α-tubulin expression ratios.Enzyme-linked immunosorbent assay (ELISA) was used to measure myeloperoxidase (MPO) in the colon tissue.g for 15 min at 4 °C. Supernatant was removed and recentrifuged at 1000 × g for 15 min. The concentration of myeloperoxidase (MPO) in the supernatant was measured using ELISA kit  according to the manufacturer’s protocol. The data were presented as pg/mg tissue.The mucosae were separated from frozen human colon samples. Then, they were carefully weighed and homogenized in lysis buffer , 1 mg/mL leupeptide, and 28 mg/mL aprotinin; pH 7.4). Homogenized tissue samples were centrifuged at 1000 × Quantitative real-time polymerase chain reaction (qPCR) was used to measure the level of interleukine-6 (IL-6) mRNA and tumor necrosis factor-α (TNF-α) mRNA.The mucosae of frozen human colon samples were separated. The method of extracting mRNA and reverse transcribing into cDNA followed the protocol described in the PrimeScript RT reagent Kit . The primers used for our investigation are described in qPCR was performed using the SYBR Select Master Mix System . The mRNA expression level was determined using StepOne Realtime PCR system with a ΔCT relative quantification model. The mRNA of the reference gene, glyceraldehyde-phosphate dehydrogenase (GAPDH) was calculated and used as a normalization factor.p-values. p-values < 0.05 were considered statistically significant. Statistical calculations were made using SPSS  19.0.Quantitative data are expressed as the mean ± SD. One-way analysis of variance was used for the calculation of The basic clinical information of the patients is presented in Histological features at low magnification 200×,  show thevs. ac-DF; re-EN vs. re-DF).At high magnification 600×, , we founvs. ac-DF; re-EN vs. re-DF) could be found.We used qPCR to compare TNF-α mRNA and IL-6 mRNA in different groups. GAPDH mRNA was used as the normalization factor. As shown in The result of Western blot  showed tvs. ac-DF; re-EN vs. re-DF).The expression of GPR43 in WBC from peripheral blood is shown in The location of GPR43 was detected with immunohistochemistry. For all of these groups, GPR43 immunoreactivity could be found in colon epithelial cells and was especially high in some crypt cells. Dotted staining could be found in all the epithelial cells. GPR43 high expressing epithelial cells were found in the crypt . In addiThe expression of myeloperoxidase (MPO) in colon mucosa was measured using ELISA. The result of ELISA  revealedOur study observed the histological feature and expression of ZO-1 in the colon of Crohn’s disease patients. ZO-1 is a tight junction protein and can be used as a marker of gut mechanical barrier ,30. CombGPR43/FFAR2 is believed to be expressed in adipose tissue, enteroendocrine cells and immune cells (particularly in neutrophils) ,14. In oIn our study, GPR43 in WBC from peripheral blood was also detected by Western blot. We found that GPR43 in WBC from the ac-food group was significantly higher than that in the other groups, and this figure B was simin vitro studies have confirmed that FFAR2/GPR43 is essential for PMN migration induced by SCFAs, and PMN from GPR43 KO mice cannot respond to SCFA [in vivo studies of GPR43 KO mice made this foreground complex. In one such study, GPR43 KO mice showed more severe inflammation and increased PMN infiltration/activation in the model of dextran sulfate sodium (DSS)-induced colitis [The health benefits of dietary fiber have long been appreciated. Dietary fibers that can influence gastrointestinal microflora are termed prebiotics . These f to SCFA ,23,24. T colitis . GPR43 K colitis . These s colitis . These s colitis . In our  colitis ,38. Alth colitis ,39,40. Det al. [There is a perspective that Crohn’s disease is a consequence of innate immune deficiency ,41, and et al.  proposedThere are some limitations to our study. Our study was retrospective, and only 7~10 patients were included in each group. Two types of enteral nutrition (Peptison and Nutrison) were consumed by the patients, but the presence of dietary fiber was not the only difference between these two types of enteral nutrition . Therefore, the difference between ac-DF and ac-EN could not be attributed solely to dietary fiber. We did not measure the SCFA in the colon or the permeability of the epithelial cells directly. Additional prospective studies are needed to reveal the true interactions of dietary fiber/SCFA with GPR43 in colitis.In conclusion, dietary fiber in enteral nutrition used by patients with active Crohn’s disease increased PMN infiltration in lamina propria. This effect was not observed in Crohn’s disease in remission. Further research is needed to evaluate whether it is useful for CD patients to improve the impaired neutrophil recruitment.


Rats of AKI were induced by kidney I/R surgery and randomly divided into three groups: I/R control receiving PBS injection; MSC group receiving normal MSC injection; hypoMSC group receiving hypoxia-preconditioned MSC injection. It was demonstrated in vitro that paracrine effects of MSCs were significantly enhanced, especially angiogenic factors. Dihydroethidium (DHE) staining showed that antioxidative activities of MSCs were significantly enhanced by hypoxia stimulation. Vascularization, apoptosis, and histological injury were all significantly improved in hypoMSC injected group compared with that in control and MSC injected groups. Finally, the renal function was also significantly improved in hypoMSC injected group compared with that in the other two groups as assessed by the serum creatinine and BUN levels.Hypoxia preconditioning has been confirmed as an effective strategy to enhance the therapeutic potentials of mesenchymal stem cells (MSCs), such as for myocardial ischemia. However, whether hypoxia preconditioning would produce beneficial effects on MSC-based renal repair has not been demonstrated. In the study, we aimed to determine the feasibility and efficacy of hypoxia preconditioning to enhance MSC-based therapy of acute kidney injury (AKI). MSCs were isolated from human adipose tissues. The paracrine effects of MSCs under normoxia and hypoxia were determined  Acute kidney injury (AKI), one of the serious nephropathy, is severely threatening human health , 2. MultIschemia-reperfusion (I/R) injury is an important cause of AKI . I/R of In recent years, transplantation of stem cells has been proposed as a promising strategy for protection of kidney after AKI , 10, 12.In present study, we aimed to determine whether hypoxia preconditioning could exert beneficial effects on MSC-based therapy of AKI. Rats of AKI were induced by kidney I/R surgery. Then, MSC or hypoxia-preconditioned MSC was injected into the left kidney cortex; PBS was used as control. The production of ROS in I/R renal tissues was determined by dihydroethidium (DHE) staining. Vascularization, apoptosis, tissue damage, and function of ischemic kidneys were evaluated by multimodality methods.Male adult SD rats were housed in cages and were fed a standard laboratory chow and water ad libitum. All procedures were consistent with the guidelines and policies of the Hospital Animal Care and Use Committee. Each procedure was optimized to minimize the pain of animals.n = 25 for each group). Animals were randomly divided into four groups: animals receiving 100 μL saline injection after AKI; animals receiving 2 × 106 human adipose-derived MSCs  injection after AKI; animals receiving 2 × 106 hypoxia-preconditioned human adipose-derived MSCs  after AKI. During the experiment, two rats died before the end. Two other rats were used so that each group still has 25 animals.A total of 75 rats were used in the study . The liquid was filtered by 80 μm mesh and then centrifuged at 800 g for 8 min. The cell pellet was resuspended in αMEM supplemented with 10% FBS. The cells were seeded onto tissue culture plates and cultured in 37°C, 5% CO2 incubator.Adipose tissues were obtained from raw human lipoaspirates. Human adipose-derived MSCs were isolated as the previous report , 26. BriThe immunophenotype of MSCs was determined by flow cytometry according to previous reports ; CD29, C6 MSCs were seeded on culture plates and cultured in normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h. Then, the medium was collected for growth factor assay; bFGF, VEGF, and HGF were determined by ELISA array. Data was expressed as mean ± SEM pictograms (pg) of the factor per 106 MSCs.10Cell viability after hypoxic treatment was detected by flow cytometry. Briefly, cells were collected and incubated with propidium iodide (PI) solution. After that, cells were analyzed by flow cytometry.2) and incubated for 24 h. After that, cells were collected for the following transplantation surgery.The acute renal ischemia/reperfusion (I/R) injury was induced by transiently clamping bilateral renal pedicles as previously reported . BrieflyμL saline or 2 × 106 MSCs or 2 × 106 hypoxia-preconditioned MSCs (n = 25/group) were injected into the left kidney cortex using a 28-gauge needle. The abdomen was then closed and rats were allowed to recover with cautious care.At the time of reperfusion, 100 n = 5 for each group). Kidneys were rapidly excised and frozen in optimal cutting temperature freezing medium for preparation of cryosections. Dihydroethidium (DHE) staining was performed on the cryosections to determine the ROS levels according to the previous report [Animals were sacrificed at day 1 after surgery (s report . Poststas report .n = 5 for each group) after surgery and fixed in 4% paraformaldehyde. 