Introduction. The aim of this study was to evaluate the serum levels of resistin and insulin-like growth factor-1 (IGF-1) and and also the potential relationship between thyroid function and levels of resistin and IGF-1 in hypothyroid and hyperthyroid patients. Methods. Fifteen cases of hypothyroid (HT), 16 of subclinically hypothyroid (SCHT), 15 of hyperthyroid (HrT), 15 of subclinically hyperthyroid (SCHrT), and 17 healthy individuals have been included in the study. Serum resistin levels were measured using enzyme-linked immunosorbent assay and IGF-1 and thyroid stimulating hormone (TSH) levels by chemiluminescence method. Results. Resistin levels in total HT group were significantly higher than in controls . In SCHrT subgroup resistin levels were significantly higher than those of controls . IGF-1 levels were significantly lower in total HT than in total HrT and control groups . Furthermore IGF-1 levels in HT subgroup were significantly lower compared to controls . In SCHT subgroup IGF-1 levels were significantly lower than those of control and SCHrT groups . There were significant correlations between IGF-1 and TSH in HT subgroup and between resistin and TSH in total HrT group. Conclusion. It was concluded that increased resistin levels are directly related to thyroid dysfunction, and GH/IGF-1 axis is influenced in clinically or subclinically hypothyroidism patients. Thyroxine (T4) and triiodothyronine (T3) hormones regulate heat production and energy utilization, and they are very important for normal growth and development. They also play important roles in regulating various homeostatic mechanisms . ThyroidIt has been understood that adipose tissue is not only a passive energy reservoir, it is but also an active endocrine tissue. Resistin is an adipocytokine which is considered to have an important role in energy metabolism and is secreted from adipocytes and macrophages. Resistin antagonizes insulin effect and causes insulin resistance especially in obese patients. Resistin is also playing a role in inflammation and is a potential biomarker in cardiovascular and many other diseases , 4.IGF-1 is a growth factor which is secreted from liver as a response to growth hormone (GH). IGF-1 has many receptors in various tissues and manifests insulin-like effect as well; it functions in many metabolic pathways including energy metabolism . There aThe aims of the present study were (1) to determine serum resistin, IGF-1, TSH, fT4, lipid, and transaminase levels together in untreated patients with hyper-and hypothyroidism and control groups for the first time and (2) to investigate potential relationship between thyroid function and serum levels of resistin and IGF-1 in HT and HrT patients.The study consisted of 15 HT patients with ages 20–76, 16 SCHT patients with ages 26–71, 15 HrT patients with ages 22–79, 15 SCHrT patients with ages 26–70, and control group consisting of 17 individuals who were detected as healthy by physical examination and laboratory findings. The total number of volunteers was 78. μIU/mL and serum free T4 (fT4) levels below 0.58 ng/dL were classified as HT; the individuals having TSH levels over 5.6 μIU/mL and serum fT4 levels 0.58–1.64 ng/dL were classified as SCHT. The individuals having TSH levels over 5.6 μIU/mL and serum fT4 levels over 1.64 ng/dL were classified as HrT; the individuals having TSH levels below 0.34 μIU/mL and serum fT4 levels 0.58–1.64 ng/dL were classified as SCHrT. The patients who have TSH levels over 5.6 μIU/mL were classified as total HT, and the patients who have TSH levels below 0.34 μIU/mL were classified as total HrT.The individuals having TSH levels below 0.34 In patient groups there was no individual who had any systemic disease. None of the patients had treatment concerning thyroid dysfunction.Detailed histories of individuals included in the study were obtained; follow-up forms were prepared. All of the tests and clinical meanings of the tests performed were explained to the individuals, and their written consents were obtained. The ethical approval of study was given by the Okmeydani Educational and Research Hospital Ethics Committee.10 mL of blood samples into plain vacuum tubes with gel were obtained after 12 hour fasting. They were centrifuged for 10 min at 2000 ×g. T-Chol, HDL-Chol, AST, ALT, TSH, and fT4 were analyzed on the same day. Undiluted serum samples were seperated into two tubes, and they were stored at −80°C for maximum 6 months. Repetative freezing and thawing were not performed. Serum T-Chol, HDL-Chol, AST, and ALT levels were determined photometrically in Beckman Coulter AU2700 auto-analyzer ; TSH ve fT4 levels were determined in Beckman Coulter UniCel DxI 800 autoanalyzer  by using access high sensitive TSH 3rd generation and access fT4 successively by chemiluminescence method. LDL-Chol levels were calculated by the Friedewald formula. AdipoGen Human Resistin ELISA kit  was used to determine resistin levels. IGF-1 levels were determined in Siemens Immulite 2000 analyzer  by solid phase enzyme marked chemiluminescence method.P < 0.05.In order to analyze data SPSS 17.0 ve GraphPad InStat 3.05 packet programs were used. Standart deviations (SD) and means of the Gaussian parameters included in the study were given in terms of groups. Both mean and min-max values and mean ± SD values were presented for the nonGaussian parameters. In comparison of more than two groups that were independent of each other (3 groups and 5 groups), Kruskal-Wallis and one way ANOVA tests were used. The groups which were determined to be different from one another in terms of parameters were compared again in binary groups. The correlation between resistin, IGF-I, and TSH was presented via Spearman's correlation coefficient. The results were evaluated in 95% confidence and significance in r = 0.463, P = 0.010)   males and 61 (78%) females. Laboratory findings and demographic properties of total HT, total HrT, and healthy control groups and HT, SCHT, HrT, and SCHrT subgroups were showed in Tables 2% males = 0.010) . There w= 0.003) . Within Significantly higher T-Chol and LDL-Chol levels in total HT group were observed in comparison to total HrT and control groups. Significantly higher T-Chol and LDL-Chol levels in HT subgroup were observed in comparison to HrT, SCHrT, and control groups. These parameters were also significantly higher in SCHT in comparison to HrT subgroup. These findings support the possible correlation between thyroid function and lipid metabolism. Serum AST levels in total HT and HT groups were detected as significantly higher in comparison to control group. Hepatocellular damage due to fatty liver caused by hypercholesterolemia in hypothyroid patients causes high AST levels. Our findings are consistent with this situation.Thyroid gland and adipose tissue functions are closely correlated with each other. Recently by the discovery of endocrine functions of adipose tissue, it has been considered that adipose tissue not only has storage function but also has active regulatory functions in energy metabolism . ResistiChanges in thyroid hormone levels have been clearly known to affect insulin secretion and sensitivity. Correlation between resistin and thyroid functions have been investigated by both human and animal studies. Syed et al.  reportedIn order to enlighten functions of resistin in human it is neccessary to accomplish detailed studies. There is very little information about relationship between thyroid functions and resistin. Furthermore existing studies present conflicting results , 6–9.Significantly higher resistin levels in total HT and total patient group were observed in comparison to control group. It was also observed serum resistin level of total HrT group was higher in comparison to control group. But that elevation was not statistically significant. Serum resistin levels of SCHrT subgroup were significantly higher than those of control group. Although they are not statistically significant, the means of resistin levels in HT, SCHT, and HrT subgroups were higher than those of control group. The study of Yaturu et al.  consisteIn the study of Krassas et al.  hyperthyIn another study consisting of 53 hypothyroid patients and 30 controls, Krassas et al.  also didIn their study consisting of 20 hypothyroid patients and 20 healthy controls, Iglesias et al.  observedIn this study higher resistin levels in total hypothyroid group conflict in comparison to control group with study of Krassas et al.  in whichConsequently, when limited number of studies about resistin examined, it was observed that resistin levels are variable in thyroid dysfunctions , 10, 12.Before now, the correlation between growth hormone excess, insulin resistance, and carbohydrate intolerance has been presented by some studies –16. It hIn another study performed with children having GH deficiency, Nozue et al. investigated effect of GH treatment on serum resistin levels . In contIn this study significantly lower serum IGF-1 levels were detected in total HT group in comparison to total HrT and control groups. Serum IGF-1 levels in HT subgroup were significantly lower in comparison to control group; serum IGF-1 levels in SCHT subgroup were significantly lower in comparison to control group and SCHrT subgroup. A negative correlation between TSH and IGF-1were also observed in HT subgroup. These results were compatible with that of Akin et al. , Schmid In this study, no correlation between resistin and IGF-1 levels was found. The reason may be limited number and age diversity of individuals in study groups. A comparement can not be made about that result because there is no study about correlation between resistin and IGF-1 in thyroid dysfunction in the literature.In conclusion, increased resistin levels are directly related to thyroid dysfunction, and changes in levels of thyroid hormones may affect synthesis and/or secretion of resistin in adipose tissue and/or macrophages. In addition, serum IGF-1 levels decrease in hypothyroid status and correlate negatively with TSH levels. GH/IGF-1 axis may be influenced in clinical or subclinically hypothyroid patients. However, it was concluded that demographic characteristics, therapeutic variations and underlying reasons of thyroid dysfunction should be taken into consideration while interpreting the resistin and IGF-1 levels in thyroid dysfunction.


