Visceral fat accumulation is one of the most important predictors of mortality in obese populations. Administration of green tea extract (GTE) can reduce body fat and reduce the risk of obesity-related diseases in mammals. In this study, we investigated the effects and mechanisms of GTE on adiposity in diet-induced obese (DIO) zebrafish.Artemia once daily (5 mg cysts/fish daily) for 40 days. Zebrafish in the three DIO groups were fed freshly hatched Artemia three times daily (60 mg cysts/fish daily). Zebrafish in the DIO + 0.0025%GTE and DIO + 0.0050%GTE groups were exposed to GTE after the start of feeding three times daily for 40 days.Zebrafish at 3.5 to 4.5 months post-fertilization were allocated to four groups: non-DIO, DIO, DIO + 0.0025%GTE, and DIO + 0.0050%GTE. The non-DIO group was fed freshly hatched ACOX1 , ACADM , and PPARA . GTE exposure also significantly decreased the visceral fat expression of SOCS3  which inhibits leptin signaling.Three-dimensional microcomputed tomography analysis showed that GTE exposure significantly decreased the volume of visceral but not subcutaneous fat tissue in DIO zebrafish. GTE exposure increased hepatic expression of the lipid catabolism genes SOCS3.The present results are consistent with those seen in mammals treated with GTE, supporting the validity of studying the effects of GTE in DIO zebrafish. Our results suggest that GTE exerts beneficial effects on adiposity, possibly by altering the expression of lipid catabolism genes and  Consequently, zebrafish are increasingly being used as models of human diseases [Zebrafish  on body weight, visceral and subcutaneous fat accumulation, and the expression of lipid metabolism genes in the liver and visceral fat in DIO zebrafish.This study conformed to the ethical guidelines established by the Institutional Animal Care and Use Committee of Mie University.Camellia sinensis) were soaked in hot water and the resulting extract was reduced to a powder by spray drying. The extract was then dissolved in hot water and mixed with an equal volume of chloroform. The aqueous phase was recovered with three volumes of ethanol and the extract was freeze-dried after removing the solvent. The composition of catechins was measured by high-performance liquid chromatography. The total catechin content in the GTE was 79.6%–81.8% ; this comprised epigallocatechin gallate (43.6%–44.4%), epigallocatechin (20.4%–20.7%), epicatechin gallate (12.0%–12.3%), epicatechin (8.1%–8.3%), gallocatechin (6.9%–7.0%), gallocatechin gallate (4.4%), and others (3.7%–3.8%). The caffeine content was 0%–0.1%.GTE was prepared and analyzed as previously described . In brieThe Zebrafish Book [Male and female zebrafish  were kept at approximately 28°C under a 14-h light and 10-h dark cycle. Water conditions of environmental quality were maintained according to ish Book .Artemia for 120 min once a day (5 mg cysts/fish daily) for 40 days. Zebrafish in the three DIO groups were fed freshly hatched Artemia for 120 min three times daily (60 mg cysts/fish daily). Zebrafish in the DIO + 0.0025%GTE and DIO + 0.0050%GTE groups were exposed to GTE for 105 min, starting 15 min after the start of feeding , three times daily for 40 days. During feeding, the tank water flow was stopped; the water in each tank was replaced with fresh water at the end of feeding. Body weight was measured on days 0, 14, 20, 27, 34, and 40. Body fat volume was measured using three-dimensional microcomputed tomography (3D micro-CT) following euthanasia on the final day of the study.Male and female zebrafish at 3.5–4.5 months post-fertilization were allocated to four groups  with five fish per 1.7-L tank. Zebrafish in the non-DIO (control group) were fed freshly hatched Experiment 1 for 21 days. After feeding on the final day, the zebrafish were euthanized, immediately transferred into tubes containing 8 mL of RNAlater , and stored at 4°C until gene expression analysis.Female zebrafish were allocated to three groups  with five fish per 1.7-L tank. All groups were fed as described in in vivo System R_mCT 3D micro-CT scanner . The following settings were used: voltage, 90 kV; current, 100 μA; magnification, ×4; slice thickness (scanning width), 50 μm; and exposure time, 2 min. Images were reconstructed and viewed using i-View type R software . The CT images were visualized and analyzed using CTAtlas Metabolic Analysis Ver. 2.03 software (Rigaku Corporation). The Hounsfield unit (HU) value of fat tissue was adjusted to between –350.0 and –145.0 in accordance with the manufacturer’s instructions. Measurement of body fat volume was limited to the abdominal cavity, and the initial point of the abdominal cavity was set at the cleithrum  using Isogen  with combination with cleanup protocol of RNeasy mini kit . cDNA was synthesized using random primers and SuperScript III Reverse Transcriptase . Total RNA was extracted from the visceral fat of fish in the DIO and DIO + 0.0050%GTE groups using an RNeasy Lipid tissue mini kit (Qiagen) and qualified using an Agilent Bioanalyzer 2100 . Because of poor RNA quality, the samples for two of five fish in the DIO group were excluded. cDNA was synthesized using a High Capacity RNA-to-cDNA Kit . Total RNA could not be extracted from the visceral fat of fish in the non-DIO group because the amount of visceral fat was too small.The liver and visceral fat were dissected from the stored fish samples prepared in PPIA , ACOX1 , ACADM (Dr03120754_m1), PPARA , and SOCS3 . The primer sets for SYBR Green were as follows: ACC : ATCATCCCACCCAAACAGAC; reverse primer (3′–5′): CCCATCACAGAAGGTGGAAC) and FASN : ATCTGTTCCTGTTCGATGGC, reverse primer (3′–5′): AGCATATCTCGGCTGACGTT). The baseline and threshold were set manually in accordance with the manufacturer’s instructions. The relative mRNA expression levels were determined using PPIA as an endogenous standard.Quantitative real-time PCR was performed on cDNA samples using a TaqMan Fast Universal PCR Master Mix (Applied Biosystems) or Fast SYBR Green Master Mix (Applied Biosystems) and an ABI Prism 7500 Fast Real-Time PCR System (Applied Biosystems) in accordance with the manufacturer’s instructions. The TaqMan gene expression assays were as follows: Experiment 1. Briefly, hatched Artemia (5 or 60 mg cysts/fish/day) were fed to the zebrafish in a 1.7-L tank as described above. For a blank control, Artemia were placed in a 1.7-L tank without zebrafish . After 90 min, the number of Artemia not eaten by the zebrafish were counted three times and subtracted from the number in the blank tank to determine the feeding volume in each tank.The feeding volume assay was conducted as previously described , with miP < 0.05 were considered statistically significant.All values are presented as means ± standard error of the mean. Statistical analysis was conducted using analysis of variance followed by Fisher’s partial least-squares difference multiple comparison test. Analyses were conducted using STATVIEW for Windows version 5.0 . Values of Artemia consumed to the amount of Artemia provided (consumption ratio) was estimated in the feeding volume assay. In male zebrafish, the consumption ratio in the DIO + 0.0025%GTE (40%) and DIO + 0.0050%GTE (37%) did not decrease compared with that in the DIO group (24%). And also in female, the consumption ratio in the DIO + 0.0025%GTE (33%) and DIO + 0.0050%GTE (38%) did not decrease compared with that in the DIO group (33%).Visual observation revealed no marked abnormalities or major differences in feeding behavior between the three DIO groups . The ratio of P < 0.05 or P < 0.01 compared with the DIO group). By contrast, there were no significant reductions in diet-induced body weight gain in males exposed to either dose of GTE, or in females exposed to 0.0025% GTE.Significant increases in body weight were observed within 14 days of the start of the experiment in male and female zebrafish in the DIO group compared with those in the non-DIO group Figure . This trTo quantify body fat volume in DIO zebrafish, we performed 3D micro-CT analysis. This allowed us to quantify the total body fat volume and to separately quantify the visceral and subcutaneous fat volumes. Figure We analyzed the mRNA expression levels of lipid metabolism genes in the liver and visceral fat tissue of female DIO zebrafish because the suppressive effects of GTE on body fat accumulation were greater in females than in males.ACOX1 , ACADM , and PPARA (a nuclear receptor protein that regulates β-oxidation) were significantly upregulated in the DIO + 0.0050%GTE group compared with the DIO group .In the liver on day 21 of the experiment, the mRNA expression levels of the mRNAs encoding p Figure . By contACOX1, ACADM, and PPARA in the DIO + 0.0050%GTE group were not significantly differ from those in the DIO group. In addition, there were no significant differences in the mRNA expression levels of FASN (fatty acid synthase) or ACC (acetyl-CoA carboxylase), which play important roles in lipogenesis. On the other hand, the mRNA expression of SOCS3 was significantly lower in the DIO + 0.0050%GTE group than in the DIO group (Figure In the visceral fat on day 21 of the experiment, the mRNA expression levels of In this study, we demonstrated that GTE significantly suppressed the accumulation of visceral fat, but not subcutaneous, in a concentration-dependent manner in zebrafish. This effect was accompanied by increase hepatic expression of lipid catabolism genes. These results are very consistent with the findings of previous studies in humans ,14 and rFASN, is downregulated in visceral fat tissue in obese mice [FASN gene expression in adipose tissue [FASN expression is not affected by a high-fat diet in wild-type rodents and is reduced in obese humans [The mechanisms underlying the suppressive effects of GTE on body fat accumulation have been investigated in rodent models. Some studies have shown that upregulated PPARs and many lipid-metabolizing enzymes stimulate fat oxidation in the liver ,35,36. Tese mice , similare tissue , wherease humans . Their rSOCS3 in the visceral fat of DIO zebrafish. SOCS3 is a negative regulator of leptin signaling and was recently proposed as an important therapeutic target for obesity [SOCS3 gene expression is increased in subcutaneous fat of obese patients [SOCS3 expression in zebrafish, which was accompanied by reduced body fat accumulation. To our knowledge, these results suggest for the first time that SOCS3 expression is correlated with body fat volume in zebrafish and is involved in the regulation of body fat volume by GTE.We also found that GTE significantly decreased the expression of  obesity . SOCS3 ppatients , and itspatients ,43. We fAnother valuable outcome of this study is that 3D micro-CT analysis can be used to analyze the effect of compounds on adiposity in DIO zebrafish. In humans, visceral fat accumulation, unlike subcutaneous fat accumulation, is strongly associated with metabolic and vascular risks, particularly diabetes, hypertension, and stroke, and thus increases the risk of mortality ,45 and iSOCS3.We showed that GTE significantly inhibits weight gain and body fat accumulation, and alters the expression of hepatic lipid catabolism genes in DIO zebrafish. Our results suggest that GTE exerts beneficial effects on adiposity, possibly by altering the expression of lipid catabolism genes and ACADM: Acyl-coenzyme A dehydrogenase, c-4 to c-12 straight chain gene; ACC: Acetyl-CoA carboxylase gene; ACOX1: Acyl-coenzyme A oxidase 1, palmitoyl gene; AgRP: Agouti-related protein; BMI: Body mass index; CT: Computed tomography; DIO: Diet-induced obesity; FASN: Fatty acid synthase gene; GTE: Green tea extract; PPARA: Peroxisome proliferator-activated receptor alpha b gene; SOCS3: Suppressor of cytokine signaling 3b gene.The authors declare that they have no competing interests.TH and YS carried out all animal studies. TH drafted the manuscript. JK helped to perform the experiments. YS, NY, MS, TY, and TT conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Takahiro Hasumura is a researcher in the research and development section of Kao Corporation. The focus of Mr. Hasumura’s research is health science.Yasuhito Shimada, MD is an assistant professor at the Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Graduate School of Medicine, Mie University. The focus of Dr. Shimada’s research is the prevention of obesity using zebrafish as a model of diet-induced obesity.Yuhei Nishimura, MD, PhD is a lecturer in the Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Graduate School of Medicine, Mie University. Dr Nishimura’s research interests are bioinformatics and pharmacoinformatics.Toshio Tanaka, MD, PhD is a professor in the Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinformatics, Graduate School of Medicine, Mie University. The focus of Dr Tanaka’s research is the identification of drug targets using pharmacogenomic methods. Dr. Tanaka has published more than 100 peer-reviewed research articles and reviews in international journals.