4 μm paraffin-embedded sections were prepared with standard protocols. Hematoxylin and eosin (H&E) staining was performed for tubular injury analysis. The examination and scores of the sections were carried out by an experienced technician who was blind to the experiment [The kidney samples were collected at 3 days and 1 week  according to previous reports . Then, tμm paraffin-embedded sections were prepared for assay. TUNEL staining was performed according to manufacturer's guideline. Then, TUNEL-stained sections were observed under light microscopy; the positive-stained cells were counted and compared among different groups.At one week, the apoptosis of renal cells was assessed based on terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. As described above, 4 Total RNA was extracted using RNAprep pure Cell/Bacteria Kit (TIANGEN) according to manufacturer's instruction. Reverse transcription was performed using standard procedures to synthesize first-strand cDNA. The gene-specific primers were designed using primer3.0 and used in the PCR amplification. PCR product was detected by agarose electrophoresis.Kidney samples were obtained and homogenized with a rotor stator. Then, tissues were lysed in Laemmli sample buffer (Bio-Rad). Proteins were collected and concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific). Proteins were loaded on a 12–15% sodium dodecyl sulfate polyacrylamide gel for electrophoresis. Then, proteins were transferred to a PVDF membrane (Roche). After blocking with 5% nonfat dried milk (in TBST), the primary antibody was added and incubated overnight at 4°C. Unconjugated antibodies were removed by washing with TBST; the membrane was further incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature . The protein bands were detected by enhanced chemiluminescence reagent (Applygen).n = 10 for each group). Serum levels of creatinine and blood urea nitrogen (BUN) were measured and compared between the four groups. Measurement of creatinine and BUN level was performed with standard protocol in our hospital.To assess the renal function, blood was taken at 1 d, 3 d, 14 d, and 28 d after the surgery for each rat , human adipose MSCs exhibited fibroblast-like morphology  expression by RT-PCR, but the factor was not upregulated in mRNA level (Data not shown). As is known, oxygen regulation of HIF-1α is essentially produced at protein level. Prolyl hydroxylases (PHD) modify HIF-1α proteins for their rapid degradation. Hypoxia leads to inhibition of PHDs and allows for the presence of HIF-1α proteins. Therefore, we further analyzed the HIF-1α expression by western blotting and found that it was increased in protein level (data not shown).After hypoxic treatment, cell viability analysis by flow cytometry showed that MSC viability was maintained at a high level. No significant cell death was found between hypoxia-treated MSCs and normal MSCs . The parP < 0.01, The increase in superoxide production after I/R is associated with augmented oxidative stress, which is a main cause of I/R injury. It has been demonstrated that factors released from stem cells could significantly scavenge superoxides in ischemic tissues . TherefoTo determine the effects of hypoMSCs on angiogenesis in ischemic kidney tissues, paraffin-embodied kidney sections were prepared 4 w after MSC transplantation. As shown in P < 0.01). Furthermore, compared with MSC injection group, apoptotic cells were further decreased in hypoMSCs injection group (P < 0.01).After I/R and MSC injection, we performed TUNEL staining to determine apoptosis in I/R kidney. After TUNEL staining, kidney sections were observed under light microscopy and positively stained cells were quantified in each group. As shown in Figures P < 0.01) while the phosphorylation of Akt was significantly raised (P < 0.01), indicating that injection of MSC significantly protected renal cells against apoptosis. Then, the antiapoptotic effects were further compared between MSC injection group and hypoMSCs injection groups. The results showed that injection of hypoMSCs further lowered cleaved csp3 expression while it improved Akt phosphorylation compared with normal MSCs (P < 0.01).Western blotting analysis was performed to provide additional evidence for cell apoptosis. Detection of apoptotic protein caspase 3 (Csp3) and prosurvival protein Akt was performed at 1 week and we obtained consistent results with TUNEL staining. As shown in P < 0.01, P < 0.01).Three days and one week after renal I/R surgery, kidney tissue specimens from each group were obtained. The tissue sections were stained by H&E staining and then assessed. Under light microscopy, significant tubular cell necrosis, cytoplasmic vacuoles, loss of brush border, and tubular dilatation could be observed in PBS injection group , indicatAt 3 days, 1 w, 2 w, and 4 w after surgery, the effects of MSC injection on renal function were evaluated. The serum levels of creatinine and BUN in each group were measured. As shown in Acute kidney I/R is associated with significant renal injury, which may be partially attributed to the lack of blood supply, mass production of superoxides, and so forth , 28. Dif in vivo application; cotransplantation with adjuvants is promising in future application; it could improve the ischemic microenvironment assisting the retention and survival of stem cells, but it fails to stimulate the therapeutic potentials of stem cells. Preconditioning before transplantation is effective to stimulate the therapeutic potentials of stem cells. Combined application of adjuvants and preconditioning would exert more beneficial effects on stem cell therapy and deserved in-depth investigation.In previous reports, several strategies have been investigated to enhance the therapeutic potential of MSCs, including (1) gene modification, that is, making MSCs overexpress cytokine genes or antiapoptotic genes, which significantly improved their therapeutic capacities or survival in ischemic tissues , 29; (2) in vivo finding that revascularization in hypoMSC-treated kidneys was the most significant (This study demonstrated that angiogenic factors, such VEGF and bFGF, were significantly enhanced in MSCs by hypoxia treatment. This may explain thenificant . We also in vivo, and significant therapeutic effects could also be obtained with MSC-conditioned medium without MSCs [Different mechanisms may exist underlying the observation that transplanted MSCs could modify all kidney effects of I/R, such as cell differentiation and paracrine effects. However, it has been demonstrated by independent groups that rare MSCs would differentiate into target cellsout MSCs , 31–33. In conclusion, the study revealed that hypoxia preconditioning significantly enhanced the therapeutic effects of human adipose-derived MSCs on AKI. Further, we confirmed that these enhanced therapeutic potentials by hypoxia preconditioning were, at least partially, attributed to the enhanced antioxidative, antiapoptotic, and angiogenic capacities of MSCs by hypoxia stimulation. The study confirmed the feasibility and efficacy of hypoxia preconditioning in MSC-based therapy for AKI and provided promising strategy in application of MSCs after renal injury.