Morphological and physiological characteristics are not sufficient to differentiate some bermudagrass genotypes because the differences between them are often subtle and subjected to environmental influences. In this study, twenty seven bermudagrass accessions and introductions, mostly from different parts of Iran, were assayed by inter-simple sequence repeat (ISSR) markers to differentiate and explore their genetic relationships. Fourteen ISSR primers amplified 389 fragments of which 313 (80.5%) were polymorphic. The average polymorphism information content (PIC) was 0.328, which shows that the majority of primers are informative. Cluster analysis using the un-weighted paired group method with arithmetic average (UPGMA) method and Jaccard’s similarity coefficient (r = 0.828) grouped the accessions into six main clusters according to some degree to geographical origin, their chromosome number and some morphological characteristics. It can be concluded that there exists a wide genetic base of bermudograss in Iran and that ISSR markers are effective in determining genetic diversity and relationships among them.Bermudagrass  Cynodon dactylon (L.) Pers. (common bermudagrass) is the most important member of the genus Cynodon because of its widespread distribution in warmer parts of the world and its use as livestock herbage and turf [and turf . Harlan and turf .Cynodon dactylon (L.) Pers. was reported to be Turkey, Iran, Afghanistan and the western part of Pakistan, which could be potentially rich in the desirable genes and genotypes [The center of diversity for some races of enotypes ,2. CharaCynodon accessions includes DNA amplification fingerprinting (DAF) [The commonly used polymerase chain reaction (PCR) based DNA marker systems are random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and more recently simple sequence repeats (SSRs) or microsatellites . The majng (DAF) , RAPD [1ng (DAF) ,19, AFLPng (DAF) ,21, ISSRng (DAF) , SSR [23ng (DAF) .In this study, we reported the feasibility of the ISSR-based PCR as an interesting approach in providing accurate molecular markers for investigating the genetic diversity among bermudagrass accessions.The fourteen selected ISSR primers produced 389 bands with an average of 27.78 bands per primer, of which 313 (80.5%) were polymorphic with an average of 22.36 bands per primer. The different ISSR primers amplified the number of bands from 20 (ISSR-14) to 40 (ISSR-11) having a range of 400 to 1200 bp fragments size. The number of polymorphic fragments detected by each primer ranged from 16 to 27. The most polymorphism was shown by ISSR-5, which showed 92.8% polymorphism. The mean of polymorphism information content (PIC) value for primers was 0.328 which ranged from 0.205 (ISSR-5) to 0.433 (ISSR-15) . Figure The average values of observed number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity (He) and Shannon’s information index (I) for all primers were 2, 1.56, 0.33 and 0.56, respectively.The similarity matrix of the 27 genotypes from ISSR data using Jaccard’s coefficient showed that the genetic relationships of genotypes were different varying between 0.057 and 0.524 (data not shown). The lowest similarity was revealed between “18-Gr” and “27-II” genotypes (0.057), whereas the highest similarity was between “3-Gs” and “6-Gs” genotypes (0.524). The latest two common bermudagrass accessions, “3-Gs” and “6-Gs”, are tetraploid and are collected from the same origin  with somr = 0.828) was observed for the un-weighted paired group method with arithmetic average (UPGMA) clustering method based on Jaccard’s similarity coefficient, suggesting that the cluster analysis represents the similarity matrix.The data obtained from ISSR analysis of 27 bermudagrass genotypes was subjected to cluster analysis. In order to recognize the best clustering and similarity coefficient methods and the cophenetic correlation coefficient, a measure of the correlation between cophenetic matrix constructed from tree and similarity matrix, was calculated for each method combination. From different methods, the highest value  that had some similar morphological characteristics, such as leaf width, length of flower, stem and leaf, and stolon color. The grouping of these accessions from the same province (Isfahan) may reflect their having a common geographic origin. Genetic similarity coefficient values in this group were from 0.128 to 0.458.Cluster C embraced two diploid accessions (“17-GN1” and “88-Khl”) and one triploid accession (“44-II”). These accessions have also similar morphological characteristics such as width of leaf and stolon color.Cynodon dactylon × Cynodon transvaalensis), Tifgreen, Tifdwarf, Tifway and Midlawn were separated from all accessions and grouped together as cluster D.Four triploid hybrids  were separated from all accessions in single clusters as labeled E and F, respectively in Principal coordinate analysis (PCoA) based on genetic similarity metrics was used to visualize the genetic relationships among accessions. The first three eigenvectors accounted for 25.61% of the total molecular variation. Since the original data are not highly correlated in PCoA, the first few PCs do not explain much of the original variation. Assessment of genetic relationships on the basis of the first three PCs could lead to misleading interpretations and therefore analysis of genetic relationships among accessions should be based on cluster analysis as well as the optimal number of PCs that explain the maximum amount of the original data.The results obtained in our study showed that ISSR markers measured sufficient polymorphism for DNA typing and can be successfully used in bermudagrass germplasm characterization and for fingerprinting purposes. Similar results were obtained in several studies using other molecular markers such as RAPD ,18.P) were used to measure the genetic diversity in bermudagrass accessions. High, medium or low polymorphism is in accordance with PIC > 0.5, 0.5 > PIC > 0.25 and PIC < 0.25, respectively [The polymorphic information content (PIC) and the polymorphism rate  or expected heterozygosity (He) is more appropriate than the actual number of alleles (Na), since the latter depend on the sample size .Bootstrapping was effectively utilized for estimating the statistical support to the internal branches of the tree. The internal tree branches that have >70% bootstrap are likely to be correct at the 95% level. However, a high bootstrap percentage still does not guarantee that long branch attractions have not biased the results. Also, in many cases, the overall tree structure provides better information than a particular branch. Wherever a priori information regarding genetic relationships is available, it is preferable to apply it in the interpretation . TherefoCynodon dactylon and Cynodon transvaalensis. In this study, we included four hybrids, which were divided into two subgroups on the UPGMA tree. Among these hybrids, “Tifgreen” and its putative spontaneous somatic mutant (Tifdwraf) were placed in two separate subgroups, whereas, “Tifway” and “Midlawn” were placed in the same subgroup. Zhang et al. and Caetano-Anolles used AFLP and DAF techniques to effectively differentiate “Tifgreen” and the putative somatic mutants [Cynodon dactylon in the taxonomic revision of the genus as listed by Harlan et al. [Hybridization is one of the most common methods used to create variation in bermudagrass . Most of mutants ,30. Theyn et al.  probablyn et al. ,31.et al. in Chinese natural bermudagrass (Cynodon dactylon) germplasm using ISSR markers [Cynodon plants and exchange of germplasm resources from different regions. This constructed dendrogram may be able to discriminate between genetically different chromosome numbers of Cynodon dactylon as reported by Anderson et al. [et al. [et al. [Cluster analysis was able to cluster bermudagrass accessions based on geographical origin, which is in agreement with previous observations of Li  markers . This shn et al. , Etemadi [et al.  and Guls [et al. .Cynodon dactylon) and four were triploid hybrids of Cynodon dactylon × Cynodon transvaalensis . Stolons of twenty-three accessions of Cynodon dactylon (L.) Pers. had been collected from Isfahan, Charmahal Bakhtiari, Gilan and Mazandaran, provinces of Iran , 200 μM of dNTPs, 1.25 μM of each primer, and template DNA of approximately 50 ng.Fourteen random ISSR primers  were used to amplify DNA fragments of the selected materials. These primers were selected based on preliminary screening, producing a higher level of polymorphism and reproducible fragment patterns . ISSR re−1) and photographed under UV light in a gel documentation system Amplifications were carried out in a Thermal Cycler  with the following PCR program: 3 min of initial denaturing at 94 °C, 40 cycles of 94 °C for 45 s, 1 min for annealing at the primer-specific melting temperature, and 72 °C for 4 min; followed by a final extension of 10 min at 72 °C. The amplified fragments were separated on a 1.5% agarose gels in 1× TBE buffer running at 65 V constant for 1.5–2 h and then stained by ethidium bromide (1 μg·mLAll clearly detectable ISSR product bands were scored as either presence (1), absence (0) or ambiguous (9) of each band for all accessions and the matrix of ISSRs data was assembled. All amplifications were repeated at least twice and only reproducible and well-defined bands were considered for analysis.pi2, where pi is the frequency of the ith allele [Polymorphism information content (PIC) was calculated using the formula PIC = 1 − ∑h allele . Jaccardh allele  to generh allele . The MXCh allele . Winbooth allele  was usedObserved number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity (He)  and Shanr = 0.828) grouped the accessions into six main clusters according to some degree to geographical origin, their chromosome number and some morphological characteristics. The enlargement of the number of primer pairs and accessions would have provided more useful information to assist the classification of the bermudograss germplasm.In summary, our results indicate that the level of polymorphism among bermudagrass accessions is appreciably high in Iran. The results of this study also suggest that the ISSR marker offers a powerful means to analyze the genetic diversity among accessions. Cluster analysis using the UPGMA method and Jaccard’s similarity coefficient (


Atherosclerotic renal artery stenosis (ARAS) is common in cardiovascular diseases and associated with hypertension, renal dysfunction and/or heart failure. There is a paucity of data about the prevalence and the role of ARAS in the pathophysiology of combined chronic heart failure (CHF) and chronic kidney disease (CKD). We investigated the prevalence in patients with combined CHF/CKD and its association with renal function, cardiac dysfunction and the presence and extent of myocardial fibrosis.The EPOCARES study  investigates the role of erythropoietin in anaemic patients with combined CHF/CKD. Eligible subjects underwent combined cardiac magnetic resonance imaging (cMRI), including late gadolinium enhancement, with magnetic resonance angiography of the renal arteries (MRA).MR study was performed in 37 patients  43.3 ± 11.2%), of which 21 (56.8%) had ARAS (defined as stenosis >50%). Of these 21 subjects, 8 (21.6%) had more severe ARAS >70% and 8 (21.6%) had a bilateral ARAS >50% . There were no differences in age, NT-proBNP levels and medication profile between patients with ARAS versus those without. Renal function declined with the severity of ARAS (p = 0.03), although this was not significantly different between patients with ARAS versus those without. Diabetes mellitus was more prevalent in patients without ARAS (56.3%) against those with ARAS (23.8%) (p = 0.04). The presence and extent of late gadolinium enhancement, depicting myocardial fibrosis, did not differ (p = 0.80), nor did end diastolic volume (p = 0.60), left ventricular mass index (p = 0.11) or LVEF (p = 0.15). Neither was there a difference in the presence of an ischemic pattern of late enhancement in patients with ARAS versus those without.ARAS is prevalent in combined CHF/CKD and its severity is associated with a decline in renal function. However, its presence does not correlate with a worse LVEF, a higher left ventricular mass or with the presence and extent of myocardial fibrosis. Further research is required for the role of ARAS in the pathophysiology of combined chronic heart and renal failure. The combination of chronic heart failure (CHF) and chronic kidney disease (CKD) is prevalent and associated with a high cardiovascular mortality and morbidity -3. Both In CHF, in several observational studies, patients with ARAS had prolonged hospital admissions and a higher mortality rate -10. ThisWe hypothesized that ARAS is prevalent in patients with combined CKD and CHF and that its presence is associated with a worse renal function, more severe cardiac dysfunction and the presence and extent of myocardial fibrosis. Therefore we assessed, in anaemic patients with combined heart and renal failure: 1. The prevalence of ARAS and 2. The association of ARAS with renal and cardiac dysfunction and the presence of myocardial fibrosis. We used cardiac magnetic resonance imaging (cMRI) for this purpose, as the quantitative values for cardiac volumes, left ventricular mass and function are more accurate than with echocardiography. In addition, intravenous gadolinium contrast material can be used both for magnetic resonance angiography (MRA) of the aorta and the renal arteries, as well as for late gadolinium enhanced (LGE) assessment of myocardial fibrosis, thereby providing information about the underlying aetiology of the cardiac dysfunction.This study is a sub study of the EPOCARES study . The study design has been published elsewhere . In shorCardiovascular Magnetic Resonance Imaging and magnetic resonance angiography (MRA) of the renal arteries were performed on a 1.5 Tesla Philips Intera . Both the heart and the renal arteries were assessed in a 45-minute protocol. The patient was placed in the supine position with a five-channel phased array coil for the cardiac analysis and a circularly polarized spine coil in longitudinal direction over the area of the kidneys below the cardiac coil for MRA of the renal