Objective. The aim of the present study was to evaluate the oxidative stress biomarkers in patients with subclinical hypothyroidism (n = 20) and health controls (n = 20). Subjects and Methods. Total cholesterol (TC), triglycerides (TGs), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), thiobarbituric acid reactive substances (TBARSs), catalase (CAT), superoxide dismutase (SOD), and arylesterase (ARE) were analyzed. Results. TC, LDL-C, TBARS, and CAT were higher in subclinical hypothyroidism patients, whereas SOD did not change. Arylesterase activity was significantly lower in the SH group, compared with the control group. Correlation analyses revealed the association of lipids (TC and LDL-C) with both oxidative stress biomarkers and thyrotropin (TSH). Thyroid hormones were correlated only with triglyceride levels. In addition, TSH was significantly correlated with TBARS, CAT, and SOD. However, no significant correlations were observed after controlling TC levels. Conclusions. We found that SH patients are under increased oxidative stress manifested by reduced ARE activity and elevated lipoperoxidation and CAT activity. Secondary hypercholesterolemia to thyroid dysfunction and not hypothyroidism per se appears to be associated with oxidative stress in subclinical hypothyroidism. Subclinical hypothyroidism (SH), defined as an elevated serum thyroid stimulating hormone (TSH) level associated with serum thyroid hormone concentrations within the reference range, is found in 4–10% of individuals from Western populations . PatientThyroid hormones are associated with the oxidative and antioxidative status of the organism. Depression of metabolism due to hypothyroidism has been reported to decrease oxidant production and thus protects tissues against oxidant damage , 10. HowLipid peroxidation (LPO) is a free radical chain reaction, which is triggered by hydroxyl radical and leads to membrane break. It facilitates the alteration in the protein structure and function and promotes generation of free radicals (FRs) . LPO is 2O2), which is then deactivated to water (H2O) by catalase or glutathione peroxidase (GPx) [The biological oxidative effects of free radicals on lipids, proteins, and DNA are controlled by a spectrum of antioxidants. Enzymatic protection against reactive oxygen species (ROS) and the breakdown products of peroxidized lipids and oxidized protein and DNA are provided by several enzyme systems such as superoxide dismutase (SOD) and catalase (CAT) . SOD catse (GPx) , 19.High-density lipoproteins (HDLs) inhibit atherosclerosis development mainly by inducing reverse cholesterol transport . HoweverGiven the high prevalence of SH in the general population, it is important to establish whether these alterations of thyroid function entail an oxidative stress and cardiovascular risk. Thus, the aim of this study was to assess the oxidative stress biomarkers and investigated their relation with lipid parameters in subjects with hypothyroidism.Forty adult subjects from clinical laboratory LABIMED, Santa Maria, RS, Brazil were recruited for the present study. They were then classified into two groups-control group: 20 healthy subjects (47.20 ± 11.73 years) and the subclinical hypothyroidism (SH) group: 20 subjects newly diagnosed (49.12 ± 10.85 years). SH was defined as an elevated thyrotropin (TSH) (>4.5 mIU/L) and normal free thyroxine (FT4) level (8.7–22.6 nmol/L) . ExclusiBlood samples were collected after 12 h overnight fasting by venous puncture into gray and red top Vacutainers  tubes. The samples were centrifuged for 15 min at 2500 ×g, and aliquots of serum were kept at −20°C for maximum of 4 weeks. An aliquot of whole blood was collected into sodium citrate (3.2%) and diluted 1 : 10 in saline solution for measurement of CAT and SOD activities. Thyroid profile was assessed by estimation of serums TSH, T3, and fT4 that were measured by chemiluminescent immunometric assay on IMMULITE 2000 . Detection limits for TSH was 0.004–14.000 mIU/L, FT4 were 3.9–77.2 pmol/L, and T3 was 0.29 nmol/L. Serum total cholesterol (TC) and triglycerides (TG) concentrations were measured using standard enzymatic methods by use of Ortho-Clinical Diagnostics reagents on the fully automated analyzer . High-density lipoprotein cholesterol was measured in the supernatant plasma after the precipitation of apolipoprotein B-containing lipoproteins with dextran sulfate and magnesium chloride as previously described . Low-denSerum thiobarbituric acid reactive substances (TBARSs) were measured according to the modified method of Jentzsch et al. . Serum w2O2 at 240 nm. An aliquot of blood was homogenized in potassium phosphate buffer, pH 7.0. The spectrophotometric determination was initiated by the addition of sample into an aqueous solution of hydrogen peroxide 0.3 mol/L. The change in absorbance at 240 nm was measured for 2 min. CAT activity was calculated using the molar extinction coefficient (0.0436 cm2/μmol), and results were expressed as U/g Hb.  Whole blood catalase (CAT) activity was determined by the method of Aebi  by measuμL of adrenalin (1 mM). This was used to determine the concentration in samples. The results were expressed as U/mg Hb. Whole blood superoxide dismutase activity was measured as described by McCord and Fridovich . In this−1 cm−1. One unit of arylesterase activity was defined as 1 μmol phenol generated/min under the above conditions and expressed as U/L serum  as the substrate. The phenol formed after the addition of a 40-fold diluted serum sample was measured spectrophotometrically at 270 nm following an established procedure . EnzymatL serum . The results were expressed as g/dL. U-test was used to compare differences between groups. Spearman correlation was assessed to evaluate the correlations between the variables. Partial correlations were performed to control the associations between variables for total cholesterol levels. Statistical significance was assumed at P < 0.05. Data were analyzed using SPSS version 11.0 software .Data are presented as mean and standard deviation (SD). The nonparametric Mann-Whitney There were no significant differences in age and body mass index (BMI) between groups. SH patients had significantly higher TSH, TC, LDL-C, and TC/HDL ratio with FT4 normal range than the control group . In SH gArylesterase activity was significantly lower in the group with SH, compared with the control group . ARE didr = 0.757, P < 0.0001), TC and CAT , LDL and TBARS , LDL and CAT , and LDL and SOD , as shown in We observed a positive correlation between TC and TBARS . However, no significant correlation was observed after controlling for TC levels. The correlations between TSH, T3, and fT4 with oxidative stress biomarkers are shown in In the present study we have demonstrated that patients with subclinical hypothyroidism had altered lipid profiles, reduced ARE activity, increased lipid peroxidation, and induction of enzymatic defense when compared with control subjects. Hypercholesterolemia is a common feature in hypothyroidism since thyroid hormones upregulate LDL-receptor expression . In a suThe level of lipid profiles is influenced by many factors. The present research has shown that thyroid hormones change the lipid profiles. Thyroid hormones may stimulate hydroxymethylglutaryl coenzyme A (HMG CoA), the key enzyme of cholesterol biosynthesis, and induce an increased synthesis of cholesterol. Additionally, the LDL-C receptor gene contains a thyroid hormone responsive element (TRE) that could allow triiodothyronine (T3) to modulate the gene expression of the LDL-C receptor resulting in an increase of LDL-C receptor synthesis. Thyroid hormones and their function are low in target tissue in SH, and researchers conjectured that SH influences lipid profiles by the above-mentioned mechanism , 35. We Thyroid dysfunctions increase LPO reactions and ROS as documented by recent studies , 40. LPO2− and H2O2 before interacting to form a more reactive hydroxyl radical (OH). These enzymes protect the red cells against O2- and H2O2-mediated lipid peroxidation [Free radical-scavenging enzymes such as SOD and CAT are the first line of cellular defense against oxidative injury, decomposing Oxidation . We havexidation . On the xidation , 44. We xidation .It is supposed that LDL particles can be protected from free radical-induced oxidation by an HDL-linked enzyme, paraoxonase 1 (PON1). PON1 is found in tissues such as liver, kidney, intestine, and also serum . It may per se does not cause oxidative stress in SH patients. On the other hand, high-plasma lipids can be considered as an oxidation substrate for the oxidative stress [In conclusion, our study shows an increase in the oxidative stress biomarkers in the circulation of patients with subclinical hypothyroidism. Oxidative stress biomarkers seem to be associated with secondary hypercholesterolemia to hypothyroidism, whereas hypothyroidism e stress . Thus, w


Reduced cognitive function is common in persons with heart failure (HF). Cardiovascular fitness is a known contributor to cognitive function in many patient populations, but has only been linked to cognition based on estimates of fitness in HF. The current study examined the relationship between fitness as measured by metabolic equivalents (METs) from a standardized stress test and cognition in persons with HF, as well as the validity of office-based predictors of fitness in this population.Forty-one HF patients enrolled in cardiac rehabilitation completed a standardized exercise stress test protocol, a brief neuropsychological battery, the 2-minute step test (2MST), and a series of medical history and self-report questionnaires.p = .03), executive function , and memory domains . Partial correlations accounting for key medical and demographic characteristics revealed greater METs was associated with the 2MST (r (32) = .41, p = .02) but not with the Duke Activity Status Index (DASI) (r(32) = .24, p = .17).Maximum METs from stress testing demonstrated incremental predictive validity for attention , but not another (DASI). Prospective studies are needed to clarify the mechanisms linking fitness and cognitive function in HF. Heart failure (HF) affects nearly six million Americans and is a leading cause of hospitalization and mortality in older adults ,2. ThougPast studies have shown that reduced cerebral blood flow ,7 and stMetabolic equivalents (METS) are often used to illustrate the intensity of tasks individuals are able to accomplish, providing a standardized definition of fitness. As such, METS are important constructs in understanding the fitness of HF patients and determining the types of activities they are able to do independently. Found to be valid estimates in other populations -18, bothGiven these findings, the proposed study had two primary goals. First, we sought to clarify the possible association between fitness levels as measured by stress testing and cognitive function in persons with HF. Second, to examine the validity of office-based estimates of fitness by correlating them to performance on stress testing. Specifically, we examined the association between stress testing and the self report Duke Activity Status Index (DASI) and the objective 2-minute step test. Based on past literature in other populations, we hypothesized that metabolic equivalents (METs) would independently predict cognitive function in multiple domains. Based on their association with other measures of fitness, we also expected that office-based measures of fitness, specifically the 2-minute step test (2MST) and the Duke Activity Status Index (DASI), would be correlated to cognitive performance.A sample of 41 older adults with HF was recruited from Summa Health System in Akron, OH. See Table A neuropsychological