To know the effect of age on survival outcome in operated and non-operated patients with colon cancer.From the Surveillance, Epidemiology, and End Results database, we identified 123,356 patients with colon cancer who were diagnosed between 1996 and 2005, grouped them as older or younger than 40 years and analyzed their 5-year cancer-specific survival (CSS) data, along with some risk factors, using Kaplan–Meier methods and multivariable Cox regression models.P<0.001), more mucinous and signet-ring histology (P<0.001), advanced AJCC stage (P<0.001), and were more likely to undergo surgery (P<0.001). For surgically treated patients, age did not significantly affect 5-year CSS . Further analysis showed that age was an independent prognostic factor in stage I–IV disease , but not for patients with unknown disease stage (P = 0.52). For non-surgically treated patients, age significantly affected 5-year CSS  in univariate analysis; and was an independent prognostic factor (P<0.001) in multivariate analysis.The younger group had significantly higher pathological grades (The CSS rate for younger CC patients was at least as high as for older patients, although they presented with higher proportions of unfavorable factors and more advanced disease. Colon cancer (CC) is one of the most common malignancies; combined with rectal cancer, it is the second leading cause of cancer-associated mortality in the United States , 2. Genewww.seer.cancer.gov/seerstat Version 8.1.2) was used to identify patients who were diagnosed with invasive CC (C18.0–19.9) between 1996 and 2005. Only patients aged between 15 and 80 years were included. Patients were excluded if they had in situ TMN staging, or had another primary tumor besides colon cancer. Histology types were limited to adenocarcinoma , mucinous adenocarcinoma (8480/3), and signet-ring cell carcinoma (8490/3). Tumor-node-metastasis classifications were based on the criteria of the American Joint Committee on Cancer (AJCC) Cancer Staging Manual , by which criteria patients were restaged. Age, sex, race, histological type, AJCC stage, and CCSS were analyzed. Because the SEER data contain no adjuvant chemotherapy information, adjuvant chemotherapy was not assessed. The primary endpoint of the study was CCSS, which was calculated as the time between diagnosis and CC-specific death. Deaths caused by CC were treated as events; living patients or deaths from other causes were censored data.The SEER program collects data on the most recent cancer incidence, prevalence, mortality, and survival data from 17 population-based cancer registries that represent approximately 28% of the US population . SEER dahttp://seer.cancer.gov/data/options.html). Requests for data access can be sent by following the instructions on that website. Permission to access the research data files was obtained with the reference number 11375-Nov2014. The study contained no personal identifying information and required no informed consent. Anonymous patient’s data were extracted from online SEER registries, which require no further institutional review approval prior to use. The study was approved by the Review Board of Nanjing Medical University, Nanjing, China.This study was based on the free public SEER database, which is an authoritative source of information on cancer incidence and survival in the United States. All relevant data were obtained from SEER*Stat  and was stratified by AJCC stages I–IV, or unknown stage. Survival curves were provided by Kaplan–Meier estimates; differences between two groups were analyzed by log-rank test. Multivariate Cox regression models were performed to evaluate risk factors for survival outcomes. All statistical analyses were completed using the statistical software Statistical Package for Social Sciences (SPSS) for Windows, version 17 . P < 0.05 was considered significant.The relation of age (younger vs older) with clinicopathological features was analyzed by chi-square (χ2) test. Comparisons of continuous variables between two groups were completed using Student’s We identified 123,356 eligible patients with CC in the SEER database during the 10-year study period (1996–2005), of whom 63,436 (51.4%) were males and 59,920 (48.6%) were females; and 4,504 (3.6%) were no older than 40 years (younger patients), and 118,852 (96.4%) were older than 40 years (older patients). The median follow up time was 69 months (interquartile range [IQR]: 22–106 months) in the younger group and 65 months (IQR: 18–100 months) in the older group.P<0.05) and less frequent among Caucasians (P<0.001) than for older patients. Younger patients also had significantly more poorly or undifferentiated tumors (P<0.001), more mucinous or signet-ring histologies (P<0.001), presented with more advanced AJCC stages , and underwent more surgery (P<0.001). The younger and older groups did not significantly differ by sex , nor did it significantly differ among all patients treated with surgery , which indicated a need for the increased incidence in this population to be further investigated with an eye to prevention and early detection. Previous studies are somewhat complicated by the lack of a standard definition of young CC patients [A sharp increase over recent decades in the number of younger patients diagnosed with CC has been reported in several countries –7. Its rpatients , 22–25. patients –28.The prognosis of CC in younger patients is controversial. Various studies have reported poorer prognosis among young patients with CC than in older patients. This has been attributed to more advanced disease at diagnosis, tumor stage , 14, 29,In general, surgeons are more inclined to use all therapeutic options in young patients as they are in better health and are more able to tolerate toxicities associated with chemotherapy and suffer from fewer postoperative complications , 30–34. Younger patients who did not undergo surgery still had better 5-year CCSS than older ones. Our analysis of the SEER data, which are nationally representative and use a large, unselected group of patients with no surgical therapy, showed that younger patients had better 5-year CCSS than older ones. Although a clinical trial showed that survival of patients with advanced CRC was significantly increased when incorporating new therapeutic agents , a meta-In summary, patients with CC who are aged 40 and younger have higher CCSS rates than their older counterparts, both after surgery by stage, and among those who are not treated with surgery. Clinicians should consider age in their assessments and treatment decisions for patients with CC.