arteries.ECG-triggered breath hold multiphase steady-state free procession (SSFP) images were acquired in the four-chamber, short-axis, and two- chamber view scans of the left ventricle. The short axis plane covered both ventricles from apex to base using 8-mm slices without interslice gap with the following scan parameters: TR/TE 4.0/2.0 ms, flip angle 50, FOV 350–400, matrix 256x256, voxel size 1.6x1.6x8.0 mm.Gadolinium-based contrast  was administered intravenously to first obtain MRA of the renal arteries at the time of injection (first pass) and 15 minutes later delayed enhancement scans of the heart. For MRA of the renal arteries a standard breath hold 3D T1 contrast-enhanced MRA technique was used. Scan parameters: TR/TE 3.7/1.33 ms, flipangle 25, FOV 430x430x75, voxel size 0.8x0.8x1.5 mm.For the delayed enhancement of the heart, breath hold inversion recovery T1 pulse images were acquired in four-chamber, short axis and left two-chamber view. Scan parameters: TR/TE 4.4/1.3 ms, flipangle 15, FOV 410x410x80, matrix 300x169, voxel size 1.4x1.4x5.0 mm. There were no complications related to the MRI procedures, and all patients tolerated the procedure well.The SSFP cine short axis scans were used to acquire measurements of the left ventricle end diastolic volume (EDV), end systolic volume (ESV), ejection fraction (EF) and wall mass . Endocardial and epicardial contours were traced manually on the stack of contiguous short-axis cine-images at end-diastole. This technique has been validated, with high accuracy and reproducibility . A trainAssessment of segmental wall motion and late gadolinium enhancement (LGE) was performed by two independent investigators, blinded for clinical data (BV and YA). The left ventricle was divided in 17 segments according to standardized nomenclature . Late enThe number of renal arteries as well as patency and presence or absence of stenosis was assessed by two independent investigators, blinded for clinical and cMRI data (JV and LM). Stenosis was graded as: no stenosis, <50% stenosis, 50-70% stenosis, 70-99% stenosis, occlusion 100% or previous stent placement. The presence of a renal artery stenosis was defined as having a stenosis >50% or previous renal revascularization, conform criteria from the Stenting in Renal dysfunction caused by atherosclerotic Renal Artery Stenosis .χ2 test, Mann–Whitney U test or the Kruskal-Wallis one-way ANOVA when appropriate. Differences were considered significant when P < 0.05. For statistical analyses the Statistical Package for Social Sciences  version 18 was used.Data are presented as medians with inter-quartile ranges (IQR) for non-normally distributed variables and means ± standard deviation (SD) for normally distributed continuous variables. Differences between groups were compared with the The original study population of the EPOCARES study comprised of 62 patients. Five patients withdrew their informed consent and one patient was excluded due to a suspected malignancy (diagnosed on routine X-ray at baseline). Of the 56 patients that eventually participated in the study, 37 patients underwent a cMRI/MRA. Nineteen patients did not undergo cMRI/MRA due to presence of cardiac implantable electronic devices n = 15), orthopnoea (n = 2), claustrophobia (n = 1) or a GFR < 30 ml/min (n = 1). These data are obtained after run in treatment with optimal medical therapy for CHF and oral iron supplementation, but before treatment with EPO. Clinical characteristics of these 37 patients are shown in Table 5, orthopOf the 37 patients that underwent cMRI/MRA, 21 patients (56.8%) had a renal artery stenosis defined as >50% stenosis. A more severe stenosis, defined as >70%, was present in 8 (21.6%) patients. A bilateral ARAS (>50%) was present in 7 (18.9%) patients. All stenosis were of atherosclerotic origin. One patient (2.7%) was previously treated bilaterally by angioplasty with stent placement. Baseline demographic, clinical and laboratory characteristics of the patients, divided up by the presence or absence of ARAS, defined as >50% stenosis, are provided in Table Data regarding cMRI are shown in Table Our study shows a high prevalence (56.8%) of atherosclerotic renal artery stenosis (ARAS) in patients with combined heart and renal failure, defined as having a stenosis >50%. When taken a stricter cut-point (> 70% stenosis) the prevalence of ARAS was still as high as 21.8% in this population. The presence of ARAS was not associated with the extent in abnormalities in left ventricular function or myocardial fibrosis based on cardiac MRI findings with late gadolinium enhancement (LGE), when compared to patients with combined CHF/CKD without ARAS. Neither did we observe a difference in the presence of an ischemic pattern of LGE. Furthermore; we found a weak association between eGFR and the severity of ARAS and observed a negative association between diabetes mellitus and ARAS. This could indicate that our small cohort may consist of two different groups of cardiorenal patients: non-diabetic patients in which ARAS is highly prevalent and diabetic patients with a much lower ARAS prevalence.ARAS is very common in patients with manifestations of non-renal atherosclerosis, particularly in patients with peripheral arterial and aortic disease. A recent literature review found a pooled prevalence of 25.3% in patients with peripheral arterial disease and 33.1% in patients with aortic aneurysm . Only fenot affect renal function [According to the present American Heart Association guidelines for the management of patients with peripheral arterial disease, the indication for percutaneous renal revascularization with stent placement (PTA) of ARAS is limited to “flash pulmonary oedema, recurrent episodes of unexplained congestive heart failure or unstable angina” . There ifunction ,19,24. Ofunction . The resNonetheless, diagnosing ARAS can be important for more reasons than to find patients suitable for PTA. One could interpret the presence of ARAS as a marker of “atherosclerotic burden” associated with a high risk of cardiovascular events, which would warrant more aggressive medical therapy . Indeed,In addition to determining the prevalence of ARAS in patients with both CHF and CKD, we determined whether there is an association between left ventricular structure and function and the presence of ARAS. Although one could hypothesize that ARAS would be associated more often, and to a greater extent, with cardiac abnormalities, such as left ventricular hypertrophy, we found no differences in left ventricular mass index, left ventricular volumes and LVEF in patients with and without ARAS. We also hypothesized that the existence of ARAS represents an “atherosclerotic burden”, representing one of the mechanisms of combined CHF/CKD. However, we found no difference in the presence of ischemic aetiology of CHF. Moreover, we could not demonstrate a difference in the presence, the location and the extent of fibrosis, as depicted by LGE. The study by Wright et al. showed more diastolic dysfunction and left ventricular hypertrophy in patients with ARAS when compared with a matched control group with similar renal dysfunction . In contCoincidentally we found a negative association between diabetes and ARAS; the patients with combined CHF/CKD without ARAS were markedly more likely to have diabetes mellitus than those patients with ARAS Figure . Some stA point of debate is the definition of ARAS, since no uniform definition exists. Previously, most studies defined ARAS as >50% stenosis ,24,30-32Some limitations of this study need to be acknowledged. The small study population, due to the complexity of the study design, consists of a selected group of stable ambulant patients, almost all using RAS inhibitors which, in addition to the exclusion of uncontrolled hypertension and patients with flash pulmonary oedema, may have led to underestimation of the prevalence of ARAS in CRS. However, despite this small study population, we believe that these data are valuable and robust, due to a paucity of data in the present literature about this subject and the reliable assessment with cardiac MRI. Furthermore, the data are baseline data from a randomized intervention study, which precludes spontaneous follow-up.In conclusion, ARAS is prevalent in patients with combined CHF and CKD and its presence does not correlate with worse left ventricular function, left ventricular volumes, mass, nor myocardial fibrosis as assessed by MRI. However, the severity of ARAS is weakly associated with renal dysfunction and ARAS is remarkably negatively associated with diabetes mellitus in this cohort. Further research is needed to investigate its role in the pathophysiology of combined chronic heart failure and chronic kidney disease and subsequent therapeutic consequences.CKD: Chronic kidney disease; CHF: Chronic heart failure; ARAS: Atherosclerotic renal artery stenosis; cMRI/MRA: Combined cardiac magnetic resonance imaging with magnetic resonance angiography of renal arteries; NT-proBNP: N-terminal pro brain natriuretic peptide; LGE: Late gadolinium enhancement; LVEF: Left ventricular ejection fraction.The authors declare that they have no  competing interests.CG, BB and KvP initiated this study. ME helped in the study design, wrote the manuscript, together with CG and BB, participated in the recruitment of patients and collection of data, together with KvP, did the statistical analyses with HH and analysed the imaging data. MJ, BK and YA analysed the cardiac data. JV and LM interpreted the MRA of the renal arteries. PD contributed to the study design and the discussion. All authors read and approved the final version of the manuscript.This investigator-initiated study was supported by the Netherlands Heart Foundation (grant number 2005B192) and by an unrestricted grant from Roche Pharmaceuticals, the Netherlands. A Heart and Stroke Foundation of Canada New Investigator award supports Branko Braam. This is an investigator-initiated study and no changes to the study protocol were made upon request of Roche, neither did Roche have insight in the data.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/12/76/prepub


Background. Ovarian pregnancy is a rare form of ectopic pregnancy. It is often difficult to distinguish from tubal pregnancy, and diagnosis and management are frequently a challenge. Case. A 33-year-old nulligravida woman presented with light vaginal bleeding and bilateral lower quadrants abdominal pain. Beta-human chorionic gonadotropin (beta-hCG) level (592 mUI/mL) and clinical and ultrasound (US) findings were suspicious for tubal pregnancy. On the third day, despite beta-hCG decrease (364 mUI/mL), she complained of severe pain in the lower abdomen, and physical examination revealed abdominal rebound tenderness. US showed a large amount of fluid in the abdominal cavity. Because of the unstable clinical condition, emergency laparoscopy and resection of left ovarian ectopic pregnancy were performed. Histology confirmed ovarian gestation. Conclusion. This case shows that ectopic pregnancy rupture may occur despite low levels of beta-hCG. Hemoperitoneum is not contraindication to laparoscopy. Ovarian pregnancy is one of the rarest variants of ectopic implantation, and incidence is estimated to be 3% of diagnosed ectopic pregnancies . The dia9/L, Hemoglobin (Hb): 12 g/dL, Hematocrit (Hct): 34%, Platelets (Plt): 216 × 109/L, and Red Blood Cells (RBC): 4.2 × 1012/L. The patient was admitted to the Gynecological ward with a diagnosis of suspected tubal pregnancy. Daily routine blood examinations, including serum beta-hCG, and US were performed showing that the patient was in a stable clinical condition.A 33-year-old nulliparous woman presented to the gynecologic emergency room of our Department complaining of light vaginal bleeding and bilateral lower quadrants abdominal pain without signs and symptoms of circulatory instability. She reported a history of 5 weeks of amenorrhea, and her previous menstrual cycles were regular. She had no history of sexually transmitted disease or pelvic inflammatory disease, nor previous gynecological surgery. Physical examination revealed initial blood pressure 110/70 mmHg, pulse 80 beats/min, and body temperature 37.5°C. On speculum examination there was a small amount of blood in the vagina but the cervical os was closed. On bimanual clinical examination she was found to have a normal size of anteverted uterus and no cervical motion tenderness. The left adnexa was tender to palpation but no adnexal mass was appreciated. Urine pregnancy test was positive, and the serum beta-chorionic gonadotropin (beta-hCG) level was 592 mUI/mL. Transvaginal ultrasound (US) examination showed an empty uterus, corpus luteum in the left ovary and a 25 mm hyperechoic mass with a central hypoechoic shadow in the left adnexa, suggestive of a tubal pregnancy . A struc12/L, and Hct to 23.5%. Transvaginal US showed a large amount of fluid in the cul-de-sac and abdominal cavity. The patient was taken to the operating room with a presumptive diagnosis of a ruptured ectopic pregnancy and hemoperitoneum. Emergency laparoscopy was performed. During laparoscopy, 600 mL of fresh blood along with dark blood clots were evacuated from the abdominal cavity. The left fallopian tube appeared to be normal. Active bleeding was observed from the left ovary, where a hemorrhagic mass suggestive of ruptured ectopic pregnancy (5 cm × 4.5 cm × 3.5 cm) was present. The gestational sac was easily shelled out of the encasing ovarian tissue (On the third day of hospitalization, despite a beta-hCG decrease to 364 mIU/mL, the patient complained of severe pain in the lower abdomen, and there was abdominal rebound tenderness during physical examination. The patient's pulse rate was 90/min, and blood pressure 95/60 mmHg. Hb levels dropped to 8.5 g/dL, the number of erythrocytes reduced to 3.17 × 10n tissue . PatholoOvarian pregnancy is a rare variant of ectopic pregnancy. Early diagnosis of ovarian pregnancy is necessary in order to avoid more serious complications and emergency invasive procedures . HoweverIn conclusion, ovarian pregnancy is a rare event that is difficult to detect, and a quantitative beta-hCG level may be an unreliable predictor of ectopic pregnancy evolution. Hemoperitoneum is not always a contraindication to laparoscopy.