battery was conducted by trained research assistants under the supervision of a licensed clinical neuropsychologist. The following tests were selected as they are clinically useful and have been used to assess cognition in HF in previous studies ,5. The fThe Modified Mini-Mental State Examination (3MS) is brief screening measure that provides an estimate of global cognitive function .Attention was assessed using Letter Number Sequencing (LNS), the Trail Making Test A (TMT-A), and the Symbol Digit Modalities Test. The LNS asks participants to repeat unordered strings of letters and numbers in both numerical and alphabetical order. Higher scores reflect better executive functioning and this task has been shown to correlate with other measures (r = -.36) ,21. In tTo test executive function performance, the Frontal Assessment Battery, the Trail Making Test B (TMT-B), and the Stroop Interference Task were used. The Frontal Assessment Battery contains six subtests which assess various aspects of executive function, including conceptualization, mental flexibility, inhibition, and sequencing. Higher scores indicate better test performance . The FABMemory tests included the California Verbal Learning Test-Second Edition (CVLT) and the Complex Figure Test (CFT). The CVLT asks participants to learn, recall, and recognize items from a 16-item word list. This test incorporates indices of delayed recall and recognition, and the average of these scores were used for analysis. This task has strong psychometric properties, including a high correlation to other verbal memory measures (r =0.63 to r = 0.96) . In the To assess language performance, the Boston Naming Test-II (BNT-II) and the Animal Naming task were used. The BNT-II requires individuals to name pictures of objects ranging from high to low familiarity . The BNTEstimated metabolic equivalents (METs) were estimated using a standardized symptom limited volitional maximal treadmill stress test. In this test, treadmill elevation increased every 60 seconds and an increase in speed every 3 minutes as to estimate an increase in workload of 15%. The stress test ended if either the participant requested or if the participant showed clinical signs of cardiovascular, neurologic, or musculoskeletal distress. Maximum METs were estimated through a combination of treadmill speed, grade, and duration .The two minute step test (2MST) requires individuals to walk in place, lifting their knees to a target midway between their kneecap and the crest of the iliac . IndividThe Duke Activity Status Index (DASI) was also administered to estimate individual's estimated peak oxygen uptake. This measure is a 12-question self-report on various aspects of physical work capacity and quality of life, asking whether individuals can complete daily tasks that are weighted based on the task's difficulty. It has been used as a valid estimate of physical ability in older adults .The Beck Depression Inventory II (BDI-II) was used to measure the presence and severity of depression . This meAll procedures were approved by the Kent State University and Summa Health Institutional Review Boards and written consent was obtained from all participants. METs were established with an exercise stress test protocol that was completed upon enrollment into cardiac rehabilitation. Within two weeks of the initial stress test, participants completed a research visit that consisted of a neuropsychological test battery, the 2MST, and series of medical history and self-report questionnaires, including the DASI. Ejection fraction was determined using medical record review.-scores  using normative data correcting for age, similar to the approach used in other studies [-scores within each cognitive domain. Consistent with convention in many clinical settings, impairment in these domains for the current study was defined as a T-score ≤ 35 relative to normative data.To facilitate clinical interpretation and to maintain directionality within the scales, all raw scores of the neuropsychological measures were transformed to T studies . Memory To examine the independent association between METs and cognitive function, a series of hierarchical regression analyses were performed with composite scores for each cognitive domain. All analyses utilized IBM SPSS Statistics version 21. For each model, age, sex , years of education, LVEF, depressive symptomatology (as assessed by the BDI-II), and diagnostic history of hypertension and diabetes , were entered into the first block. METs was then entered into the second block of each model to determine its incremental predictive validity above and beyond demographic and medical characteristics. Finally, a series of partial correlations adjusting for the aforementioned demographic and medical characteristics was conducted to examine the association of METs with the 2MST and peak oxygen uptake as estimated by the DASI.The current sample obtained an average maximum of 6.24 SD = 3.11) METs during the stress test. See Table 1 METs duF = 2.39, p = .04) and executive function  = 2.91, p = .02). After controlling for age, sex, education, LVEF, depressive symptomatology (as assessed by the BDI-II), and history of hypertension and diabetes, METs demonstrated incremental predictive validity for attention , executive function , and memory domains . In each case, higher METs levels were associated with better cognitive test performance. No such pattern emerged for language abilities . Notably, bivariate correlations also revealed higher METs were associated with better performance on the 3MS (r(39) = .38, p = .01). See Table Demographic and medical factors were associated with test performance for attention  = .41, p = .02). No relationship emerged between METs estimated from stress test and peak oxygen uptake as estimated by the DASI (r(32) = .24, p = .17).To clarify the association between different estimates of fitness level, partial correlations accounting for the effect of medical and demographic characteristics revealed increased METs was associated with better 2MST performance (r(32) = .34, p = .047) and executive function (r (32) = .40, p = .02) even after accounting for age, sex, education, LVEF, depressive symptomatology, and diagnostic history of hypertension and diabetes. The 2MST was not associated with memory (r(32) = .13, p = .46) or language performance (r(32) = .15, p = .41). Peak oxygen uptake as estimated by the DASI was not independently associated with cognitive function in any of the domains: attention (r(32) = .02, p = .90), executive function (r(32) = .05, p = .78), memory (r(32) = -.02, p = .91), language (r(32) = .17, p = .34).Consistent with this and the above pattern, partial correlations showed lower 2MST performance was also associated with worse attention  but not another (i.e. DASI). Several aspects of these findings warrant brief discussion.Results showing that METs predicted cognitive function above and beyond ejection fraction are consistent with past work showing fitness is an important contribution to cognition in other healthy and medical populations ,43. TherAs noted above, HF patients frequently experience cognitive decline , which cWe also examined the relationship between METs and two office-based measures of fitness to help clinicians identify those HF patients at risk for cognitive impairment. Found to be valid estimates in other populations -18, bothThe current findings are limited in several ways. Studies are needed to clarify the mechanisms that link fitness levels and cognitive function in older adults with HF, particularly studies utilizing neuroimaging and direct measures of CBF. Future research would also benefit from prospective examination of fitness levels and cognitive function to better understand this relationship, particularly the possibility that programs like cardiac rehabilitation that may improve cognitive function in persons with HF. An additional limitation to the current work includes relatively small sample size and larger studies are needed to confirm the current findings.In summary, maximum METs is an independent predictor of cognitive function in HF patients. Future work is needed to clarify the underlying mechanisms for this relationship, particularly prospective studies to determine whether structured exercise programs may improve cognitive function in persons with HF.HF: Heart failure; METs: Metabolic equivalents; 2MST: 2 minute step test; DASI: Duke activity status index; LVEF: Left ventricular ejection fraction; 3MS: Modified mini mental status exam; LNS: Letter number sequencing; TMT-A: Trail making test A; TMT-B: Trail making test B; CVLT: California verbal learning test; CFT: Complex figure test; BNT-II: Boston naming test-II; CBF: Cerebral blood flowThe authors declare that they have no competing interests.SG contributed to study concept and design, acquisition of subjects, data analysis and interpretation, and manuscript preparation. MA participated in study concept and design, acquisition of subjects, data analysis and interpretation, and manuscript preparation. MS, RC, NR, LS, LC, RJ, JH, JR, LO, and JG contributed to contributed to study concept and design, data analysis and interpretation, and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/13/29/prepub


Recently, a complex (X/C) hepatitis B virus (HBV) recombinant, first reported in 2000, was proposed as a new genotype; although this was refuted immediately because the strains differ by less than 8 % in nucleotide distance from genotype C. Over 13.5 % (38/281) of HBV isolates from the Long An cohort in China were not assigned to a specific genotype, using current genotyping tools to analyse surface ORF sequences, and these have about 98 % similarity to the X/C recombinants. To determine whether this close identity extends to the full-length sequences and to investigate the evolutionary history of the Long An X/C recombinants, 17 complete genome sequences were determined. They are highly similar (96–99 %) to the Vietnamese strains and, although some reach or exceed 8 % nucleotide sequence difference from all known genotypes, they cluster together in the same clade, separating in a phylogenetic tree from the genotype C branch. Analysis of recombination reveals that all but one of the Long An isolates resembles the Vietnamese isolates in that they result from apparent recombination between genotype C and a parent of unknown genotype (X), which shows similarity in part to genotype G. The exception, isolate QL523, has a greater proportion of genotype C parent. Phylogeographic analysis reveals that these recombinants probably arose in southern China and spread later to Vietnam and Laos. Hepadnaviridae, has a circular, partially dsDNA genome of about 3200 nt , the prototype member of the family HBV recombination was first detected in sequential samples from infected individuals and among integrated sequences from cases of hepatocellular carcinoma (HCC) . There aRecently, star program (http://www.vgb.ucl.ac.uk/starn.shtml) and the National Centre for Biotechnology (NCBI) HBV genotyping tool (http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi). These analyses revealed that 71 and 14 % of the study subjects are infected with genotypes C and B, respectively, but 13.5 % (38/281) of the isolates were not assigned to a specific genotype. Comparison to sequences in GenBank using the blast program  with the Vietnamese strain VH24 (GenBank accession no. AB231908)  theIsolate Ql523 had one different breakpoint and contained more of the putative genotype C parent (from position 1670 to 610) than the other 16 isolates. The remaining part was identical to that of the other Long An sequences. This suggests that isolate QL523 and the other isolates are the result of two independent