Parental smoking during pregnancy is associated with lower birthweight and gestational age, as well as with the risks of low birthweight (LBW) and preterm birth. The present study aims to assess the association of parental smoking during pregnancy with birth outcomes in urban and rural areas.This was a secondary analysis of data collected in the Indonesia Family Life Survey, between 1993 and 2007, the first national prospective longitudinal cohort study in Indonesia. Retrospective data of parental smoking habits, socioeconomic status, pregnancy history and birth outcomes were collected from parents with children aged 0 to 5 years (n = 3789). We assessed the relationships between the amount of parental smoking during pregnancy with birthweight (LBW) and with gestational age (preterm birth).We found a significant reduction in birthweight to be associated with maternal smoking. Smoking  was associated with a decrease in the gestational age and an increased risk of preterm birth. Different associations were found in urban area, infants born to smoking fathers and both smoking parents (>20 cigarettes/day for both cases) had a significant reduction in birthweight and gestational age as well as an increased risk of LBW and preterm birth.Residence was found to be an effect modifier of the relation between parental smoking during pregnancy, amount of parental smoking, and birth outcomes on their children. Smoking cessation/reduction and smoking intervention program should be advised and prioritized to the area that is more prone to the adverse birth outcomes. Tobacco smoking is a global public health problem. Cigarette smoke not only affects smokers but also contributes to the health problems of nonsmokers . BirthweMany chemicals in maternal smoking and second-hand smoke pass from the pregnant woman to the fetus through the placenta ,5. NicotThe National Socio-Economic Survey and Basic Health Research, a series of population-based surveys in Indonesia, revealed that smoking prevalence among adults 15 years or older has increased from 26.9% in 1995, 31.5% in 2001, 34.4% in 2004, 34.2% in 2007 to 34.7% in 2010. This largely reflects an increasing smoking prevalence among males, from 53.4%, 62.2%, 63.1%, 65.6% to 65.9% during this period. As smoking among adult males has been steadily increasing, so too has consumption among women, from 1.7%, 1.3%, 4.5%, 5.2% to 4.2%, with almost a 300% increase from 1.3% in 2001 to 5.2% in 2007 . SmokingIn Indonesia, health disparities across the urban–rural gradient exist. Rural residents compared to urban, have poorer health outcomes and is one of influencing factors contributing to health inequalities in Indonesia . Urban–rhttp://www.rand.org/labor/FLS/IFLS.html). The author granted access to the dataset by registering for access to the IFLS data download link. The sample represents about 83% of the Indonesian population, covering 13 major provinces out of a total of 27 provinces ”. “Do you still have the habit of smoking cigarettes or have you totally quit? [Still going on/have quit]”. We then constructed two variables: (a) paternal smoking status and (b) maternal smoking status. Each variable was classified into smokers, quitters, and non-smokers. To get information about the paternal and maternal smoking status during pregnancy, we pieced together information on “At what age (years) did you start to smoke cigarettes on a regular basis?” and “How long  have you totally quit from smoking cigarettes?” From the first question, we obtained information on whether the father or mother who smoked was smoking during pregnancy, by simultaneously looking at the age they began to smoke, their age, and their child’s age. From the second question, we obtained information on whether the father or mother who quit smoking smoked during pregnancy, by simultaneously looking at the duration of quitting smoking and their child’s age. We then constructed two variables: (a) paternal smoking during pregnancy and (b) maternal smoking during pregnancy. We classified each variable into smokers and non-smokers. We then combined paternal and maternal smoking during pregnancy into one key explanatory variable called “parental smoking during pregnancy”, which was categorized into: (a) neither parent smoked, (b) only the father smoked , (c) only the mother smoked ; and (d) both parents smoked.For parental smoking amount, the question was asked for mother and father separately, such as “How much do/did you approximately consume now/before totally quitting? [Total amount in cigarettes a day]”. We then constructed two variables: (a) paternal smoking amount and (b) maternal smoking amount. We classified paternal smoking into: (a) none, (b) 1-10 cigarettes/day, (c) 11-20 cigarettes/day, and (d) >20 cigarettes/day. Due to the small percentage of mothers who smoked > 10 cigarettes/day, maternal smoking amount was classified into three categories only: (a) none, (b) 1-10 cigarettes/day, and (c) >10 cigarettes/day. We then combined paternal and maternal smoking amount into one key variable called “smoking amount in both parents”. We considered three categories: (a) none, (b) 1-20 cigarettes/day, and (c) >20 cigarettes/day.2, only mother ≥ 25 kg/m2, only father ≥ 25 kg/m2, and both parents ≥25 kg/m2), household income , and residence . The information about the father’s education, maternal employment status, parental BMI, household income, and residence were obtained after the child was born. We used those variables as covariates for birthweight and gestational age because we assumed that those variables did not exhibit much difference before and after the child was born.We considered the following as covariates for birthweight and gestational age: sex of infant (boy and girl), birth order , maternal age at delivery , father’s education , maternal employment status (not working and working), parental BMI . Multiple logistic regression analyses controlled for covariates estimated the odds ratios (ORs) and 95% confidence intervals of LBW and preterm birth. To evaluate the functional relation between birth outcomes as a continuous and binary dependent variable and parental smoking amount, we applied a Generalized Additive Model (GAM). The models were adjusted for birth order. By default, the smoothing parameter for each B-spline term is chosen to yield four degrees of freedom. We set the statistical threshold for significance at 0.05.For sensitivity analyses, we re-ran the analyses to assess whether the birth outcomes results related to parental smoking would change substantially if we included/omitted adjustment to various covariates and interactions to ensure that they produce approximately the same results. We also tested the final model by including/omitting adjustment to various covariates to the stratified analysis. We found that a stratified model is sensitive to maternal age in the rural area but not in urban area.2 (79.9%), a low income family (24.9%) or in a rural area (58.7%). We found similar results for infants born to smoking mothers and parents who both smoke . Mothers who smoked > 10 cigarettes per day had a significant fourfold increase in LBW risk compared with nonsmoking mothers . Compared with the nonsmoking parents, the risk of LBW significantly increased in for parents who (combined) smoked >20 cigarettes/day .Due to small percentage of mothers who smoked and both parents who smoked, we only put birth order as a covariate for the association between parental smoking amount and birth outcomes of their children. The association between paternal smoking amount and birthweight was evident in the group of fathers who smoked >20 cigarettes/day, with a 151 g decrease in birthweight. The association of the maternal smoking amount with birthweight and risk of LBW appeared to be dose-dependent. All infants of the maternal smoking groups had lower birthweights, ranging from 117 g (1-10 cigarettes/day) to 351 g (>10 cigarettes/day). The largest estimate of association between the smoking amount in both parents and birthweight was among parents who smoked > 20 cigarettes/day (combined), with an average birthweight decrease of 156 g. The fathers who smoked >20 cigarettes/day had a significantly higher risk of having LBW infants than those fathers who did not smoke . Compared with the nonsmoking parents, children with smoking mothers were 3.37 times more likely to be born at preterm gestation . Children with smoking parents were 3.10 times more likely to be born at preterm gestation than those with nonsmoking parents .Table p = 0.001). The risks of preterm birth were significantly higher in mothers who smoked 