The objective of this study was to investigate the association between serum ucOC levels and insulin resistance in humans with type 2 diabetes mellitus.Recent We measured serum ucOC levels in 129 patients with type 2 diabetes. Insulin resistance was assessed using the euglycemic hyperinsulinemic clamp technique. The insulin resistance indices used were the M value, which is the total body glucose disposal rate, and the M/I value, which is the M value adjusted for the steady state plasma insulin level. ucOC levels were not correlated with the M value  or the M/I value .We found no association between ucOC levels and insulin resistance in patients with type 2 diabetes mellitus. Among rodents -5. Most  rodents ,2. Howev rodents . Focusin rodents . In cont rodents . These cTherefore, we examined whether serum ucOC levels are associated with insulin resistance in patients with type 2 diabetes using M and M/I values, which are gold standard indices for measuring insulin resistance, using the euglycemic hyperinsulinemic clamp technique.A total of 129 subjects were selected from participants admitted our diabetes center at Osaka City University Hospital. Patients with serum creatinine levels >1.2 mg/dL (106 μmol/L) and other active medical diseases were excluded. Of the 129 patients selected, 65 were taking the following medications: insulin (n = 15), sulfonylureas (n = 29), α-glucosidase inhibitors (n = 5), biguanides (n = 1), insulin secretagogues (n = 3), thiazolidinedione (n= 2), and combination therapies of these drugs (n = 10). The study protocol was approved by the local ethics committee, and informed consent was obtained from all participants prior to study initiation.U test was used to compare ucOC levels between male and female subjects. P values of <0.05 were considered statistically significant.HbA1c (%) levels were estimated as National Glycohemoglobin Standardization Program equivalent values (%) and were calculated by the formula HbA1c (%) = HbA1c  + 0.4%, considering the relative expression of HbA1c (%) as measured by standard laboratory methods and previous Japanese standard materials . ucOC le-1·min-1 and 4.0  mg·kg-1·min-1·mU-1·L×100, respectively. ucOC levels were not correlated with either the M value  (Figure Clinical characteristics of the subjects are summarized in Table This is the first study, as per our knowledge, to examine the association between ucOC levels and insulin resistance using the euglycemic clamp technique, which is the gold standard method for evaluating insulin resistance in humans. We found no significant correlation between ucOC levels and insulin resistance in patients with type 2 diabetes. These findings suggest that it is premature to conclude that ucOC plays a role in glucose metabolism in humans.The HOMA-IR is easily calculated from fasting insulin and glucose levels and is commonly used for the evaluation of insulin resistance in clinical practice. However, some attention needs to paid to the interpretation of these values as indices of insulin resistance because they mainly depend on the balance between hepatic glucose output and insulin secretion, which is maintained by a feedback loop between the liver and pancreatic β-cells . In partAdministration of recombinant ucOC into mice clearly enhanced both insulin secretion and sensitivity -5. HowevThe present study had several limitations. First was the cross-sectional study design and the relatively-small sample size. Second, there was no information regarding medications for metabolic bone diseases, including vitamin K, which up-regulates gamma-carboxylation of OC. Third, we did not measure total OC levels. However, the ucOC/OC ratio may be a more relevant measure. Finally, although we hypothesized that ucOC could affect insulin sensitivity via adiponectin, serum adiponctin levels were not determined.In conclusion, we found no association between ucOC levels and insulin resistance in patients with type 2 diabetes as per the euglycemic hyperinsulinemic clamp technique. Further studies are required to confirm the impact of ucOC, OC, and ucOC/OC ratio on insulin resistance in humans.OC: Osteocalcin; ucOC: Undercarboxylated form; HOMA-β: Homeostasis model assessment–β; HOMA-IR: Homeostasis model assessment of insulin resistance; BMI: Body mass index; SBP: Systolic blood pressure; FPG: Fasting plasma glucose; TC: Total cholesterol; HDL: High density lipoprotein; TG: Triglyceride; sCre: Serum creatinine.The authors declare that they have no competing interests.Mori conceived of the research hypothesis and performed the statistical analyses, and wrote the manuscript. Emoto assisted in conception of the research hypothesis and in writing the manuscript. Motoyama and Lee collected the data. Yamada, Morioka, Imanishi, Shoji and Inaba reviewed and edited the manuscript. All authors read and approved the final manuscript.


Lantana camara Linn and Lantana montevidensis Briq (Verbenaceae) found in tropical and subtropical areas around the world are popularly known as “camará” or “chumbinho.” In popular medicines, both plants are used as antipyretic and carminative and in the treatment of respiratory system infections. In this study, the antibacterial activity of the ethanolic extracts of L. camara and L. montevidensis leaves and roots against gram-positive and gram-negative strains standard and multi-resistant bacteria isolated from clinical material are presented. In order to determine the minimal inhibitory concentration (MIC), the microdilution method was used. The extracts demonstrated antibacterial activity against all tested bacteria, but the L. montevidensis fresh leaves extract present the best result against P. aeruginosa (MIC 8 μg/mL) and against multi-resistant E. coli (Ec 27) (MIC 16 μg/mL). These results drive new researches with both species in order to isolate the constituents responsible for the activity.The use of medicinal plants with therapeutics properties represents a secular tradition in different cultures, mainly in underdeveloped countries.  The use of medicinal plants with therapeutical purposes represents a secular tradition in different cultures. Vegetal species have been shown effective to treat many infections, such as tumors ConsiderLantana is a genus of about 150 species of perennial flowering plants popularly used as antirheumatic, stimulant, antibacterial, biologic control, and as ornamental plant.[6The Verbenaceae family comprises 100 genera and about 2600 species distributed in tropical and subtropical regions around the world. Lantana l plant.6Lantana camara Linn, typical in Americas and Africa, and Lantana montevidensis Briq, native to Brazil and Uruguay are popularly known as “camará”, “cambará” or “chumbinho.” They are shrubs introduced in many countries as ornamental plants and considered as invasive species in many parts of the world.[he world. In populhe world.in vitro antibacterial activity of ethanolic extracts from L. camara and L. montevidensis leaves and roots using the microdilution method to assay the susceptibilities of five bacteria strains (American Type Culture Collection - ATCC) and two multi-resistant strains isolated from clinical material.The purpose of this study was to evaluate the L. camara and L. montevidensis were collected in March, 2009, from the Small Aromatic and Medicinal Plants Garden of the Natural Products Research Laboratory (LPPN) at Universidade Regional do Cariri (URCA), city of Crato, Ceará state, Brazil. The exsiccate was deposited at the Herbarium Caririensis “Dárdano de Andrade Lima”, Biology Department, under registry numbers 1662 and 1619, respectively, for L. camara and L. montevidensis.The leaves of L. camara (240 g of the fresh leaves and 185 g of the roots) and L. montevidensis (400 g of the fresh leaves and 714 g of the roots) were placed in a flask containing cold ethanol and left in this position for 72 h at ambient temperature. A rotary vacuum pump extractor was used to remove the ethanol from the extracts (under reduced pressure). The extracts were weighted and stored.Ethanolic extracts were prepared using the cold extraction method. Staphylococcus aureus (ATCC 12692), Proteus vulgaris (ATCC 13135), Pseudomonas aeruginosa (ATCC 15442), Víbrio cholareae (ATCC 15748), Escherichia coli (ATCC 2992) and two multiresistant strains obtained from clinical material: E. coli  and S. aureus .The antibacterial activities of the extracts were investigated by employing a microdilution method, recommended by NCCCLS M7-A6. The assa8 UFC/mL . After this, the suspension was diluted to 1 × 106 UFC/mL in 10% BHI. 100 μL of each dilution were distributed in 96-well plates plus extracts in different concentrations, achieving 5 × 105 UFC/mL as the final concentration of the inoculum.Brain Hear Infusion Broth (BHI 3.8%) was used for bacterial growth . The inoculum was an overnight culture of each bacterial species in BHI broth diluted in the same media to a final concentration of approximately 1 × 10Extracts were dissolved in distilled water and dimethyl sulfoxide (DMSO) to a concentration of 103 μg/mL. Further serial dilutions were performed by addition of BHI broth to reach a final concentration in the range of 512 a 8 μg/mL. All experiments were performed in triplicate, and the microdilution trays were incubated at 35 ± 2°C for 24 h. Antibacterial activity was detected using a colorimetric method by adding 25 μL of resauzurin staining (0.01%) aqueous solution in each well at the end of the incubation period. The minimal inhibitory concentration (MIC) was defined as the lowest essential oil concentration able to inhibit the bacteria growth, as indicated by resauzurin staining .L. camara and L. montevidensis presented excellent results against the pathogenic microorganisms tested [Several new antibacterial agents are currently being developed in response to the emergence of bacterial resistance to existing drug. New vegetal sources presenting antimicrobial activity and low toxicity could be a viable alternative, with low cost and easily accessible to poor communities where the species are found. The antis tested .L. camara leaves extract was active against P. vulgaris and V. cholerae (MIC 128 μg/mL for both strains); in addition the root extract was effective against P. vulgaris and P. aeruginosa . The leaves and roots L. montevidensis extracts were active against P. vulgaris and P. aeruginosa (MIC 8 μg/mL) and two strains of E. coli (MIC 16 μg/mL for the multiresistant strain) as shown in The extracts presented antibacterial activity against clinically relevant pathogens (gram positive and gram negative). Lantana species showed that they were able to inhibit the growth of gram-positive bacteria strains.[P. aeruginosa. This is relevant information as Navon-Venezia (2005)[P. aeruginosa, for example).Previous studies using extracts from  strains. However,ia (2005) reportedLantana species secondary metabolism as reported in phytochemical studies.The results that we present here are relevant, as the literature has shown that gram-positive bacteria are more sensitive to antibiotics. The gram-negative bacteria display some particularities that inhibit antibiotics penetration, as the lipopolyssacaride layer that determines the permeability and susceptibility to antibiotics. We sugge