recombination events involving the same parental strains.The spatial dispersion of the X/C recombinants was estimated by ancestral state reconstruction, using a Bayesian Markov chain Monte Carlo (MCMC) phylogenetic framework Fig. . For reaIn order to investigate the probable origin of the C-like parental strain of the X/C recombinants, the corresponding regions were compared to HBV genotype C sequences of known geographical origin, using a Bayesian MCMC phylogeographic framework Fig. . Surprisχ2=3.2, P>0.05).As stated in Methods, the 38 unassigned isolates are derived from 281 study subjects recruited as asymptomatic hepatitis B surface antigen carriers . TherefoThe major findings of this study are that the recombinant Long An sequences are highly similar (96–99 %) to the Vietnamese strains and some of them reach or exceed 8 % difference from all known genotypes, while the remainder differ by less than 8 % from genotype C. However, they all cluster in the same clade, separating from the genotype C branch of a phylogenetic tree based on complete genome sequences Fig. . FurtherThis unusual recombinant was first reported in 2000 as aberrant strains  and with response to treatment , then manually corrected with the sequence editor BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Maximum-likelihood trees were reconstructed under the General Time Reversible model of nucleotide substitution, with proportion of invariable sites and gamma-distributed rate heterogeneity (GTR+I+Γ), using the software paup* version 4.0b10  the full-length sequences (3215 nt), and (ii) the S ORF (681 nt) of the viruses. The sequences were aligned to 198 HBV sequences of all known genotypes retrieved from GenBank, using the program n 4.0b10 . The robpaup*, for both the full-length and the S gene sequences.Pairwise genetic distances between the Long An and reference isolates were also calculated under the GTR+I+G model of evolution, using the program P value cut-off was set to 0.05, a Bonferroni correction was applied, phylogenetic evidence was required, breakpoints were polished, alignment consistency was checked for and overlapping signals were disentangled. SEQEN parametric simulations were used. Specific settings for each program were as follows: for RDP, no reference sequence was selected, and percentage of identity between recombinant sequences was set from 0 to 100. For Geneconv, sequence triplets were scanned, each indel was treated as a polymorphism and the g-scale was set to 1. For MaxChi, gaps were stripped and variable sites per window were set to 70. For Chimera, variable sites per window were set to 70. For Bootscan, the window size was set to 200 bp, step size to 20 bp and neighbour-joining trees used. The number of bootstrap replicates was 100, the cut-off percentage was 70 % and model options were set to the Kimura 2-parameter model . The trees were reconstructed under the GTR+G model of nucleotide substitution. The positions of identified breakpoints were confirmed by reducing the window step size to 20 bp in the regions flanking the breakpoints.The recombinant breakpoints of suspected mosaic genomes were mapped using the program SlidingBayes (n=17); Vietnam , after discarding trees corresponding to a 10 % burnin. The MCCT was edited with the program FigTree version 1.1.2.Genotype X/C recombinants have been found in southern China, Laos and Vietnam. In order to investigate the spatial dispersion patterns of the viruses and attempt to identify the geographical origin of the recombinant form, phylogeographic analyses were conducted according to the Bayesian MCMC method developed by on 1.5.2 . Dated pBayesian MCMC phylogeographic analyses were also performed under the aforementioned conditions for the C-like (positions 1670–3100) and G-like fragment (positions 200–1250) of the X/C recombinant form only, in order to identify the most likely origin of the corresponding parental strains. The C-like fragment was compared to HBV genotype C1 sequences from China , Indonesia (AB033557), Japan , Malaysia (GQ924657) and South Korea (AY641558–AY641563 and X14193); genotype C2 sequences from Cambodia (AB117758), India (DQ315781–DQ315783), Japan (AB049610), Myanmar , Thailand , Malaysia  and Vietnam ; genotype C3 sequences from New Caledonia (X75656 and X75665); and genotype C4 from Australia (AB048704 and AB048705). The G-like fragment was compared to HBV genotype G sequences from Brazil (EF464097), France (EF634480 and EF634481), Germany (AF405706 and DQ207798), Italy (EF514346–EF514349), Japan (AP007264), Mexico (AF369533), the Netherlands (DQ403176 and GU565217) and the USA (AB056513 and AB064313); HBV C/G recombinants from Thailand (DQ078791 and FJ361772); HBV B/G recombinants from Taiwan (AB555499) and Japan (AB549213); and HBV G/A recombinants from Canada (EU833889 and EU833890).The nucleotide sequence data reported in this paper have been submitted to the GenBank/EMBL/DDBJ databases under accession numbers FR714490–FR714506.


Elevation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) is prominent in acute dengue illness. The World Health Organization (WHO) 2009 dengue guidelines defined AST or ALT≥1000 units/liter (U/L) as a criterion for severe dengue. We aimed to assess the clinical relevance and discriminatory value of AST or ALT for dengue hemorrhagic fever (DHF) and severe dengue.We retrospectively studied and classified polymerase chain reaction positive dengue patients from 2006 to 2008 treated at Tan Tock Seng Hospital, Singapore according to WHO 1997 and 2009 criteria for dengue severity. Of 690 dengue patients, 31% had DHF and 24% severe dengue. Elevated AST and ALT occurred in 86% and 46%, respectively. Seven had AST or ALT≥1000 U/L. None had acute liver failure but one patient died. Median AST and ALT values were significantly higher with increasing dengue severity by both WHO 1997 and 2009 criteria. However, they were poorly discriminatory between non-severe and severe dengue  and between dengue fever (DF) and DHF . There was significant overlap in AST and ALT values among patients with dengue with or without warning signs and severe dengue, and between those with DF and DHF.Although aminotransferase levels increased in conjunction with dengue severity, AST or ALT values did not discriminate between DF and DHF or non-severe and severe dengue. Dengue is a global public health problem, as the incidence of the disease has reached hyperendemic proportions in recent decades. Infection with dengue can cause acute, febrile illness or severe disease, which can lead to plasma leakage, bleeding, and organ impairment. One of the most prominent clinical characteristics of dengue patients is increased aspartate and alanine aminotransferase liver enzyme levels. The significance of this is uncertain, as it is transient in the majority of cases, and most patients recover uneventfully without liver damage. In this study, we characterized this phenomenon in the context of dengue severity and found that, although liver enzyme levels increased concurrently with dengue severity, they could not sufficiently discriminate between dengue fever and dengue hemorrhagic fever or between non-severe and severe dengue. Therefore clinicians may need to use other parameters to distinguish dengue severity in patients during early illness. Dengue is a mosquito-borne arboviral infection endemic to most tropical and subtropical countries Elevated AST and ALT levels have been associated with bleeding In 2009, the World Health Organization (WHO) revised its dengue guidelines and proposed severe organ impairment as one category of severe dengue in addition to severe plasma leakage and severe bleeding All laboratory-confirmed dengue patients identified from our hospital microbiology database and treated using a standardized dengue clinical care path at the Department of Infectious Diseases, Tan Tock Seng Hospital (TTSH), Singapore from 2006 to 2008 were retrospectively reviewed for demographic, serial clinical and laboratory, radiological, treatment, and outcome data. These cases were positive by real-time polymerase chain reaction (PCR) 9/liter, bleeding, and plasma leakage Cases were categorized using serial clinical and laboratory data from the entire clinical course as dengue fever (DF), DHF, or dengue shock syndrome (DSS) using WHO 1997 classifications Cases were also categorized as dengue without warning signs (WS), dengue with WS, or severe dengue using WHO 2009 classifications We performed a subgroup analysis for median maximum AST and ALT values stratified by febrile (days 1–3 of illness), critical (days 4–6), and convalescent (days 7–10) phases as defined by WHO 2009 We excluded severe dengue due to isolated elevation of AST or ALT≥1000 U/L from our definition of severe dengue outcome, as this would be a confounder in assessing the relevance of AST or ALT levels in defining dengue severity. Patients had AST/ALT taken at presentation and then throughout hospitalization at the physician's discretion. Maximum AST and ALT values recorded at a median of 4 days of illness (interquartile range [IQR]: 3–5 days) were used in this analysis. Those with pre-existing liver diseases were excluded. At TTSH, the ULN for AST is 41 U/L; for ALT, it is 63 U/L for males and 54 U/L for females.We assessed the clinical relevance of elevated AST or ALT levels using four liver failure criteria—two for acute liver failure, and two that determine prognosis from chronic liver disease. The American Association for the Study of Liver Diseases (AASLD) recommends defining acute liver failure in a patient as: international normalized ratio (INR)≥1.5, any degree of altered mental status, and illness <26 weeks in duration without pre-existing cirrhosis The Mann-Whitney U and Kruskal-Wallis tests were used to determine statistical significance for continuous variables, and chi-square or Fisher's exact test for categorical variables. Statistical tests were conducted at the 5% level of significance. Receiver operating characteristic (ROC) curves showing the area under the curve (AUC) were generated to determine the discriminatory performance of aminotransferase values. All statistical analyses were performed using Stata 10 .This was a retrospective study involving data collection from medical records. All patient data were anonymized during analysis. This study was approved by the Institutional Review Board, National Healthcare Group, Singapore [DSRB E/08/567].From 2006 to 2008, 690 dengue PCR positive cases were reviewed. Males comprised 493 (71%) of the cases, and the median age of the cohort was 35 years (IQR: 27–43 years). A Charlson comorbidity index ≥3, which predicts increased one-year mortality Median length of illness from onset to hospital presentation was 4 days (IQR: 3–5 days), while median length of hospital stay was 5 days (IQR: 4–6 days). Intravenous fluid was administered to 641 (93%) and platelet transfusion to 86 (12%). Intensive care unit (ICU) admission was required in 3 patients, and death occurred in 1 patient due to pneumonia.Overall, 595 (86%) had AST above the ULN, and 316 (46%) had ALT above the ULN. Seven patients (1.0%) had severe dengue according to WHO 2009 criteria concurrent with AST or ALT≥1000 U/L while three additional patients had severe dengue due to AST or ALT≥1000 U/L only. Of the former seven patients, 86% had severe plasma leakage, 29% had severe bleeding, and none had severe organ impairment other than isolated AST or ALT≥1000 U/L. Among the 3 patients admitted to the ICU, AST or ALT values were above the ULN but below 1000 U/L.No patients in our cohort developed acute liver failure under AASLD or King's College criteria. With Child-Pugh scoring, 2 (0.3%) belonged to Child-Pugh class C. With MELD scoring, predicted three-month mortality