1-10 cigarettes per day  and >10 cigarettes per day . With the nonsmoking group as the baseline, the risk of preterm birth increased with the number of cigarettes smoked daily for both parents > 20 cigarettes/day .The association between the paternal smoking amount and gestational age was evident in the group of fathers who smoked >20 cigarettes/day, with a significantly decrease in gestational age (0.44 weeks). The association of maternal smoking amount with gestational age and the risk of preterm birth appeared to be dose-dependent. The maternal smoking group had shorter gestational ages, ranging from 0.72 weeks (1-10 cigarettes/day) to 1.01 weeks (>10 cigarettes/day). The largest estimated association between the smoking amount in both parents and gestational age was found in the group of >20 cigarettes/day, with a significant decrease in gestational age (0.47 weeks). The association between paternal smoking and preterm birth was more obvious in infants whose fathers smoked >20 cigarettes/day  in urban area and 16 g higher (p = 0.168) in rural area than that of infants born to nonsmoking parents. The mean birth weight of infants born to smoking mothers was 14 g lower (p = 0.878) in urban area and 262 g lower (p < 0.001) in rural area than that of infants born to nonsmoking parents. The mean birth weight of infants born to smoking parents was 186 g lower (p = 0.049) in urban area and 28 g lower (p = 0.608) in rural area than that of infants born to nonsmoking parents. However, no significant associations were found between LBW and parental smoking during pregnancy in four groups.From the stratified analyses in Table p < 0.001) in urban area and 78 g lower (p = 0.058) in rural area than that of infants born to nonsmoking fathers. The association of maternal smoking amount with birth weight and risk of LBW appeared to be dose-dependent in both urban and rural areas, ranging from -192 g (1-10 cigarettes/day) to -299 g (>10 cigarettes/day) in urban area and from -57 g (1-10 cigarettes/day) to -365 g (>10 cigarettes/day) in rural area. The mean birth weight of infants born to the smoking amount in both parents >20 cigarettes/day were 223 g lower (p < 0.001) in urban area and 99 glower (p = 0.010) in rural area than that of infants born to nonsmoking parents. The odds ratios (ORs) for LBW of infants born to fathers who smoked >20 cigarettes/day were 2.89 (p = 0.001) in urban area and 1.52 (p = 0.160) in rural area, respectively. The OR for LBW of infants born to mothers who smoked >10 cigarettes/day in urban and rural area was 1.13 (p = 0.905) and 6.23 (p = <0.001), respectively. The ORs for LBW of infants born to the smoking amount in both parents >20 cigarettes/day were 2.66 (p = 0.001) in urban area and 1.90 (p = 0.036) in rural area, respectively.The mean birth weight of infants born to fathers who smoked >20 cigarettes/day was 231 g lower (p = 0.086) in urban area and 1.31 weeks shorter (p = <0.001) in rural area. The mean gestational age of infants born to smoking parents was 1.09 weeks shorter (p = 0.003) in urban area and 0.2 weeks shorter (p = 0.440) in rural area. The ORs for preterm birth of infants born to smoking mothers were 1.87 (p = 0.307) in urban area and 5.14 (p = 0.003) in rural area, respectively. The ORs for preterm birth of infants born to smoking parents were 4.70 (p = 0.002) in urban area and 1.49 (p = 0.638) in rural area, respectively.From the stratified analyses in Table p = 0.001) in urban area and 0.21 weeks shorter (p = 0.195) in rural area than that of infants born to nonsmoking fathers. The association of maternal smoking amount with gestational age appeared to be dose-dependent in rural area, but not in urban area. All infants of the maternal smoking groups had shorter gestational age, ranging from 0.75 weeks (>10 cigarettes/day) to 0.98 weeks (1-10 cigarettes/day) in urban area and from 0.5 weeks (1-10 cigarettes/day) to 1.21 weeks (>10 cigarettes/day) in rural area. The largest estimate effect of the smoking amount in both parents on gestational age was found among group of >20 cigarettes/day, decreased in gestational age of 0.72 weeks (p < 0.001) in urban area and -0.28 weeks (p = 0.068) in rural area. The ORs for preterm birth of infants born to fathers who smoked >20 cigarettes/day were 2.80 (p = 0.001) in urban area and 1.60 (p = 0.143) in rural area, respectively. There was a dose-dependent association between maternal smoking amount and risk of preterm birth both in urban and rural areas. The ORs for preterm birth of infants born to mothers who smoked 1-10 cigarettes/day were 3.11 (p = 0.007) in urban area and 2.16 (p = 0.115) in rural area, respectively and >10 cigarettes/day in urban and rural area were 4.34 (p = 0.034) and 6.76 (p < 0.001), respectively. The ORs for preterm birth of infants born to both parents’ smoking amount >20 cigarettes/day were 2.79 (p = 0.001) in urban area and 1.97 (p = 0.045) in rural area, respectively.After adjusting for birth order, the mean gestational age of infants born to fathers who smoked >20 cigarettes/day was 0.71 weeks shorter  that is specifically designed to analyse data when the impact of predictors on the outcome variables are nonlinear. We showed that paternal smoking amount, maternal smoking smoking, and smoking amount in both parents were all closely associated with birth outcomes, except for maternal smoking amount, which was not associated with birthweight and LBW. The sharp decrease in birthweight between 15-50 cigarettes/day and in gestational age between 12-35 cigarettes/day as well as the gradual increased in LBW between 25-75 cigarettes/day and in preterm birth between 1-50 cigarettes/day for smoking fathers and both smoking parents provides a partial explanation that heavy cigarette smoking can endanger unborn babies. The shortened gestational age between 1-5 cigarettes/day and between 20-35 cigarettes/day as well as the modest increased in preterm birth between 1-10 cigarettes/day for smoking mothers also suggest that even a light cigarette smoking can have an outsize effect on the pregnancy duration.Our study confirms different detrimental effects on birth outcomes of smoking during pregnancy by different residence. Paternal smoking did not decrease birth weight and did not have any significant influences on the LBW, gestational age, or preterm birth of their children in both urban and rural area. Previous studies have supported our finding -19. The Maternal smoking, especially mothers who smoked >10 cigarettes/day was not significantly associated with birth weight, gestational age, and the risk of LBW and preterm birth in urban area, but the associations were significant in rural area. There was a significant higher percentage of smoking mothers, non-educated mothers, working mothers, low family income in rural area than that in urban area. This is consistent with the nationally representative surveys in Indonesia showed that female adults smoking prevalence was higher in rural areas than the urban areas ,23,46. ANumerous studies have demonstrated that maternal smoking and/or maternal exposure to passive smoke during pregnancy is associated with adverse birth outcomes, however further evidences for smoking behaviors by both parents on birth outcomes have rarely been analyzed. There is insufficient scientific research on smoking and health in the Indonesian population. To our knowledge, this study is among the first studies in Indonesia to assess the association of paternal smoking, maternal smoking, and both parents smoking during pregnancy with birth outcomes on their children in urban-rural settings.The findings of the present study should be viewed with some limitations in mind. First, reports of parental smoking during pregnancy were retrospective. While studies show high reliability in self-reported smoking status , nonetheA significant reduction in birthweight and gestational age, as well as an increased risk of LBW and preterm birth were found to be associated with maternal smoking and the heavy amount of parental smoking. Smoking cessation/reduction should be advised to pregnant women to reduce morbidities in their neonates. In urban areas, there continues to be a clear need for health care professionals to counsel both smoking expectant mothers with smoking spouses, especially when their husbands smoked >20 cigarettes/day regarding the adverse consequences of smoking during their pregnancy and offer behavioral counseling. Not only does maternal smoking during pregnancy, directly and indirectly, influence the well-being of the children  but also has key health consequences for the expecting mother.