Cissus quadrangularis (PECQ) impact on the development of osteopenia in type 1 diabetic rat model has been evaluated. Diabetic rats were treated orally with two doses of  PECQ. Another group of diabetic rats were treated with subcutaneous injection of synthetic human insulin. The cortical and trabecular bone thickness and bone strength were significantly decreased in diabetic rats. Treatment with two doses of  PECQ significantly prevented these changes in diabetic rats. However,  PECQ treatment (two doses) did not alter the glycemic levels in these diabetic rats. Increased levels of serum alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), and hydroxyproline were noted in diabetic rats when compared to normal control rats. The two doses of  PECQ treatment further improved the serum ALP levels and significantly decreased the serum levels of TRAP and hydroxyproline. The effects of  PECQ treatment on histological, biomechanical, and biochemical parameters are comparable to those of insulin. Since  PECQ improves the bone health in hyperglycemic conditions by enhancing the cortical and trabecular bone growth and altering the circulating bone markers, it could be used as an effective therapy against diabetes-associated bone disorders.Petroleum ether fraction of  Diabetes mellitus (DM) is a combination of metabolic disorders characterized by impaired metabolism of carbohydrates, proteins, and fat resulting from insulin deficiency . SkeletaCissus quadrangularis (CQ) is a climbing shrub, which belongs to the Vitaceae family. It is usually seen in hot climate in various states of India, Sri Lanka, Malaya, Java, and West Africa [ CQ has been shown to have an ability to accelerate the healing of bone fracture [ CQ [ CQ has shown the presence of phytoestrogen steroids [ CQ has been shown to have potent antiosteoporotic activity [ In vitro study has shown that the petroleum ether fraction of CQ (PECQ) enhances the proliferation and differentiation of rat bone marrow mesenchymal stem cells [ PECQ on defective fetal skeletal ossification in maternal diabetes has been studied (ahead of print). Though the pharmacological effect of CQ on bone health has been studied extensively, no attempt has been made to study the efficacy of CQ on osteopenia associated with DM. In the presented study, we evaluated the effect of CQ against bone histology, biomechanical changes, and circulating bone markers in type 1 diabetic rats.t Africa . In Ayurt Africa . CQ has fracture . Experimure [ CQ , 30, 31.steroids . Recentlactivity . In vitrem cells . Previou CQ were air-dried and grinded into powder. Using a soxhlet apparatus, 1.3 kg CQ powder was subjected to extraction with 95% ethyl alcohol. The ethanol extract obtained (125 g) was further suspended in water. Then, it was partitioned with petroleum ether (b.p. 60–90°C) solvent. The total yield (9.1% w/w) of PECQ was stored at 4°C until use. Every day fresh suspension of PECQ was prepared by using carboxymethyl cellulose (CMC).The fresh stems ofIn the present study, 3-month-old male Wistar rats (180–220 g weight) were used. After obtaining approval from the Institutional Animal Ethical Committee, rats were placed in the Central animal research facility of Manipal University according to guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Proper ventilation with temperature control was maintained on a 12 hr dark and 12 hr light schedule throughout the experiment. Rats were fed a standard balanced diet and water. PECQ at daily doses of 500 mg/kg and 750 mg/kg body weight. The dose was selected based on the previous study [After one week male Wistar rats were induced diabetes with streptozotocin (STZ) injection intraperitoneally (40 mg/kg body weight), which was dissolved in 0.1 M citrate buffer, pH 4.5. Control rats were injected 0.1 M citrate buffer. The blood glucose levels were analyzed seven days after injection using Glucometer (AccuChek Active). Animals demonstrating hyperglycemia (>250 mg/dL) were treated orally withus study . The dian = 30) were allocated to 5 groups each containing 6 rats. Rats in normal control (NC) Group A received 0.5% CMC; Group B, diabetic control group (DC), received 0.5% CMC; Group C, the diabetic + CQ1 group (DC + CQ1), received 500 mg/kg body weight dose of PECQ; Group D, the diabetic + CQ2 group (DC + CQ2), received 750 mg/kg body weight dose of PECQ; and Group E, the diabetic + INS group (DC + INS), rats received INS. The blood glucose levels were analyzed at regular intervals of the experimental period. Following the completion of experiment, the animals were sacrificed under anesthesia by cervical dislocation. Before sacrificing the animals, blood was collected for estimation of serum ALP, TRAP, and hydroxyproline. Right femora were collected for histomorphometrical analysis of trabecular bone and cortical bone. Left femora were stored at −70°C for testing the biomechanical properties. Right tibia was collected for measuring the dry weight.Experimental rats (μm thickness) of the lower end of the femur were taken on rotary microtome and then processed for eosin and hematoxylin staining. The stained sections were used for analysis of the thickness of trabecular bone in the metaphyseal and epiphyseal regions, by using Olympus Cellsens Imaging Software .Left femora were dissected and the soft tissue separated. Tissues were fixed in the PLP fixative for 24 hr at 4°C. Then, the femora were subjected to decalcification using EDTA-glycerine solution. After 20 days of complete decalcification, tissues were dehydrated and placed in paraffin wax. The longitudinal sections . Briefly, the left femora were brought to room temperature from −70°C and wiped with tissue paper. In the material testing machine, the bone was placed horizontally on two supports and load was applied in the middle of the shaft, at a speed of 5 mm/min until the bone was fractured.ALP and TRAP levels were estimated by spectrophotometric method, using commercially available kits (Agappe diagnostics). Serum hydroxyproline levels were analyzed by Neuman and Logan method .Right tibia were collected and dissected free of soft tissue. Bone tissues were kept in a hot-air oven at 110°C for 48 hr and were weighed in digital balance as described previously .P < 0.05.Results were expressed as the mean ± standard error of mean. Data was analyzed by using Graphpad Prism (version 5.1). One-way ANOVA followed by Bonferroni's multiple comparison test was used to evaluate differences between groups. Statistical significance was considered at  PECQ treatment did not alter the blood glucose levels in diabetic rats when compared to diabetic nontreated rats  throughout the experiment. The two doses ofP < 0.001; Figures  PECQ significantly improved the trabecular bone thickness in the DC + CQ1 , DC + CQ2 (P < 0.01), and DC + INS (P < 0.001) groups when compared to DC rats (Mean maximum flexor load (N) required to produce break in the femur of NC, DC, DC + CQ1, DC + CQ2, and DC + INS groups was 96.53 ± 5.37, 53.2 ± 5.03, 76.47 ± 4.4, 81.42 ± 6.24, and 91.53 ± 4.79 newtons, respectively. Mean maximum flexor load was significantly less in the diabetic nontreated rats , DC + CQ2 (P < 0.05), and DC + INS (P < 0.001), when compared to DC rats , DC + CQ2 (P < 0.001), and DC + INS (P < 0.001) groups when compared to NC group , DC + CQ2 (P < 0.01), and DC + INS (P < 0.01) groups in comparison to diabetic nontreated rats (P < 0.05), DC + CQ2 (P < 0.05), and DC + INS (P < 0.01) groups in comparison to DC group  signaling is found to be downregulated in both humans and animal models with type I DM , 57. Decd IGF-II . Based o PECQ on bone changes in diabetic rats observed in the present study could be due to the phytoestrogen steroids present in it [ PECQ and INS showed further increase in the ALP levels compared to diabetic group. This result indicates that PECQ enhances osteoblast proliferation and thus facilitates the bone formation. An in vitro study has demonstrated the stimulatory effect of E2 on differentiation of bone marrow mesenchymal stem cells, in hyperglycemic conditions [ PECQ on ALP activity could be due to estrogen-mimicking action of phytoestrogen steroids present in it [Earlier studies have demonstrated the beneficial effect of synthetic estrogen E2 against the bone loss in hyperglycemic state , 26. Thent in it , 33, whint in it . It has nt in it . In the nditions . The bennt in it , 33. PECQ and INS significantly decreased the TRAP levels compared to diabetic animals. The observed effect of the PECQ on the TRAP activity could be due to the direct action of the phytoestrogen steroids present in it [Compared to normal control group, serum TRAP levels are significantly increased in diabetic animals. Serum TRAP is a biomarker of osteoclast activity. The increased levels of this protein indicate that the reduced bone mass in the diabetic rats is also due to excessive bone resorption. With respect to serum TRAP activity, our results are consistent with previous findings . Howevernt in it , 33. Thint in it . PECQ and INS significantly decreased the serum hydroxyproline levels. Gopalakrishnan et al. in their in vitro study demonstrated the positive effect of E2 on mineralization and histochemical staining for collagen in the bone marrow stromal cells [ PECQ could be due to the estrogen-like action of phytoestrogen steroids [Serum hydroxyproline is a breakdown product of collagen. In the present study, the hydroxyproline levels were significantly increased in the diabetic rats indicating the inhibitory effect of diabetes on bone collagen. This result is consistent with that of the previous study . Treatmeal cells . The possteroids , 33 on c PECQ is effective in improving histological, biomechanical, and biochemical changes of bone in diabetic rats. Though exact mechanism of action of PECQ has not been ascertained, the observed effect of PECQ could be due to its osteogenic, antioxidant, and anti-inflammatory properties. However, in this context, extensive studies are required to confer the exact mechanism of PECQ on bone cells in hyperglycemic conditions.Preliminary results of the present study indicate that