of 6% were identified in 68 (10%) patients in our cohort and 19.6% in 2 (0.3%) patients. The same two patients who were Child-Pugh class C also had a predicted 19.6% three-month mortality using MELD scoring; they both had DSS and severe dengue.p<0.001); median ALT values were 49 U/L, 53 U/L, and 73.5 U/L (p = 0.002). p = 0.01), and median ALT values were 52 U/L, 60 U/L, and 74 U/L (p = 0.05).Median AST and ALT values for dengue without warning signs, dengue with warning signs, and severe dengue  were 83.p<0.001). Median ALT values were 56 U/L, 73 U/L, and 97.5 U/L (p = 0.01). For patients with DF, DHF, and DSS, median AST values were 105 U/L, 130 U/L, and 129 U/L (p<0.001), and median ALT values were 68 U/L, 78 U/L, and 85.5 U/L (p = 0.008).In a separate analysis of our serology-positive cohort (n = 1487), median AST values for dengue without WS, dengue with WS, and severe dengue were 84 U/L, 114 U/L, and 147 U/L (p = 0.29) and for dengue without WS, dengue with WS, and severe dengue, they were 1.60, 1.68, and 1.78 (p = 0.10), respectively.In other hemorrhagic fevers, higher AST∶ALT ratios correlated with disease fatality The majority of our patients' maximum AST and ALT values were recorded during febrile (n = 258) and critical (n = 377) phases of acute dengue illness. By WHO 2009 dengue severity classification, the median AST and ALT values were significantly higher for severe dengue compared to dengue with and without warning signs during both the febrile and critical phases but not the convalescent phase . By WHO In order to determine the reliability of AST and ALT values in defining dengue severity, ROC curves for AST and ALT against severe dengue excluding isolated transaminitis were determined . The AUCThe box plots in Our analysis showed that liver aminotransferase levels were associated with but did not adequately differentiate between dengue severity. Although median AST and ALT values were significantly higher in those with DHF/DSS versus DF, and severe dengue versus non-severe dengue, very few (1.0%) had AST or ALT≥1000 U/L. Notably, none developed liver failure, and death occurred in only 1 patient (0.1%). The majority of patients recovered uneventfully.The lack of acute liver failure in our study was not unusual, as the incidence of acute liver failure in dengue patients was 1.1% in studies by Trung and Kuo While some studies have shown that AST and ALT values differ between DF and DHF Trung et al. showed significant differences comparing other febrile illness, dengue without plasma leakage, and dengue with plasma leakage with and without shock during critical and convalescent phases for AST but during critical phase for ALT only The impact of co-infection with hepatitis viruses or concomitant hepatotoxic drugs was not assessed in our retrospective study, although we did exclude those with known liver comorbidities. Kuo et al. found that hepatitis B or C did not increase the extent of liver aminotransferase elevation in a retrospective adult dengue study in Taiwan The etiology of elevated aminotransferase levels during acute dengue illness is unclear since AST is expressed in the heart, skeletal muscle, red blood cells, kidneys, brain, and liver, while ALT is secreted primarily by the liver Our retrospective study has some limitations. Aspartate and alanine aminotransferase values were tracked according to clinical judgment rather than at regular intervals during illness. We did not have dengue serotype data for each patient, but in 2006, DENV-1 was predominant in Singapore with a switch to DENV-2 in 2007–2008 In patients with DHF/DSS or severe dengue, early diagnosis and proper management may improve outcome in most patients without comorbidities. However, in resource-limited countries, patients with severe disease may present late to the hospital with shock, with or without organ impairment at the time of admission. Our study highlights that early diagnosis and proper management of dengue patients may lead to excellent prognosis without organ injury.In conclusion, elevated aminotransferase levels were associated with DHF/DSS and severe dengue in our cohort of adult patients with confirmed dengue. However, no threshold values discriminated between DF and DHF or between severe dengue and non-severe dengue.


This retrospective study was undertaken to determine the incidence of kidney dysfunction (KD) and to identify potential risk factors contributing to development of KD in orthopaedic population following an elective or emergency surgery.A total of 1025 patients were admitted in our institution over a period of one year with various indications. Eight hundred and ninety-three patients (87.1%) had a surgical procedure. There were 42 (52.5%) male and 38 (47.5%) female with a mean age of 72 years (range: 47 to 87 years). We evaluated the following potential risk factors: age, comorbidities, shock, hypotension, heart failure, medications , rhabdomyolysis, imaging contrast agents and pre-existing KD.The overall incidence of KD was 8.9%. Sixty-eight patients developed acute renal injury (AKI) and 12 patients developed acute on chronic kidney disease (CKD). In sixty-six (82.5%) patients renal function was reversed to initial preoperative status. Perioperative dehydration (p = 0.002), history of diabetes mellitus (p = 0.003), pre-existing KD (p = 0.004), perioperative shock (p = 0.021) and administration of non-steroid anti-inflammatory drugs (NSAIDs) (p = 0.028) or nephrotoxic antibiotics (p = 0.037) were statistically significantly correlated with the development of postoperative KD and failure to gain the preoperative renal function.We conclude that every patient with risk factor for postoperative KD should be under closed evaluation and monitoring. The overall incidence of kidney dysfunction (KD) after elective or emergency orthopedic surgical procedures is reported to reach 9.1% . The risHowever, certain patient populations are at much higher risk. Factors that are identified to increase the risk of AKI include age, past history of kidney disease, left ventricular ejection fraction less than 35%, cardiac index less than 1.7 L/min/m2, hypertension, peripheral vascular disease, diabetes mellitus, emergency surgery, and type of surgery .Surgical procedures that are at higher risk for development of AKI include those involving coronary artery by-pass, cardiac valve reconstruction, open aortic aneurysm bypass, and liver transplantation -13. PatiThere are limited data on how to assess postoperative KD following major orthopaedic procedures. Our hypothesis is that several factors associated with these procedures such as the increased surgical time, the need for blood transfusion, and the use of perioperative antibiotics and analgesics may contribute to the development of acute kidney injury ,2. The pWe retrospectively analyzed the medical records of 1025 admissions to our institution in a period of 13 months between February 2008 and March 2009. The study protocol was approved by the Scientific Council and the Ethics Committee of Levadia General Hospital, Greece. Patients that underwent an elective or emergency orthopaedic procedure were eligible for inclusion to the study. Patients that were admitted for conservative treatment were excluded. There were 586 male and 439 female with a mean age of 64 years . Demographics, indications for admission, comorbidities and length of stay in hospital  were assessed and all collected data are presented in Table All patients received medical intervention according to a pre-determined protocol. This involved a medical and social history and a physical examination. Investigations included full blood count, urine analysis, glucose, urea, creatinine, electrolytes, electrocardiogram, chest X-ray and other appropriate radiographs of musculoskeletal system.The patients who developed KD [acute or acute on chronic kidney dysfunction (CKD)] post-operatively, were evaluated by a senior nephrologist and were classified according to the final outcome of renal function into (i) complete resolution, (ii) failure to return to prior kidney function, (iii) end-stage renal disease and (iv) death. Potential risk factors for KD were pulled from medical charts including age, perioperative hypotension or shock, and presence of comorbidities such as diabetes mellitus, coronary heart disease, heart failure, and CKD. The type and duration of use of potential nephrotoxic agents , angiotensin-II receptor antagonists (AT-II), diuretics, antibiotics (aminoglycosides), non-steroidal anti-inflammatory (NSAID) and opiates) were also recorded.All cases with documented perioperative complications were prospectively followed-up in order to reveal potential correlations between the development of KD and associated comorbidities. KD according to Acute Kidney Injury Network (AKIN) criteria has been defined as the increase in serum creatinine ≥ 0.3 mg/dL or increase to ≥150% (1.5- to 3-fold) from baseline ,18.Also the outcome regarding the possibility to return to prior renal function was estimated. As “full recovery of renal function” was defined as the return of lab values to normal. “Incomplete recovery” was defined the improvement of renal function but still remaining over 120% of initial values. “Stable” was defined as no recovery and “Progression of renal dysfunction” as defined by an increase in Crea or decrease in GFR values comparing to the baseline .Data is expressed as mean ± standard deviation (S.D.) or median  for continuous variables and as percentages for categorical data. The Kolmogorov - Smirnov test was utilized for normality analysis of the parameters. Univariate analyses were made by using the chi-square test or alternatively the Fisher exact test to analyze the relation between the outcome variable and the qualitative variables, whereas the Student t-test or Mann–Whitney U-test and One-way ANOVA or Kruskal - Wallis were used to analyze the relation between the outcome variable and the quantitative measures respectively.All the estimated potential risk factors, such as the age, the comorbidities (diabetes mellitus), shock, hypotension, heart failure, medications , rhabdomyolysis, imaging contrast agents and pre-existing KD, whether or not they demonstrated significant associations with outcome variable  in univariate analysis were included in the model, and stepwise elimination  was used to arrive at the final model. Goodness of fit was evaluated using the Hosmer-Lemeshow statistic.A p-value of <0.05 (two sided) was used to denote statistical significance, though associations reaching borderline significance (0.05 < p < 0.1) were also identified as being of potential interest. All analyses were carried out using the statistical package SPSS vr15.00 .From February 2008 to March 2009 in a total of one thousand and twenty-five patients which admitted in our institution, eight hundred and ninety-three patients (87.1%) had a major orthopaedic procedure. Specifically, there were 663 cases with lower extremities fractures, 124 cases with upper extremities fractures, 1 case with compartment syndrome, 56 cases with Total Hip Arthroplasty and 49 cases with Total Knee Arthroplasty. Of them, four hundred and sixty-six (52.2%) patients had numerous pre-existing comorbidities including, diabetes mellitus in 122 cases, heart failure in 76 cases, chronic obstructive airways disease in 20, and pre-existing CKD in sixteen cases. One hundred and five patients received Nephrotoxic agents, whereas dehydration at the time of admission was noted in 137 cases. Perioperatively 5 cases developed shock (3 hypovolemic and 2 cardiogenic) and one procedure was complicated with rhabdomyolysis after a severe crash injury of the lower extremity. Demographic data and perioperative complications of all surgical patients are summarized in Table KD occurred in 80 (8.9%) cases. There were 42 (52.5%) male and 38 (47.5%) female with a mean age of 72 years . 