Intrahepatic cholestasis of pregnancy is always accompanied by adverse fetal outcomes such as malfunctions of respiration. Farnesoid X receptor (FXR) plays a critical role in the homeostasis of bile acids. Thus, we are determined to explore the effects of farnesoid X receptor (FXR) and five bile acids on respiratory rhythm generation and modulation of neonatal rats. Spontaneous periodic respiratory-related rhythmical discharge activity (RRDA) was recorded from hypoglossal nerves during the perfusion of modified Krebs solution. Group 1–6 was each given GW4064 and five bile acids of chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), cholic acid (CA) as well as ursodeoxycholic acid (UDCA) at different concentrations to identify their specific functions on respiratory rhythm modulations. Group 7 was applied to receive FXR blocker Z-guggulsterone and Z-guggulsterone with the above bile acids separately to explore the role of FXR in the respiratory rhythm modulation. Group 8 was given dimethyl sulfoxide (DMSO) as controls. Apart from UDCA, CDCA, DCA LCA and CA all exerted effects on RRDA recorded from hypoglossal nerves in a concentration-dependent manner. Respiratory cycle (RC), Inspiratory time (TI), Expiratory Time (TE) and Integral Amplitude (IA) were influenced and such effects could be reversed by Z-guggulsterone. FXR may contribute to the effects on the modulation of respiratory rhythm exerted by bile acids. Conversion of cholesterol to bile acids plays an important role in the cholesterol homeostasis and serves as the main pathway for cholesterol catabolism As one of the members in the nuclear hormone receptor superfamily, FXR is highly expressed in liver, kidney, adrenal glands and intestine. As the target genes of FXR like bile salt export pump (BSEP) and small heterodimer partner (SHP), FXR is able to maintain the homeostasis of cholesterol and bile acids via regulating their expressions by binding to the FXR response element (FXRE) located on the promoters of these genes However, excessive serum concentrations of bile acids are toxic to the cells. As a multifactorial liver disorder in clinical scenarios, intrahepatic cholestasis of pregnancy is associated with abnormal bile acid levels in serum, causing not only maternal itching but also high infant morbidity and mortality Rhythmic breathing is essential for blood oxygenation and survival. Respiratory movements shall be spontaneously produced from birth onwards. Within the brainstem, a complex neural network plays a critical role in periodic respiratory generation. Numerous studies show that the respiratory rhythm seems to be generated in the medulla, whereas the exact place of respiratory rhythm generation remains controversial Our previous study showed that there was a significant change in the respiratory function in animals with high serum concentration of bile acids. Bile acids in the serum can have direct effects on respiratory functions besides reflex responses. Still, the underlying mechanisms remain debatable Neonatal Sprague-Dawley rats (0-3d) were obtained from the Laboratory Animal Center of Southern Medical University (SCXK 2006-0015). All the experimental procedures were in compliance with the National Institutes of Health Guidelines for Care and Use of Laboratory Animals and received approval from the Bioethics Committee of Southern Medical University.Z-guggulsterone was purchased from Santa Cruz Biotechnology , Chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), ursodeoxycholic acid (UDCA) were purchased from Shanghai Yiji Co., Ltd . Lithocholic acid (LCA) and cholic acid (CA) were purchased from Shanghai Guidechem Co., Ltd .2, 2.4 mmol/L; MgSO4, 1.3 mmol/L; NaHCO3, 26 mmol/L; glucose, 30 mmol/L). The cerebrum was quickly removed as well as the cerebellum by transacting at the intercollicular level. The above procedures were completed within 2–3 min. The cranial and the spinal nerves were retained as much as possible. The preparation which included the medial region of the nucleus retrofacialis (mNRF), the pre-Bötzinger complex and part of the ventral and dorsal respiratory group with the hypoglossal nerve roots was then placed in a 0.5–2 ml bath and perfused at a rate of 4–5 ml/min with modified Krebs solution (pH 7.35–7.45) equilibrated with 95% O2 – % CO2 gas. The temperature of the perfusion medium was kept at 27°C Under deep ether anaesthesia, the head and vertebral column of Sprague-Dawley rats from birth to 3 days of age were isolated and placed in a dissection chamber filled with the modified Krebs solution  were used. Filled with modified Krebs solution, the electrodes were connected through Ag-AgCl wires to pre-amplifiers. Spontaneous periodic respiratory-related rhythmical discharge activity (RRDA) was recorded from hypoglossal nerves and displayed on the screen. The output of the pre-amplifier was fed to a recorder. Respiratory cycle (RC), inspiratory time (TI), expiratory time (TE) and integral amplitude (IA) of respiratory-related rhythmical discharge activity (RRDA) were then analyzed. Drugs were dissolved in the modified Krebs solution and applied by perfusion −1∼10−5 mg/mL CDCA, 10−1∼10−5 mg/mL DCA, 1∼10−4 mg/mL LCA, 1∼10−3 mg/mL CA and 1∼10−3 mg/mL UDCA). After RRDA was stably recorded from hypoglossal nerves for at least 10 minutes during which data recorded was used as controls, the bile acids at different concentrations were then applied. The bile acids were washed out after experiments were finished. Group 7 was conducted to observe the effects of FXR blocker Z-guggulsterone (20 µM) and combination of Z-guggulsterone and the above four bile acids . After RRDA was stably recorded for at least 10 min during which the data recorded was used as controls, Z-guggulsterone was firstly applied. After 15 minutes of observation, Z-guggulsterone was washed out. The combination of Z-guggulsterone and the above four bile acids was then applied after full recovery of RRDA. Group 8 was conducted with DMSO  as control. As the time when the effects of bile acids or other agents took place varied in each experiment, we waited for about 10min after the effects first showed up to make sure our results were stable and convincible and recorded the data for at least 15min during which the middle 10min of data was used to analyze.This experiment was divided into 8 groups: Group 1 was conducted to observe the effects of GW4064 (3 µM) on RRDA recorded from hypoglossal nerves. Group 2–6 was each conducted to observe the effects of five different bile acids  on respiratory rhythm generation and modulation. The above five bile acids were dissolved in dimethyl sulfoxide (DMSO) at different concentrations  and GW4064 (50 mg/kg) were performed on neonatal Sprague-Dawley rats (0-3d). The injections were conducted 6 times with an interval of 40 min. The brains were separated 30min after the last injection and were prepared for RT-PCR.Total RNA was isolated from tissue samples with Trizol (Invitrogen) according to the manufacturer's instructions. RNA was reverse transcribed by M-MLV Reverse Transcriptase  according to the manufacturer's protocol. Sequence-specific primers for FXR and SHP were, respectively, shown in Real-time PCR was performed with qPCR Master Mix  on a Real-Time PCR detection instrument using the SyBr Green detection protocol as outlined by the manufacturer. Briefly, the amplification mixture consisted of 0.5 µM