PAH are causal to phenylketonuria (PKU), an autosomal recessive disease characterized by neuropsychiatric symptoms including intellectual disability. We examined whether there is an association between common single nucleotide polymorphisms (SNPs) of PAH and memory performance in the Japanese population.Phenylalanine hydroxylase (PAH) is the enzyme that metabolizes phenylalanine, an essential amino acid required for catecholamine synthesis. Rare mutations in PAH .Subjects were 599 healthy adults . The Wechsler Memory Scale-Revised (WMS-R) was administered to all participants to assess memory performance. Genotyping was performed for 6 selected tagging SNPs of Analyses of covariance controlling for sex and education years, indicated a significant association between a SNP (rs2037639) and age-corrected verbal memory index of WMS-R  which remained significant after correction for multiple testing ( p = 0.0013 < 0.0017 = 0.05/30tests). Individuals with the GG genotype showed a significantly lower mean verbal memory score, compared with those individuals carrying the AA/AG genotype . A haplotype block containing two markers of rs2037639 and rs10860936 was associated with verbal memory index .PAH are associated with verbal memory in healthy adults. Unknown functional polymorphisms in PAH or those in other genes nearby might affect memory performance.Our findings suggest that common genetic variations in  PAH encodes phenylalanine hydroxylase [EC 1.14.16.1] that converts phenylalanine to tyrosine which is a precursor of the neurotransmitter dopamine and other catecholamines [PAH were registered as responsible for PKU (http://www.pahdb.mcgill.ca/) [Accumulating evidence suggests that genetic factors influence human complex behavior, including neurocognition throughout the life span ,2. Studiolamines ,6. More PAH confer susceptibility to schizophrenia, a disease characterized by pervasive neurocognitive deficiencies including distinct memory impairment [Interestingly, previous studies ,8 reportpairment , as wellPAH might be associated with neurocognitive function, memory performance in particular. The aim of the present study was to examine the possible association of single nucleotide polymorphisms (SNPs) of PAH with memory performance in healthy adults. To our knowledge, there has been no report that examined such an association.Based on these observations, we hypothesized that common genetic variations in Subjects were 599 healthy Japanese adults . Mean education years of the participants were 15.2 ± 2.7 years. All subjects were biologically unrelated and were recruited via advertisements of free local magazines in the western part of Tokyo Metropolitan and our website announcement. All subjects underwent structured interview using the Japanese version of the Mini International Neuropsychiatric Interview (M.I.N.I.) ,11 and aThe Japanese version of Wechsler Memory Scale-Revised (WMS-R) ,14 was aPAH, encompassing 2 kilobase (kb) of sequence upstream and downstream , using the ‘Tagger’ application of the program Haploview 4.2 (http://www.broad.mit.edu/mpg/haploview/) (version 3.0 release R2) [2 threshold of 0.8 with a minor allele frequency (MAF) threshold of 0.1. Genotyping of the SNPs was performed using the TaqMan allelic discrimination assay  with 10 ng human DNA and 1.5 μl 2× qPCRTM Mastermix Plus Quick Gold Star . Polymerase chain reaction was performed with an initial cycle at 95°C for 10 minutes followed by 50 cycles of 92°C for 15 seconds and 60°C for 1 minute in GeneAmp PCR System 9700 (Applied Biosystems). The allele-specific fluorescence was discriminated with an ABI PRISM 7900 Sequence Detection Systems (Applied Biosystems). Genotype data were determined blind to the WMS-R scores. Ambiguous genotype data were excluded from the analysis. The call rate of non-missing genotypes was over 97%.Preparation of genomic DNA from venous whole blood of subjects was performed according to standard procedures. We selected 6 tagging SNPs  throughout 2 test. Demographic characteristics between genotype groups was compared by using ANOVA or χ2 test, as appropriate. The ANCOVA was performed to test the effect of genotype on each score of the 5 indices of WMS-R, controlling for sex and education years. Paired comparisons for verbal memory scores of each genotype were carried out with the ANCOVA controlling for sex and education years. Haploview 4.2 was used to check deviation from the Hardy-Weinberg equilibrium (HWE) for each SNP, and to calculate measures of linkage disequilibrium (LD). Haplotype blocks were constructed using the default block search algorithm of Gabriel et al.[http://pngu.mgh.harvard.edu/~purcell/plink) [http://www.psycho.uni-duesseldorf.de/abteilungen/aap/gpower3/) was used [The statistical package for the social sciences (SPSS) version 11.0  was used for the analysis of variance (ANOVA), analysis of covariance (ANCOVA) and χel et al.. Haplotyl/plink)  was usedwas used . StatistThere were no significant group differences in age or smoking status between the genotype groups   using the PLINK software revealed that haplotype block 2, which contained two markers (rs2037639-rs10860936), was significantly associated with verbal memory index  of the 6 tag SNPs in PAH. The association between SNP rs2037639 and verbal memory remained significant after correction for multiple testing. Haplotype-based analysis revealed that a haplotype containing two markers (rs2037639-rs10860936) was significantly associated with verbal memory index. These results suggest that common functional variants in PAH impact verbal memory performance in healthy adults. Alternatively, there might be a possibility that functional variants in other genes nearby, which would be in linkage disequilibrium with rs2037639 or the haplotype, may play a role. Since there were no PKU patients in our sample, the obtained result cannot be ascribed to mutations responsible for PKU. Further, considering the carrier frequency for PKU of about 1/150 that corresponds to the PKU incidence among Japanese of 1/70000 to 1/ 120000 [We examined whether common genetic variations of / 120000 ,22, the Of note, the SNP rs2037639 was previously reported to be associated with schizophrenia in a male Bulgarian sample  [PAH are related with impaired working memory in humans [Pahenu2) [PAH and cognition. Since there is no direct evidence of functional effects of the SNPs, the mechanism underlying the relationship is currently unclear. The SNP (rs2037639) may have some functional effects such as regulation of transcription of PAH. Alternatively, the SNP might be in linkage disequilibrium with an unknown functional polymorphism. The functional effect could alter phenylalanine level, which then alters tyrosine level and activation of tyrosine hydroxylase. Finally the PAH genotypes could affect dopamine level in the brain [There is evidence that rare PKU mutations in n humans ,24 and cPahenu2) . Howeverhe brain ,27, whiche brain . In addihe brain -31, posscatechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). Krach et al. reported that the functional COMT Val158Met polymorphism links neural activation pattern during episodic memory tasks [et al. found that tolcapone, the COMT inhibitor, enhanced working memory assessed by the letter–number sequencing tasks in healthy subjects with the Val/Val genotype [et al. found that the MAOA variable number of tandem repeats (VNTR) polymorphism, which is located in its promoter region and controls the transcriptional activity of the gene, was associated with working memory-related performance, indicating that n-back performance measured by reaction time was poorer in individuals with MAO-L  than in individuals with MAO-H  [PAH variants could affect synthesis of catecholamines.Previous genetic studies have shown an association of neurocognition with genes encoding enzymes regulating catecholamine metabolism, such as ry tasks . In theigenotype . On the ctivity) . Since Ppost-hoc power analysis to evaluate the statistical power for the association analysis between verbal memory index and the PAH rs2037639 genotypes, the current sample size had a 80% power (β = 0.2) to detect mean difference of 3.3, 4.9, and 5.4 between AA vs. AG, AA vs. GG, and AG vs. GG, respectively, at the α (two-tailed) = 5% level. Further studies with a larger sample size might be required to detect smaller differences. The mechanism by which polymorphisms of PAH influences memory performance is currently unclear, i.e., there is no direct evidence of functional effects of the examined SNPs. Future animal and human studies are therefore warranted to elucidate the mechanism.There are limitations in the study. The present sample size may have been small, which is subject to type II errors. When we performed a PAH and memory performance assessed with WMS-R in Japanese healthy adults. We obtained evidence suggesting that genetic variation of PAH  are associated with verbal memory. Considering the carrier frequency for rare mutations responsible for PKU, our data suggest that common functional polymorphisms in PAH impact verbal memory. Alternatively, functional polymorphisms in other genes nearby may play a role in genetic basis for verbal memory.We performed an association study between genetic variations of ANOVA: Analysis of variance; ANCOVA: Analysis of covariance; COMT: Catechol-O-methyltransferase; DSM- IV: Diagnostic and statistical manual of mental disorders, 4th edition; HWE: Hardy-Weinberg equilibrium; LD: Linkage disequilibrium; MAF: Minor allele frequency; MAO: Monoamine oxidase; MINI: Mini international neuropsychiatric interview; PAH: Phenylalanine hydroxylase; PKU: Phenylketonuria; SNP: Single nucleotide polymorphism; WMS-R: Wechsler memory scale-revised.The authors declare that they have no competing interests.TT and HK designed the study and TT wrote the draft of the manuscript. TT, DS, HH, KH, MO and SF performed psychiatric assessments for the participants. JM, AN and YK administered the WMS-R. TT and NY performed the genotyping. TF supervised the writing of the paper. HK supervised the project and gave critical comments on the manuscript. All authors contributed to and have approved the final manuscript.Demographic characteristics.Click here for file


Cognitive impairment is prevalent in older adults with heart failure (HF) and associated with reduced functional independence. HF patients appear at risk for reduced driving ability, as past work in other medical samples has shown cognitive dysfunction to be an important contributor to driving performance. The current study examined whether cognitive dysfunction was independently associated with reduced driving simulation performance in a sample of HF patients.18 persons with HF  completed echocardiogram and a brief neuropsychological test battery assessing global cognitive function, attention/executive function, memory and motor function. All participants then completed the Kent Multidimensional Assessment Driving Simulation (K-MADS), a driving simulator scenario with good psychometric properties.t-tests showed that HF patients performed worse than healthy adults on the driving simulation scenario. Finally, partial correlations showed worse attention/executive and motor function were independently associated with poorer driving simulation performance across several indices reflective of driving ability .The sample exhibited an average Mini Mental State Examination (MMSE) score of 27.83 (SD = 2.09). Independent sample The current findings showed that reduced cognitive function was associated with poor simulated driving performance in older adults with HF. If replicated using behind-the-wheel testing, HF patients may be at elevated risk for unsafe driving and routine driving evaluations in this population may be warranted. Heart failure (HF) has become one of the most prevalent forms of cardiovascular disease with an estimated 600,000 persons newly diagnosed each year . HF is tRecent work also shows that cognitive impairment in HF is associated with reduced driving independence . Indeed,It is possible that reduced cognitive function in HF patients may impede their ability to perform complex driving behaviors . DrivinBecause of the prevalence of impairments in frontal systems function  it would be expected that HF patients exhibit driving errors that involve organization, planning, and reasoning such as choosing the travel route and travel times, monitoring distance, adjusting speed, among other executive skills . This paThe above findings suggest that HF patients may be at risk for reduced driving ability, though no study to date has examined the relationship between cognitive function and driving performance among patients with HF. The current study sought to determine whether reduced neuropsychological test performance was associated with poorer driving performance using driving simulation technology in a sample of older adults with HF. To further evaluate risk of poor driving ability in this population, we also compared simulated driving performance between HF patients and healthy young adults.A sample of 18 participants with HF was recruited from an ongoing NIH study examining neurocognitive function among older adults with HF. Strict inclusion and exclusion criteria were implemented in order to capture the independent effects of HF on neurocognitive outcomes. The inclusion criteria were age of 50-85 years, English as a primary language, and a diagnosis of New York Heart Association (NYHA) class II or III at the time of enrollment. Additional inclusion criteria also required a valid driver’s license and currently driving. Prior to study entry, all patients were screened for neurological and psychiatric conditions that may influence cognitive function or restrict their performance of instrumental activities of daily living. Specifically, individuals were precluded from study entry if they had a history of neurological disorders , head injury with >10 minutes loss of consciousness, severe psychiatric disorder , past or current substance abuse/dependence, and renal failure.Participants averaged 67.72 (SD = 8.56) years of age, and were 11.1% female. Medical chart review also revealed that the sample had an average left ventricular ejection fraction of 39.38 (SD = 13.25). See Table The STISIM Driving Simulator (Build 2.08.03) by Systems Technology Inc. was used to assess driving ability. It is a computer based, interactive drive simulator software package, and has been configured to control: (a) a high fidelity steering wheel with two analog levers for left/right turn indication; (b) an analog pedal set consisting of an analog brake pedal and another pedal for gas; and (c) a 46” High Definition LCD television at an average presentation distance of 4.5 feet. The STSIM driving software was used to devise the driving scenarios that all participants completed. The STSIM has been shown to correlate with on-road driving tests ,31.HF participants first completed a practice scenario with the driving simulation technology. This was conducted in order for drivers to become comfortable with the simulator and practice the tasks they will be required to perform during the test trial such as accelerating, decelerating, following the speed limit signs, navigating turns, stopping at stop signs/traffic signals, and maintaining lane position. The practice scenario is nearly 3 miles long, lasted approximately 15 minutes, and assessed driving performance in multiple settings, including city, country, and highway environments.Participants then completed the Kent Multidimensional Assessment Driving Simulation (K-MADS) driving scenario, which is distinct from the practice simulation. All participants were instructed to drive as safe as possible as they normally would on the road. The K-MADS is a roughly 7 mile long driving scenario that takes approximately 20-25 minutes to complete. It has good psychometric properties  . The K-MThe K-MADS yields several indices indicative of driving performance that were utilized in the current study, including total collisions, number of stop signs missed, number of centerline crossings, number of road excursions, % of time over the speed limit, and% of time out of the lane.A brief battery of neuropsychological tests was administered to assess cognitive function across multiple domains. All neuropsychological tests used in the current study exhibit strong psychometric properties, including excellent reliability and validity. The domains and neuropsychological tests administered are as follows:The Mini Mental State examination (MMSE) was used to assess global cognitive status. It is a widely used, brief cognitive screening tool that provides an estimate of global cognitive function, tapping aspects of attention, memory, language, and spatial abilities ,33.Trail Making Test A and B, Digit Symbol Coding, Letter Number Sequencing, and the Stroop Color Word Test were used to assess attention/executive function. Trail Making Test A requires participants to sequential order a series of numbers as quickly as possible and it is a reliable and valid measure of complex visual scanning and psychomotor speed . Trail MThe California Verbal Learning Test-II (CVLT-II) total recall, short delay free recall, and long delay free recall was used to assess memory function . IndividThe Grooved Pegboard is a reliable measure used to assess fine motor dexterity ,42. PartThe Kent State University and Summa Health System Institutional Review Board (IRB) approved the study procedures and all participants provided written informed consent prior to study enrollment. The investigation conforms with principles outlined in the Declaration of Helsinki. As part of the larger NIH study procedures, a medical record review was performed and participants completed demographic and medical history self-report measures. A brief neuropsychological test battery was also administered to all HF patients as part of the larger study protocol to assess cognitive function. Upon completion of cognitive testing, HF participants were then recruited to complete a single time, 45-minute, driving assessment using the driving simulation technology.Two cases exhibited missing data for LVEF and those values were handled using simple mean imputation. For descriptive purposes and to facilitate clinical interpretation of cognitive test performance in the sample, raw scores for the neuropsychological test measures were transformed to T-scores (a distribution with a mean of 50 and standard deviation of 10) using normative data adjusting for age, and in the case of memory, gender. A T-score cutoff of ≤ 35 was used to characterize impairment in the cognitive domains. This cutoff was chosen because it is reflective of performances 1.5 SD below the mean relative to normative standards and it is a commonly used cutoff for the clinical identification and diagnosis of cognitive impairment .t-tests first examined driving simulation performance in this sample of HF participants compared to performance among healthy adults from a validation study [For the main analyses, three separate composite scores were computed for attention/executive function, memory, and motor function that consisted of the mean of the raw scores of neuropsychological measures within each cognitive domain. To maintain directionality for attention/executive, scores for neuropsychological tests measured in units of time  were multiplied by -1 so that lower scores reflect worse performance. Independent samples on study  on the fA series of partial correlations adjusting for age, gender , education, and LVEF were then conducted to examine the relationship between attention/executive function, memory, and motor function with the specific driving indices, including total collisions, number of stop signs missed, number of centerline crossings, number of road excursions, % of time over the speed limit, and % of time out of the lane.The sample exhibited an average MMSE score of 27.83 (SD = 2.09). Specifically, 27.8% of the sample scored below a 27 and 11.1% scored below an MMSE score of 25. When using a T-score cutoff of ≤ 35 to characterize impairment in attention/executive function, memory, and motor functioning, 5.6% of the sample exhibited impairments in attention/executive function, 11.1% for memory, and 16.7% showed impairments in motor function.Reduced performance on the driving simulation was also prevalent with many HF participants recording at least one collision . Similarly, HF participants also frequently crossed the centerline and deviated from the lane and speed limit on several occasions. Refer to Table t-test was conducted to examine driving simulation performance between the HF participants and healthy young adults. Independent samples t-test revealed that HF participants performed significantly worse than the healthy young adults on all of the driving simulation indices examined: Number of collisions (t(113) = -2.40, p = .02; M (SD) = 2.00(1.28) versus 1.30 (1.11)), number of stop signs missed (t(113) = -2.14, p = .045; M(SD) = .78(.81) versus .35(.60)), number of centerline crossings (t(113) = -2.40, p = .05; M(SD) = 5.83(3.96) versus 3.82(3.13)), and number of road excursions (t(113) = -2.44, p = .03; M(SD) = 4.83(6.39) versus 1.12(1.93)).Independent samples r(12) = -.56, p = .04) and % of time out of lane (r(12) = -.54, p = .045). In each, case worse attention/executive function was associated with reduced driving ability in each of these indices. A similar pattern also emerged for motor function: total number of collisions (r(12) = .57, p = .04), % of time over the speed limit (r(12) = .65, p = .01), and a trend for number of stop signs missed (r(12) = .47, p = .09). There was also a trend between reduced MMSE scores and greater number of stop signs missed (r(12) = -.50, p = .07). Memory performance demonstrated no associations with any of the driving indices. See Table Partial correlations adjusting for age, gender, education and LVEF revealed significant associations between attention/executive function with number of centerline crossings   and reduWe found that HF participants performed significantly worse than healthy adults on the simulated driving scenario and that reduced attention/executive and motor function were associated with poorer driving simulation performance. Specifically, attention/executive function correlated with lane deviations and centerline crossings while motor function was associated with errors that require motor coordination . Consistent evidence shows that executive function is a key cognitive process required for driving ability in dementia populations . On-roadThis pattern of findings is concerning, as the multiple brain regions  responsible for these cognitive processes are susceptible to damage in HF patients as a result of ischemia . Thus, sInterestingly, the current study found no association between memory testing and simulated driving performance. The literature is inconsistent regarding the effects of memory on driving ability ,53. It iTaken together, the association between driving simulation performance with frontal systems function, but not memory, is consistent with the extant evidence in HF that demonstrates executive function to be the most important cognitive ability for functional independence in this population . It is pThe generalizability of the current findings is limited in several ways. First, cross-sectional data was analyzed and prospective studies are needed to confirm the link between cognitive decline and reduced driving ability in HF patients. Additionally, although simulated driving offers many advantages such as cost-effective and maintains the safety of the participant and the larger community ,58, futuIn brief summary, the current study found that HF patients performed worse on a driving simulation scenario than healthy adults and reduced cognitive function was associated with poorer driving simulation performance in this sample. Such findings suggest that HF patients may be at-risk for driving impairment. Implementation of brief cognitive screening in clinical care settings may help to identify HF patients that may be unsafe for driving and thus allowing for earlier clinical intervention, including referral for a formal driving evaluation or advisement to family members to monitor patients’ driving. Future studies examining driving performance in HF patients using on-road testing are needed to confirm these findings and determine the most effective approach for identification of driving impairment in clinical settings.The authors declare no competing interests.Study concept and design: MA, MBS, JG. Data acquisition: MA, MJC, JG. Analysis and interpretation of data: MA, JG. Manuscript preparation: MA, MBS, MJS, JG. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2318/13/58/prepub