68 patients of 80 developed AKI and 12 patients developed acute on CKD. The demographic data and medical characteristics of patients who developed KD are shown in Table Eight patients (10%) with KD had a low serum urea concentration (<17 mg/dL), at the time of KD diagnosis, 34 (42.5%) had a normal concentration  and 38 patients (47.5%) had a raised serum urea concentration (>52 mg/dL). Seventeen (21.2%) had a low serum creatinine concentration (<0.7 mg/dL), forty-three (53.7%) had a normal creatinine (0.7-1.30 mg/dL) and 20 patients (25%) had a raised creatinine value. Mean GFR was 81,71 mL/min (ranging from 23 to 343 mL/min).Sixty-six (82.5%) of 80 patients, recovered their renal function prior to surgery Table . The meaThere was not statistical significant difference in mortality in patients with sustained AKI compared with patients with transient AKI. There was a statistically significant correlation of perioperative dehydration (p = 0.002), history of diabetes mellitus (p = 0.003), pre-existing renal dysfunction (p = 0.004), perioperative shock (p = 0.021) and administration of non-steroid anti-inflammatory drugs (NSAIDs) (p = 0.028) or nephrotoxic antibiotics (p = 0.037) with the development of irreversible renal filtration. On the other hand, heart failure, and administration of ACE-I, AT-II and diuretics were not statistically significantly correlated with perioperative KD. The sample size was found small and inappropriate for any correlation between the use of opiates, iodinated contrast agents or the presence of rhabdomyolysis and the development of KD.Furthermore, we found a significant main effect of the estimated potential risk factors in renal function after an episode of AKI. A patient with diabetes mellitus has an 18.2-fold risk of irreversible KD, comparing with a patient without the disease (p = 0.003). Patient with perioperative shock of any etiology have 8.5 times greater risk for KD (p = 0.02). Also, there is an 8.5% higher risk of KD with an increase of age by one year (p = 0.05).Thirty-nine (48.7%) patients showed full recovery of their renal function, 19 (23.8%) incomplete recovery, 15 (18.7%) were stable and 7 (8.8%) patients showed a progression of renal dysfunction. Three cases (21.4%) from the group with pre-existed KD (n = 14) developed ESRF (two with pre-existing KD and one with diabetes mellitus), while one patient (7.1%) died.This study demonstrates an incidence of 8.9% of KD in a group of patients that had undergone major orthopaedic procedures in our hospital over a period of one year. Also an assessment of numerous of potential risk factors of postoperative KD was carried out.Definition of acute renal failure is still controversial and sometimes confusing . AuthorsChronic kidney disease (CKD) as defined in 2002 by the Kidney Disease Outcomes Quality Initiative (K/DOQI) of the National Kidney Foundation is either kidney damage or decreased kidney function for 3 or more months . NumerouIt is known that the overall incidence of KD after elective or emergency orthopedic surgical procedure is up to 9.1% . MoreoveIt is generally recognized that risk factors for the development of KD in this population include dehydration, increasing age, diabetes mellitus, shock, heart failure, medications , rhabdomyolysis, iodinated contrast agents and pre-existing KD. In a recent study by Bennet et al. , authorsOptimal fluid balance, as well vigilance and caution especially for patients with high risk of KD, are crucial. This statement is in agreement with other authors ,2,28,31.Measures to prevent preoperative KD include close evaluation of patients with risk factors and monitoring by a specialist. A consideration of opioid-reducing strategies such as early fracture fixation, regular simple analgesia  and regional nerve blockade is necessary. Also an appropriate volume of fluid was required to optimize cardiovascular status, reflecting that the current standard care renders patients under-resuscitated before, during and after surgery.We have shown an 8.9% rate of KD in patients undergone major orthopaedic procedures. We have highlighted the risk factors and showed the correlation thereof with the development of KD peri-operatively. We believe that close monitoring of this selective group of patients and limitation of use of nephrotoxic agents could lead to a significant decrease of KD in these cases. However, we acknowledge that larger patients sample is needed in order to identify other clinical and laboratory factors that may contribute to KD and thus improve our quality of everyday practice.The authors confirm that there are no conflicts of interest for the above manuscript.KK, CD, GM, SAP and SG participated in the design of the study, data acquisition and analysis and writing of this manuscript. SG, KK and VS participated in the analysis and writing of this paper. VS participated in the analysis and also in revising critically the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2369/13/101/prepub


Mirabilis jalapa L. root (EEM) in normal and diabetic mice. After induction of diabetes with streptozotocin, both normal and diabetic mice were singly or repeatedly for 28 days administrated with EEM at doses of 2, 4, 8 g/kg, respectively. Before induction of diabetes, mice were administrated with EEM at doses of 2, 4, 8 g/kg for 14 days and were injected with streptozotocin and continued on EEM administration for another 28 days. Both after and before induction of diabetes, repeated administration with 4, 8 g/kg EEM continually lowered blood glucose level, decreased serum insulin level and improved insulin sensitivity index, and lowered serum total cholesterol, triglyceride levels and triglyceride content in liver and skeletal muscle, and increased glycogen content in these tissues; but repeated administration had no influence on those indexes of normal mice. Single administration with EEM  showed hypoglycemic effect in oral glucose tolerance test in normal and diabetic mice. Single administration with EEM had no hypoglycemic and hypolipidemic effects on normal and diabetic mice. These results suggest that EEM possesses both potential insulin sensitivity, hypoglycemic, and hypolipidemic effects on diabetes.The present study investigated the insulin sensitivity, hypoglycemic, and hypolipidemic activities of ethanolic extract of  Type 2 diabetes mellitus, a metabolic disorder with manifestations of hyperglycemia and hyperinsulinemia, is one of the commonest chronic diseases worldwide . Though Mirabilis jalapa L. belongs to the family Nyctaginaceae and is known as “four o'clock,” “maravilha,” “belle de nuit,” “buenas tardes,” “dondiego de noche,” “jalap,” “noche buena,” or “Tzu Mo Li” in different areas. Mirabilis jalapa L. is widely used to treat dysentery, diarrhea, muscular pain, and abdominal colics in different countries [Mirabilis jalapa L. is widely distributed and commonly used with its root and has been used as traditional Chinese medicine and ethnic drug to treat diabetes [Mirabilis jalapa L. root contains alkaloids, glycosides, carbohydrates, and phytosterols by phytochemical analysis [Mirabilis jalapa L. root [Mirabilis jalapa L. root on streptozotocin-induced diabetic Wistar albino rats.ountries –9, and iountries –12. In Cdiabetes , 14, condiabetes , genitoudiabetes . Mirabilanalysis . Accordi L. root . Trigone L. root , 20 and  L. root , 22. Onl L. root  reportedMirabilis jalapa L. root (EEM) on both diabetic and normal mice after induction with streptozotocin with both singly and repeatedly administration, and on diabetes before induction with repeatedly administration, and compared with glibenclamide as a reference standard. Oral glucose tolerance test was also measured on normal and diabetic mice after single EEM administration.In this study, the experiments were designed to detect the insulin sensitivity and hypoglycemic and hypolipidemic activities of ethanolic extract of Mirabilis jalapa L. was collected from Chongqing city of China. They were taxonomically authenticated by Dr. Guo-yue Zhong, and a voucher specimen of the plant (TRI7374) has been deposited in Chongqing Academy of Chinese Materia Medica.The root of Mirabilis jalapa L. root (2 kg) was refluxed with 10 volumes of 70% ethanol three times, one hour for each time. The combined ethanolic extract was filtered using a Millipore filter  to remove particulate matter. The ethanolic extract was concentrated and dried into solid residues in vacuo . The residue was stored at 4°C, and the desired dose  was reconstituted in distilled water daily, just before administration.The root was dried in shade and powdered in a grinder. For the preparation of EEM, dried powder of Mirabilis jalapa L. root has recently been studied for its chemical composition by Piyali et al. [i et al. . The aquMirabilis jalapa L. root [μm); mobile phases: acetonitrile water ; flow rate: 0.8 mL/min. Peaks were analyzed spectroscopically at 265 nm with a UV-visible-light detector. The EEM solutions were quantified by spiking with a known amount of standard (trigonelline) and also by comparing the area under curve. The repeatability of the method was evaluated by injecting the solution of EEM and standard solution for three times, and the relative standard deviation percentage was calculated.High performance liquid chromatography method has been developed for the analysis of trigonelline in  L. root . High-pe Male KM mice (18–22 g) were purchased from Third Military Medical University, China. The animals were given a standard pellet diet and water ad libitum and were maintained under standard environmental conditions . Streptozotocin was purchased from Sigma Co. Ltd, St. Louis, MO, USA. Glibenclamide  and metformin hydrochloride  were purchased from Wuhan Yuancheng Technology Development Co. Ltd, China. Insulin radioimmunoassay kit was provided by Beijing North Institute of Biological Technology, China. All other chemicals were obtained from local sources and were analytical grade. Throughout the test, experimental mice were processed in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines.n = 10 in each group). The normal control group received distilled water, and normal treated groups received glibenclamide at a dose of 0.76 mg/kg and EEM at doses of 2, 4, and 8 g/kg, respectively. The selected doses of EEM were according to the literature [ Sixteen-hour-fasted normal mice were both randomly assigned to five different groups , serum insulin, and total cholesterol (TC) and triglyceride (TG) levels. The liver and skeletal muscle were removed and immediately frozen in liquid nitrogen.The repeated administration procedure is as follows: mice were treated as single administration once daily for 28 days. Body weights were weighed every two weeks. At 14th day, 16-hour-fasted blood sample was collected from the tail for blood glucose measurement. At 28th day, mice were anaesthetized and 16-hour-fasted eye socket blood of mouse was collected for measurement of blood glucose, serum insulin, and TC and TG levels. The liver and skeletal muscle were also collected and frozen in liquid nitrogen.n = 10 in each group). The diabetic control group received distilled water, and diabetic treated groups received glibenclamide at a dose of 0.76 mg/kg and EEM at doses of 2, 4, and 8 g/kg, respectively. The single and repeated administration procedures were the same as in The animals were fasted for 16 h prior to the induction of diabetes. Streptozotocin was freshly prepared in 0.1 mol/L citrate buffer solution (pH4.5) and was intraperitoneally injected to mice with a single dose of 60 mg/kg. After three days, 16-hour-fasted blood