primers, 10 µl of qPCR Master Mix, and 1.5 µl template DNA in a total volume of 20 µl. Samples were amplified with the following program: initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation for 15 s at 95°C and annealing/elongation for 60 s at 60°C. All PCRs were run in triplicate, and control reactions without template were included.All experiment data was processed by SPSS 17.0. All experiment results were shown using sample mean ± standard deviation and student's t tests were used to test statistical significance. For comparisons in the RT-PCR test, data were analyzed using a one-way analysis of variance (ANOVA), followed by LSD test. P<0.05 indicates a significant difference.See Bile acids are acknowledged as physiological ligands of FXR. Under our experimental conditions, all four bile acids  and GW4064 except UDCA were able to increase the mRNA of FXR. As one of the target genes of FXR, SHP was also required in the homeostasis and regulation of bile acids. The mRNA levels of SHP were increased by CDCA, CA, DCA, LCA and GW4064. Similarly, UDCA failed to increase the mRNA of SHP. Although the mRNA levels of FXR increased by CDCA did not reach a significant difference compared to Control, CDCA markedly increased the mRNA of SHP. SeeGW4064 is a highly specific synthetic FXR agonist. As our results indicted, GW4064 (3 µM) significantly decreased RC by 17.19%, TI by 20.89% and TE by 16.91%. And such effects were completely reversed by co-administration of GW4064 and Z-guggulsterone (20 µM). −1 mg/mL decreased RC by 25.37% (P<0.05), TE by 27.20% (P<0.05). And 10−3 mg/mL CDCA decreased TI by 19.33% (P<0.05), IA by 12.11% (P<0.05). 10−5 mg/mL CDCA decreased RC by 22.94% (P<0.05), TI by 21.50% (P<0.01) and IA by 41.75% (P<0.05). SeeAs CDCA is the most potent agonist for FXR, we first investigated the influence of CDCA on RRDA. When applied alone, CDCA at the concentration of 10−4 mg/mL increased TE by 25.37% (P<0.05). And 10−5 mg/mL DCA decreased TI by 10.24% (P<0.05), IA by 13.61% (P<0.05). SeeWe then moved to examine how DCA would affect the RRDA. We found that DCA at the concentration of 10−1 mg/mL decreased RC by 37.25% (P<0.01), TE by 41.60% (P<0.01). And 10−2 mg/mL LCA decreased RC by 8.22% (P<0.05), TE by 10.02% (P<0.05). 10−3 mg/mL LCA decreased RC by 25.19% (P<0.05) and TE by 27.99% (P<0.05). SeeAfter we had a look at how CDCA and DCA influenced the RRDA, we showed that LCA at the concentration of 10P<0.05), TI by 20.61% (P<0.05), TE by 48.14% (P<0.05) and IA by 21.14% (P<0.01). And 10−1 mg/mL CA decreased TI by 24.83% (P<0.01), IA by 17.58% (P<0.01). 10−2 mg/mL CA decreased TE by 32.51% (P<0.05) and increased IA by 10.70% (P<0.05). SeeAs one of the known agonists of bile acids for FXR, CA at the concentration of 1 mg/mL decreased RC by 46.31.064% (−3 mg/mL) was separately applied with no significant influences on RC(s)?TI(s)?TE(s)?IA ( µV×s) of RRDA recorded from hypoglossal nerves compared to controls. Thus, data was not illustrated in this article.Under our experimental conditions, UDCA at different concentrations  was applied. As a result, no significant differences compared to the control were observed. Then, Z-guggulsterone was washed out. When full recover of the RRDA was achieved, we used the combination of Z-guggulsterone and the four bile acids. And again, there were no significant differences. See , After RRDA was stably recorded from hypoglossal nerves, DMSO  was applied to observe its effects. As a result, no significant differences compared to the control were observed. in vitro preparation of brain slice proves to be useful experimental tools as its spontaneous periodic nerral activity recorded from cranial nerves corresponds to the respiratory rhythm produced in the brain stem of intact animals. Although the periodic rhythm of this in vitro preparation has some significant differences compared to normal respiratory rhythm in the intact animal due to reasons such as absence of the afferent inputs from the periphery, the preparation is still considered valuable for physiological and biochemical investigations of the respiratory rhythm generation and modulation in mammalian central nervous system. Thus, we used the in vitro brainstem medulla slice of neonatal Sprague-Dawley rats in our study.Smith and his co-workers According to our previous studies, the decrease of the blood pressure resulted from classical depressor reflex by stimulation of pneumogastric nerves resembled that resulted from intravenous injection of diluted bile acids in terms of the total blood pressure reaction time. The reaction time here we referred to marks the period between onset of the effect and the time point when the effect diminished. However, changes in respiratory rhythm are not the same situation. After stimulation of pneumogastric nerves, the total respiratory reaction time lasted nearly equal to the total blood pressure reaction time. But intravenous injection of diluted bile acids caused a 9–10 fold longer respiratory reaction time, as well as higher peak amplitude. We then analyzed that the significant changes in respiratory functions caused by intravenous injection of diluted bile acids compared to pneumogastric nerves stimulation could be due to the additive effects of bile acids and reflex responses. This indirectly proved that bile acids could be involved in the regulation of respiratory rhythm while no exact elucidation of the underlying mechanism was available. And moreover, injection of UDCA led to no significant changes in blood pressure or respiratory rhythm. Also, the changes in blood pressure caused by intravenous injection of bile acids were not in accordance with those in respiratory rhythm seesee−4 mg/mL increased TE by 25.37%. Due to our limitations, possible explanations including the signaling networks and mechanisms on how bile acids exerted their specific effects via FXR in the brain could not be given. For purposes of accurate mechanisms, further studies are required.Based on our experiment, GW4064, as a well acknowledged agonist of FXR, and all of the four bile acids CDCA, LCA, CA, DCA except UDCA exerted significant effects on the spontaneous periodic respiratory-related rhythmical discharge activity (RRDA) recorded from hypoglossal nerves in a concentration-dependent manner, altering changes on four aspects of respiratory cycle (RC), inspiratory time (TI), expiratory time (TE), and integral amplitude (IA) Taken all the above together, it is interesting to speculate that the bile acids may affect respiration in two separate ways, the respiratory frequency and the respiratory pattern. And by respiratory pattern we mean that while the respiratory frequency stays the same, the inspiratory and expiratory time change. With RC, TI and TE as our measurement indicators, DCA only affect the respiratory pattern while CDCA, LCA, CA have not only influence on the frequency, but also on both the inspiratory and expiratory time. Almost in all concentrations of CA, LCA and CDCA, the TI and TE tend to decrease in a concentration-dependent manner, resulting in higher respiratory frequencies. This again proves the involvement of bile acids in the regulation of respiration, exerting their effects on respiratory frequency, pattern and amplitude.As a member of nuclear hormone receptor superfamily, farnesoid X receptor (FXR) plays a pivotal role in maintaining the homeostasis of cholesterol and bile acids. FXR carries out most of its functions by altering the expression of its target genes such as bile salt export pump (BSEP) through binding to the FXR response element (FXRE). The influences of CA, DCA, LCA and CDCA on respiratory rhythm could be reversed by the FXR blocker Z-guggulsterone, indicating the role of FXR in the respiratory regulation. As natural activators of FXR, CDCA, CA, DCA and LCA bind to FXR with decreasing potency see In this experiment, we confirm that CA, DCA, LCA, CDCA and GW4064 are all able to regulate respiratory rhythm except UDCA. And activation of FXR is probably involved in such process as FXR inhibitor Z-guggulsterone completely reversed the effects of the above bile acids and GW4064