Pseudomonas fluorescens (strain DSM 8341) was used as positive control for fluoroacetate dehalogenase activity. Two isolates were identified by 16S rRNA gene sequencing as Pigmentiphaga kullae (ECPB08) and Ancylobacter dichloromethanicus (ECPB09). These bacteria degraded sodium fluoroacetate, releasing 20 mmol L−1 of fluoride ion after 32 hours of incubation in Brunner medium containing 20 mmol L−1 of SF. There are no previous reports of fluoroacetate dehalogenase activity for P. kullae and A. dichloromethanicus. Control measures to prevent plant intoxication, including use of fences, herbicides, or other methods of eliminating poisonous plants, have been unsuccessful to avoid poisoning by fluoroacetate containing plants in Brazil. In this way, P. kullae and A. dichloromethanicus may be used to colonize the rumen of susceptible animals to avoid intoxication by fluoroacetate containing plants.The objective of this paper was to report the isolation of two fluoroacetate degrading bacteria from the rumen of goats. The animals were adult goats, males, crossbred, with rumen fistula, fed with hay, and native pasture. The rumen fluid was obtained through the rumen fistula and immediately was inoculated 100  Palicourea marcgravii, P. aeneofusca, P. juruana, and P. grandiflora); Bignoniaceae ; and Malpighiaceae  [P. marcgravii is the most important Brazilian toxic plant and M. rigida is the most important toxic plant in Northeastern Brazil [P. marcgravii, A. bilabiata [M. rigida [The most important group of poisonous plants in Brazil is one that causes “sudden death” during physical effort, responsible for nearly 500 000 cattle deaths each year. It is composed of 13 species of plants, belonging to 3 families: Rubiaceae ( sepium) . P. marcn Brazil . Sodium ilabiata , 13, and. rigida . In the . rigida ; it disr. rigida . Microbi. rigida , but als. rigida . Sodium . rigida . Acinetobacter, Arthrobacter, Aureobacterium, Bacillus, Pseudomonas, Weeksella, and Streptomyces were isolated [Ancylobacter, Burkholderia, Cupriavidus, Paenibacillus, Staphylococcus, Stenotrophomonas, and Ralstonia [Several soil bacteria have the ability to degrade sodium fluoroacetate. In central Australia, seven fluoroacetate degrading bacteria genera including isolated . Recentlalstonia . Howeveralstonia , 23. As ad libitum. They were housed in the Veterinary Hospital of the Universidade Federal de Campina Grande in the State of Paraíba, Northeastern Brazil, and had no access to areas with sodium fluoroacetate-containing toxic plants.The animals were male adult goats, crossbred, with rumen fistula, and fed with hay, native pasture, and water The rumen fluid was obtained through the rumen fistula and immediately inoculated in test tubes.μL of rumen fluid in tubes containing 9 mL of mineral medium (Brunner) added with vitamins (http://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium457.pdf) and 20 mmol L−1 sodium fluoroacetate (SF) (Sigma-Fluka) as single carbon source. This medium will be here designated as Brunner medium. Samples were incubated at 39°C in an orbital shaker. After 48 hours, 1 mL of the first growth was transferred to test tubes containing 9 mL of Brunner medium and incubated under the same conditions described above.Bacterial isolation started by the inoculation of 100 − selective electrode (Thermo Electron Corporation) in 24-well plates containing 500 μL of culture and 500 μL of Total Ionic Strength Adjustment Buffer-TISAB . The fluoride ion released by the microbial SF degradation was expressed in millimoles, the defluorination rate of 20 mmol L−1 corresponding to the release of 20 mmol L−1 F−.The SF defluorination was measured with a F−1 to 10−9. To obtain pure colonies the highest dilution that presented SF defluorination was plated on Brunner agar (Brunner medium with agar 1%) and incubated at 39°C for 72 hours. Subsequently, each colony was used to inoculate three test tubes containing 9 mL of Brunner medium, which were monitored for SF defluorination. Pseudomonas fluorescens (strain DSM 8341) was used as positive control for fluoroacetate dehalogenase activity, cultivated under the same conditions except that the incubation was at 28°C [Samples presenting SF defluorination were cultivated in serial dilutions from 10 at 28°C . Nine mLμM of 27f and 1492r universal primers [μL. In the negative control, the DNA volume was substituted by ultrapure water. The amplified products were applied into 1% agarose gel and submitted to electrophoresis. DNA was stained with ethidium bromide and bands visualized with an imaging system (UVP-Bioimaging Systems).Genus identification for bacteria displaying defluorination activity was done by polymerase chain reaction (PCR) amplification and sequencing of the 16S rRNA gene. DNA extraction was performed with Brazol (LGC Biotechnology) according to the manufacturer's specifications. 16S rRNA gene was amplified in buffer containing 0.5  primers , 2U of TThe sequencing reaction was performed with BigDye kit according to manufacturer's recommendations (Applied Biosystems) and the product sequenced in the Genetic Analyzer 3500 XL sequencer (Applied Biosystems).http://pbil.univ-lyon1.fr/cap3.php). DNA sequences were analyzed by basic local alignment search tool (BLAST) available on the website of the National Center for Biotechnology Information (NCB—http://www.ncbi.nlm.nih.gov/BLAST). Species identification was based on maximum score, identity and coverage values. The Greengenes database and workbench were used to corroborate species identification (http://greengenes.lbl.gov). The Bootstrap consensus tree was generated with MEGA5 software using the neighbor-joining statistical method (http://www.megasoftware.net/mega.php) [16S rRNA gene sequences were assembled with the CAP3 Sequence Assembly Program (ega.php) . Pigmentiphaga and Ancylobacter species, respectively (P. kullae sequence was the most similar (score = 2237) to ECPB08, although other 16S rRNA sequences had the same coverage and identity parameter values. Similarly, the 16S rRNA sequences from Ancylobacter vacuolatus and Ancylobacter sp. strain WPCB135 were the most similar (score 2388) to ECPB09, but the differences in alignment parameters for other Ancylobacter 16S rRNA sequences were only marginal. Consequently, it was not possible to infer from the score values which species would be phylogenetically the closest to ECPB09 (JQ345721).The 16S rRNA gene sequences from the two fluoroacetate degrading isolates ECPB08 (JQ345720) and ECPB09 (JQ345721) were similar to the 16S rRNA genes from ectively . On the Pigmentiphaga and Ancylobacter, respectively  and ECPB09 (JQ345721) were phylogenetically closely related to the genera ectively . Indeed,rain K24 , and is ymorphus . AlthougPigmentiphaga was initially proposed by Blümel et al. [Pigmentiphaga kullae (strain K24) as its unique specie, which degrades xenobiotic compounds. In 2007, Yoon et al. [Pigmentiphaga daeguensis (strain K110T), isolated from wastewater collected from a dye works in Korea. Later, Chen et al. [Pigmentiphaga litoralis (strain JSM 061001) and P. soli from a tidal flat sample in the South China Sea and from soil in South Korea, respectively.The genus l et al.  with Pign et al.  describen et al.  and Lee n et al.  isolatedMicrocyclus by Ancylobacter, with a single species, A. aquaticus [Ancylobacter dichloromethanicus (strain DM16), isolated from contaminated soil [Raj and Maloy  proposedquaticus . Since tted soil . This sp−1 of fluoride ion after 32 hours of incubation in Brunner medium with 20 mmol L−1 of SF (Burkholderia sp. The Pseudomonas fluorescens control strain (DSM 8341) reached the same level of defluorination, and there was no release of fluoride ions when the Brunner medium was incubated without bacteria, due to the stability of the strong carbon-fluorine bond in the fluoroacetate [Pigmentiphaga or Ancylobacter species.Both ECPB08 JQ345720) and ECPB09 (JQ345721) grew in culture medium containing SF as the sole carbon source. These bacteria displayed SF degradation activity, releasing 20 mmol L−1 of SF . These r5720 and oacetate . There aPigmentiphaga no dehalogenase gene has yet been described, a dichloromethane dehalogenase sequence from A. dichloromethanicus is deposited in GenBank [The defluorination ability of both isolates can be due to the expression of a fluoroacetate dehalogenase gene or to another unspecific dehalogenase. Although for  GenBank . Liu et  GenBank  confirme GenBank . Similar GenBank  who foun GenBank .Bacillus and Flavobacterium species, were isolated from soil in Australia, even in the absence of sodium fluoroacetate in the environment [Pigmentiphaga and Ancylobacter species were isolated from goat rumen, although the animals were not fed plants that produce this component. This finding suggests that the fluoroacetate dehalogenase gene is rather ubiquitous and that its expression may represent a selective advantage for microorganism, possible due to the ability of the enzyme to degrade other related compounds, as mentioned before.Sodium fluoroacetate degrading bacteria, mostly ironment . In thisMascagnia rigida is present are more resistant to poisoning than those kept in areas without these plants [M. rigida poisoning can be induced by the administration of repeated low nontoxic doses of the plant or by transfaunation of rumen fauna from resistant to susceptible animals (unpublished data). These results suggest that either changes in rumen microflora or sustained high expression levels of the SF dehalogenase gene from the endogenous rumen microbiota are responsible for this tolerance. On the other hand, it has been reported that animals grazing in areas where e plants . Recent Butyrivibrio fibrisolvens with a gene encoding fluoroacetate dehalogenase from Moraxella species, which was efficient to prevent fluoroacetate poisoning in sheep. In an alternative approach, the two isolates, Pigmentiphaga sp. and Ancylobacter sp. are candidates to be used in colonizing the rumen to avoid intoxication by fluoroacetate containing plants. Control measures to prevent plant intoxication, including use of fences, herbicides, or other methods of eliminating poisonous plants, have been unsuccessful to avoid poisoning by fluoroacetate containing plants in Brazil . An alte