sample was collected from the tail for measurement of fasting blood glucose level. Mice with fasting blood glucose level higher than 11.1 mmol/L were considered diabetic ones and were randomly divided into five different groups , two reference drugs, glibenclamide (2.5 mg/kg) and metformin (50 mg/kg), and three doses of EEM  were orally administered to groups of 10 mice each. Two hours later, glucose (2.0 g/kg) was orally administrated to each mouse. Blood samples were taken from tail vein at 0 (just before the glucose administration), 15, 30, 60, and 120 min for the assay of glucose. Serum insulin concentration was measured by commercial kits according to the manufacturer's instructions. Insulin sensitivity index (ISI) = log (1/fasting plasma glucose × serum insulin).TC and TG levels were determined by spectrophotometry with commercial kit provided by Nanjing Jiancheng Bioengineering Institute, China.g at 4°C for 15 min. Supernatants (upper phase contained extracted TG) were collected and evaporated with vacuum centrifuge. The TG content was determined by spectrophotometry with commercial kit .Glycogen content was determined by spectrophotometry with commercial kit provided by Nanjing Jiancheng Bioengineering Institute, China. TG content in liver and skeletal muscle was measured by the method of Hu et al. . The froP < 0.05 was considered significant.All data were expressed as mean ± SD. Statistical analysis was performed with one-way ANOVA followed by Tukey post hoc test for multiple comparisons. Mirabilis jalapa L. root . But fasting blood glucose level did not change after single administration of EEM at doses of 2, 4, and 8 g/kg either in normal or diabetic mice . Progressive reduction was observed after 28 days of repeated 4, 8 g/kg EEM administration (P < 0.01). Both 14 and 28 days of 2 g/kg EEM administration resulted in no reduction in blood glucose level. Oppositely, the blood glucose level of untreated diabetic mice was continually increased during the experimental period. Changes of blood glucose level after repeated EEM and glibenclamide administration in normal and diabetic mice were shown in P < 0.01) when compared to untreated normal mice. Two hours after single administration of all doses of 2, 4, and 8 g/kg EEM and glibenclamide failed to reduce serum insulin level and to improve ISI both in normal and diabetic mice Significant increased serum insulin level but decreased ISI were in diabetic mice (P < 0.01). But 28-day administration of 2 g/kg EEM and glibenclamide had no effect on serum insulin level and ISI.As showed in P < 0.01) when compared to untreated normal mice. Two hours after single administration of all doses of 2, 4, and 8 g/kg EEM and glibenclamide failed to reduce serum TC and TG levels both in normal and diabetic mice (Streptozotocin injection caused significant increases in serum TC and TG levels (tic mice .P < 0.01).Changes of serum TC and TG levels in normal and diabetic mice during 28 days of repeated EEM and glibenclamide administration were shown in P < 0.01). Single administration of 2, 4, and 8 g/kg EEM and glibenclamide also did not produce any significant change in glycogen and TG contents in liver and skeletal muscle in diabetic mice.Changes of glycogen and TG contents in liver and skeletal muscle of normal and diabetic mice after single administration of EEM and glibenclamide were shown in P < 0.01). Administration of 2 g/kg EEM for 28 days did not produce any significant change in glycogen and TG contents in liver and skeletal muscle. However, repeated administration of 4, 8 g/kg EEM significantly increased glycogen content and reduced TG content in liver and skeletal muscle compared with untreated diabetic mice (P < 0.01). Repeated administration of glibenclamide significantly increased glycogen content (P < 0.01), but did not affect TG content in liver and skeletal muscle compared with untreated diabetic mice.Changes of glycogen and TG contents in liver and skeletal muscle of normal and diabetic mice during 28 days of EEM and glibenclamide administration were shown in P < 0.01) (P < 0.01). In the diabetic mice, EEM  also showed significantly inhibitory effect on the increased blood glucose level after glucose load (P < 0.01) (P < 0.01). In normal mice, EEM  significantly inhibited the increase in blood glucose level after glucose load ( < 0.01) , while l < 0.01) , while lWhen 2, 4, and 8 g/kg EEM and glibenclamide were administered for two weeks before induction of diabetes with streptozotocin and for a further 28 days afterwards, essentially similar effects were observed on blood glucose, serum insulin, ISI, serum TC, serum TG, glycogen, and TG contents in liver and skeletal muscle to those seen when EEM and glibenclamide were administered after induction of diabetes (data not shown).β cells and had a high blood glucose level, which are in accord with the results of previous investigations [Blood glucose level was increased significantly from the 3rd day in untreated diabetic mice. This increment may be due to reduced glucose clearance apparently arising from a defect in glucose transport  and/or tigations , 27. Sinβ cells.When studying a chronic disease such as diabetes, it is more pertinent to test the maintenance of lower blood glucose level with long-term treatment rather than the acute hypoglycemic effect after a single administration. In this study, we found that repeated administration EEM continually decreased blood glucose level in diabetic mice both after and before induction with streptozotocin. In diabetic liver and skeletal muscle, glycogen content was lower and TG content was higher than those of normal mice, which accords with other reports , and thiSeveral medicinal plants have been reported to restore activity of key enzymes of glucose and glycogen metabolism which are strongly disturbed in streptozotocin-induced diabetic mice , 33. HypMirabilis jalapa L. root contains phytocompounds, such as alkaloids, glycosides, carbohydrates, and phytosterols [Mirabilis jalapa L. roots contain plentiful trigonelline. As one of the components of Mirabilis jalapa L. root, trigonelline reduced blood glucose level in animal and human [α levels, and liver fatty acid synthase activity and increased carnitine palmitoyl transferase, glucokinase activities, and glucokinase/glucose-6-phosphatase ratio in the liver [The philosophy behind the medicinal uses of traditional Chinese medicine is holistic in nature, which differs from western medicines, and all chemical components present in a particular traditional Chinese medicine are believed to be of therapeutic importance. It was generally recommended to separate and measure the active chemical ingredients for standardization and quality control of herbal products. What is more, the confirmation of main constituent in herb and herbal extract helps to clarify its pharmacological effects. Previous studies reported that the osterols , 17. Phynd human –22 and ind human , 39. Whahe liver , 40. TheMirabilis jalapa L. root can be used to treat type 2 diabetes with hyperlipidemia. Mirabilis jalapa L. root may be developed as an oral hypoglycemic agent or functional food for diabetic patients with hyperlipidemia and for persons with high risk of diabetes. This finding represents an experimental confirmation of the Chinese traditional use of this plant for diabetic treatment. Consequently, consumption of Mirabilis jalapa L. root may prevent the complication of hyperglycemia associated with diabetes. Finally, the precise mechanism(s) and site(s) of this activity and the active constituent(s) of Mirabilis jalapa L. root still need to be determined in addition to toxicological studies in further experiments.In conclusion, our study demonstrates that


E. coli and S. aureus compared with individual silver nanoparticles, rGO nanosheets or their nanocomposites.A sandwich-like antibacterial reagent (Ag/HNTs/rGO) was constructed through the direct growth of silver nanoparticles on the surface graphene-based HNTs nanosheets. Herein, various nanomaterials were combined by adhesion effect of DOPA after self-polymerization. Ag/HNTs/rGO posses enhanced antibacterial ability against  Halloysite nanotubes (HNTs), a kind of aluminosilicate clay, exhibit novel physical and chemical properties due to predominantly hollow tubular structure, thereby providing opportunities for advanced applications in many fields. A significant advantage of HNTs is their easiness to obtain and low price in the comparison with other nanomaterials, like carbon nanotubes123Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus). However, the antibacterial performance of individual graphene nanosheets is limited compared with silver which are easy to agglomerate contributing to the decrease of the antibacterial effects. In early approaches, graphene-AgNPs-based nanocomposites were fabricated by a number of workers which show enhanced antibacterial properties15212223Antibacterial reagents, including antibiotics1415178Bioadhesive proteins of marine organisms, such as mussels , have atHerein, HNTs were introduced to graphene-AgNPs-based nanocomposites combining the adhesive ability of DOPA to fabricate a sandwich-like nanomaterial based on the interpenetrative nanocomposites. This new approach is designed to reinforce the synergistic effects between graphene and HNTs in the interest of the direct growth of silver nanoparticles. 2123−1, which ascribes to C = O stretching vibrations in the carboxyl group of GO after the oxidation of natural graphite. However, after the construction of Ag/HNTs/rGO, the characteristic peak around 1720 cm−1 vanished and the whole curve of Ag/HNTs/rGO was almost identical to that of natural graphite. The two small peaks around 2930 cm−1 and 2840 cm−1 of Ag/HNTs/rGO are supposed to be in the consequence of the partial destruction of carbon skeleton of natural graphite during the oxidation process, which tends not to be restored thoroughly by reduction. These results indicated that GO nanosheets were reduced to rGO by DOPA during the fabrication process of Ag/HNTs/rGO.Fourier transform infrared spectroscopy (FTIR) of Ag/HNTs/rGO was performed to justify whether GO nanosheets was reduced to rGO during the construction process of the sandwich-like nanomaterial. It can be seen from 2 at the temperature of liquid nitrogen. The specific surface area (SBET) for Ag/HNTs/rGO showed an obvious enhancement with a value 287.10 m2/g. Nevertheless, the SBET value for Ag/HNTs and Ag/rGO were 59.6 m2/g and 128.92 m2/g, respectively. That is to say, a high-surface-area substrate was synthetized as our initial design.To investigate the surface area change of Ag/HNTs/rGO, the specific surface area (BET) was measured using the adsorption of NE. coli and Gram positive bacterial strains, S. aureus as bacterium models. Minimal inhibitory concentration (MIC) was observed by the tube double dilution method to evaluate the effectiveness of as-prepared nanomaterials. In the tube double dilution method, MIC is defined as the lowest concentration (in μg/ml) of the antimicrobial agent that prevents visible growth of a microorganism under defined conditions. The MIC results of the sandwich-like nanomaterial, Ag/HNTs/rGO and the controls including Ag, Ag/HNTs, rGO/Ag is shown in The antibacterial properties of Ag/HNTs/rGO was studied against Gram negative bacterial strains, E. coli or S. aureus without treated by Ag/HNTs/rGO are observed as small white dots and almost fill the whole plate as shown in E. coli and S. aureus demonstrate the effectiveness and popularity of the antibacterial properties of Ag/HNTs/rGO.Additionally, the sandwich-like nanomaterial showed excellent antibacterial performance through bacteriostasis rate test. It can be seen in E. coli cells were