New technologies are diffusing into medical practice swiftly. Hand-held devices such as smartphones can record short-duration  ECGs, but their effectiveness in identifying patients with paroxysmal atrial fibrillation (AF) is unknown.2 score≥1 point: mean 2.3 points) who had no documentation of atrial tachycardia (AT)/AF or antiarrhythmic or anticoagulant drug use at baseline. All were subsequently newly diagnosed with AT/AF by a cardiac implantable electronic device (CIED) over one year of follow-up. Using a computer simulation approach , we estimated the detection rate for paroxysmal AT/AF via daily snapshot ECG monitoring over various periods, with the probability of detection equal to the percent AT/AF burden on each day.We used data from the TRENDS study, which included 370 patients , and was higher in those with AT/AF burden ≥0.044 hours/day compared to those with AT/AF burden <0.044 hours/day .The estimated AT/AF detection rates with snapshot monitoring periods of 14, 28, 56, 112, and 365 days were 10%, 15%, 21%, 28%, and 50% respectively. The detection rate over 365 days of monitoring was higher in those with CHADS2 score and AT/AF burden.Daily snapshot ECG monitoring over 365 days detects half of patients who developed AT/AF as detected by CIED, and shorter intervals of monitoring detected fewer AT/AF patients. The detection rate was associated with individual CHADSNCT00279981ClinicalTrials.gov  One-third of atrial fibrillation (AF) patients have no obvious symptoms, and the prevalence of paroxysmal AF is 25–60% of total AF cases –5. Asympcountries, so patients can readily use them over long periods of time  or high risk [a CHADS2 score of ≥2]) and AF burden , and the rate for detecting AT/AF was calculated within each group. A significance level of P< 0.05 was used, and all statistical analyses were performed using SAS, version 9.2 .Continuous variables are presented as the mean ± SD and categorical variables are presented as percentages. Continuous variables were compared using a 2 score, or blood pressure levels between the included and not included patients.For the 370 participants, the median of average AT/AF burden, the maximum daily AT/AF burden, and the percentage of days with AT/AF were 0.044 hours/day (range = 0.0003–23.993), 5.5 hours/day (range = 0.1–24), and 1.6% (range = 0.3–100), respectively. Their distribution is shown in 2 score and low/high AF burden, is shown in 2 score of ≥2 than for those with a CHADS2 score of 1 . The detection rate of snapshot monitoring for each time window was higher among the patients with high AT/AF burden than among those with low AT/AF burden . In those with high AT/AF burden, the prolongation of snapshot monitoring up to 365 days increased the detection rate to 91%, while a corresponding value in those with low AT/AF burden was 14%. When we used the cutoff value of 5.5 hours/day in defining high or low AF/AT burden, a threshold associated with a 2-fold increase in thromboembolic events shown in the original TRENDS study [The detection rate of snapshot monitoring over various periods , calculated based on low/high CHADSDS study , results2 score ≥1 and no history of AT/AF at baseline. The detection rate increased with longer snapshot monitoring, up to 50% when used over 365 days, and shorter intervals of monitoring detected even fewer AT/AF patients. Only 10% of patients with AT/AF were captured by 14 days of snapshot ECG monitoring, and only 15% with 28 days of monitoring. The use of snapshot ECG monitoring to guide clinical care should be implemented cautiously, with the recognition that this method of detection likely underestimates AF episodes.Using a computer simulation approach in the TRENDS study, we estimated the sensitivity of daily snapshot ECG monitoring over various periods in detecting newly diagnosed AT/AF among the patients with a CHADSReliable and accurate detection of AF is important for decisions on starting or changing anticoagulation or antiarrhythmic therapy in those with stroke risk factors . The digital revolution and rapid development of smartphones, mobile connectivity, and social networking have the potential to transform therapeutic approaches . Using aAt least two studies have assessed the sensitivity of intermittent ECG monitoring for detecting AT/AF, comparing it with the findings from concomitant continuous ECG monitoring . Ziegler et al. suggested that among 547 pacemaker patients with symptomatic AT/AF, the sensitivity (31%-71%) and negative predictive value (21%-39%) were low in several approaches with intermittent monitoring, such as annual, quarterly, or monthly 24-hour Holter ECGs, 7- or 30-day long-term recordings, or symptom-based monitoring . CharitoAn intensification of intermittent ECG monitoring on a number of different days could improve the sensitivity of intermittent ECG monitoring in detecting AF ,22. Amon2 score ≥2 points (53%) than in those with a CHADS2 score of 1 point (38%). The detection rate also differed by individual AT/AF burden. Only 14% of patients with AT/AF burden <0.044 hours/day were captured, even by 365 days of snapshot ECG monitoring. However, we need to consider that there is still much debate as to whether such a short AF episode actually implies an increased risk of stroke and warrants screening of a population with low AF burden [The detection rate of snapshot ECG monitoring, when it was performed over 365 days, was higher in those who had a CHADSF burden ,27,28. BF burden .This study has some limitations. First, the AT/AF burden data from the device was aggregated on a daily basis, without permitting us to choose specific times within the day and duration of each AT/AF episode to simulate our monitoring or to analyze daytime vs. nighttime onset of AT/AF. Snapshot ECG recording during sleep is not feasible, and thus the detection rate of snapshot ECG monitoring in the current study may be overestimated. Second, the results do not reflect all clinical scenarios since there was no consideration for recordings during symptomatic episodes. However, symptoms believed to be caused by AF have been shown to correlate with the arrhythmia in only 17% of episodes .The studAmong the patients with stroke risk factors, daily snapshot ECG monitoring with up to 365 days of follow-up detects AT/AF in half or less than half of patients whose AT/AF is detectable by CIED. The data from the current study raise concerns about the accuracy of daily snapshot ECG monitoring as a tool for detecting AF in those with stroke risk factors. We need to determine the most appropriate applications of snapshot ECG monitoring , balancing technological possibilities, patient burden, and costs.