further analyzed by using TEM. As shown in E. coli cells were evenly fuscous with well-defined membranes. However, after treated with Ag/HNTs/rGO for 16 h, the E. coli cells were ruptured, pale along with the release of cytoplasm and a large portion of the cells was decomposed as can be seen in The morphology changes of 600) of E. coli suspension treated with different prepared reagents in test tubes for 5 h and 12 h were determined, which is a widely used method to determine the growth of bacteria for the assessment of antibacterial abilities28600 values followed the order: control > rGO > Ag > Ag/HNTs > Ag/rGO > Ag/HNTs/rGO, which is almost the same with the MIC of them. Silver nanoparticles which grow directly from Ag/HNTs, Ag/rGO, and Ag/HNTs/rGO showed lower OD600 after both 5 h and 12 h cultivation. This phenomenon can be on account of the decrease of conglomeration of silver nanoparticles. Additionally, each group of data presents a good stability with a very minor anova. It is important to emphasize that the sandwich-like nanomaterial, Ag/HNTs/rGO, showed the lowest OD600 especially after 12 h cultivation in contrast to other antibacterial reagents. These results indicated that Ag/HNTs/rGO possesses a better ability in resisting bacteria growth agreeing strongly with the MIC results, which probably benefits from enhanced surface area of Ag/HNTs/rGO.The optical density at 600 nm  were purchased from Sinopharm Chemical Reagent Co., Ltd., and were used as received. Halloysite clay from Henan Province (China) was milled and sieved to obtain halloysite nanotubes (HNTs). Tris (hydroxymethyl) aminomethane (AP) and dopamine (AP) were purchased from Sigma Aldrich. Polyethersulfone  was obtained from BASF, Germany. All the other chemicals  were obtained from Tianjin Kermel Chemical Reagent Co., Ltd., China, and were used without further purification. The used water is deionized water.GO nanosheets were prepared by oxidizing natural graphite based on an improved method2 area with about 1 mm thickness.As prepared Ag/HNTs/rGO particles, 0.3 g, were added in 5 mL N, N-Dimethylacetamide (DMAc) and dispersed thoroughly by stirring. Then PES, 0.9 g, was added under stirring and the stirring process continued at least 24 h. After PES completely dissolved in the solution, the mixture was poured into a home-made glass groove with about 3 mm deepness. The solvent was evaporated thoroughly under an open environment at room temperature and the Ag/HNTs/rGO film was peeled off from the glass carefully. The resulted Ag/HNTs/rGO has a 20 × 60 mm−1 resolution with Thermo Nicolet IR 200 spectroscope . Typically, 64 scans were signal-averaged to reduce spectral noise. The spectra were recorded in the 400–4000 cm−1 range using KBr pellets. A FEI model TECNAI G2 transmission electron microscope  was used to study the morphology of Ag/HNTs/rGO. The samples were dispersed in solvent with the aid of ultrasound. The suspended particles were transferred to a copper grid (400 meshes) coated with a strong carbon film and dried. Energy Dispersive System (EDS) was carried out in a JEOL JSM-7500F FE-SEM with samples sputtered with gold.FTIR spectra were performed at 2 cm5 CFU/mL of bacteria and incubated overnight at 37°C for 8 h. Growth of the cells was determined by observing the turbidity of the culture. The lowest concentration, at which no visual turbidity could be observed, represented the MIC of the antibacterial material.Minimal inhibitory concentration (MIC): Serial dilutions of the antibacterial materials were performed in Mueller-Hinton broth which were inoculated with 5 × 105 times was cultured on an agar plate. After incubated at 37°C for 16 h, the numbers of colonies were counted and the bacteriostasis rate was determined by dividing the number of colony-forming units (CFU) which are killed by antibacterial reagent by the number of CFU of the control group.A 100 μL of the above bacterial suspension after being diluted 10E. coli cells treated with Ag/HNTs/rGO solution for 30 min were fixed with 2.5% glutaraldehyde. The cells were washed with PBS and then postfixed with 1% aqueous OsO4 (Fluka) for 1 h and washed again twice with PBS. The cells then were dehydrated through ethanol series  and embedded in Epon/Araldite resin (polymerization at 65°C for 15 h). Thin sections (90 nm) containing the cells were placed on the grids and stained for 1 min each with 4% uranyl acetate (1:1 acetone/water) and 0.2% Raynolds lead citrate (water), air dried, and examined under a transmission electron microscope .Transmission Electron Microscopic (TEM) Measurements: Y.T.Z. and B.Z. conceived and designed the study. Y.T.Z. and J.D.L. wrote the manuscript. L.Y. performed the experiments. All the authors contributed to discussion and reviewed the manuscript.


To identify clinical characteristics associated with internal neurofibromas in children with NF1, as a means of ensuring the early identification of patients at high risk for malignant peripheral nerve-sheath tumors developed from preexisting internal neurofibromas.P ≤ 0.15) were entered into a multiple logistic regression model after checking for potential interactions and confounding. Multiple imputation was used for missing values.We used data from two NF1 populations, in France and North America, respectively. The French database comprised 1083 patients meeting NIH diagnostic criteria for NF1 and the Neurofibromatosis Institute Database of North America comprised 703 patients. Patients younger than 17 years of age were eligible for our study if they had been evaluated for internal neurofibromas using computed tomography and/or magnetic resonance imaging. Clinical characteristics associated with internal neurofibromas by univariate analysis  met our inclusion criteria. Their mean age was 7.7 ± 5.0 years and there were 192 (53.8%) males. Internal neurofibromas were present in 35 (9.8%) patients. Internal neurofibromas developed earlier in females than in males and their prevalence increased during adolescence. Factors independently associated with internal neurofibromas were age (OR = 1.16 [1.07-1.27]), xanthogranulomas (OR = 5.85 [2.18-15.89]) and presence of both subcutaneous and plexiform neurofibromas (OR = 6.80 [1.52-30.44]).Several easily recognizable clinical characteristics indicate a high risk of internal neurofibromas in children with NF1 and, therefore, a need for very close monitoring. Ten pThe purpose of this study was to identify clinical characteristics associated with internal neurofibromas in children with NF1. Such clinical characteristics could serve to ensure the early identification of children with NF1 who require particularly close monitoring for internal neurofibromas.The study was performed using data from two NF1 populations, one in France and the other in North America. Patients meeting the diagnostic criteria for NF1 established at the National Institutes of Health Consensus Development Conference were incThe study was approved by the Île-de-France IX Ethics committee Paris, France. Informed consent was obtained from all patients. The study adhered to Declaration of Helsinki guidelines.The demographic information  and clinical characteristics recorded in the databases were collected during routine clinical assessments at neurofibromatosis clinics  and/or magnetic resonance imaging (MRI). The presence of internal neurofibromas was coded 1 in the databases.P values <0.05 were considered statistically significant.Quantitative variables are described as mean ± standard deviation (SD). Age was not converted to a categorical variable. Qualitative variables are described as number (%). All tests were two-tailed and P values smaller than 0.15 in the univariate analyses were entered into a multiple logistic regression model. The final model included the variables independently associated with the presence of internal neurofibromas.The characteristics of patients with and without internal neurofibromas were compared in univariate analyses. Odds ratios (ORs) were estimated with their 95% confidence intervals (95% CIs), using logistic regression models. ORs were adjusted for age because the prevalence of several NF1 characteristics varies with age (number of café-au-lait spots and cutaneous and subcutaneous neurofibromas). Potential interactions were assessed by pairwise analyses and confounding by fitting multiplicative models. Variables yielding We first conducted an analysis without the individuals who had missing data . We then estimated the missing values for the co-variates independently associated with the internal neurofibromas in the final model, using the multiple-multivariate-imputations-by-chained-equations procedure in STATA, with thWe conducted all statistical analysis using STATA Statistical Software  and LogXact-8 software .TableTableP = 0.10). To facilitate the interpretation of the model, the usual assessment of interaction effects is done by entering an additional variable composed of the product of the two variables[The presence of Lisch nodules was strongly associated with the other variables and was not independently associated with internal neurofibromas in the multivariate analysis. The study of potential interactions between variables showed a effect modification between plexiform neurofibromas and subcutaneous neurofibromas  and 20 to 40 years . MPNSTs were the main cause of death (60%)[NF1 has been reported to be associated with a 15-year decrease in life expectancy. We receth (60%). Thus, tth (60%).P = 0.001). In addition, if effective treatments are found, they will be more likely to prevent the growth of internal neurofibromas than to reduce their size[Internal neurofibromas grow faster in young children than in adults. Volumeteir size. TherefoMany factors support the internal validity of our study. First, all patients had a definitive diagnosis of NF1. Selection bias seems unlikely, as the prevalence of NF1 characteristics were consistent with those reported previously in other populations of NF1 patients,26. Our P = 0.08, P = 0.22, and P = 0.17, respectively), possibly because statistical power was limited[Internal neurofibromas were present in 35 (9.8%) patients, in keeping with previous data. In a study of 53 children with NF1 who underwent MRI of the entire spine, 7 (13.2%) patients had internal spinal neurofibromas. In our  limited. Further limited and Nort limited, respect limited. AlthougIn sum, we identified easily recognizable clinical characteristics that were associated with internal neurofibromas in children with NF1. These risk factors could be used to ensure the early identification of NF1 patients who require particularly close monitoring for internal neurofibromas. In the future, this approach may allow the initiation of treatments at the time of fastest growth of internal neurofibromas, when they are most likely to be effective.aOR: Adjusted odds ratio; CI: Confidence interval; CT: Computed tomography; MPNST: Malignant peripheral nerve sheath tumor; MRI: Magnetic resonance imaging; NF-1: Neurofibromatosis 1; SC-NF(s): Subcutaneous neurofibroma(s); SD: Standard deviation.SBG, PW, DR, SF, OC, SB, LVA, VMR, SHR, ES have non-financial interests that may be relevant to the submitted work.SBG and PW made substantial contributions to the study conception and design. SBG, PW, SF, SHR, VMR, LVA, ES, DR, and SB made substantial contributions to data acquisition or analysis and to interpretation of the data. ES performed the data analysis under the supervision of SBG. SBG, PW, and ES were involved in drafting the manuscript or revising it critically for important intellectual content. All authors gave final approval of the version submitted for publication.Grant from the programme hospitalier de recherche clinique , French Ministry of Health.