Seventeen sets of model-predicted impedance and mean pulmonary arterial pressure (mPAP) show good agreement with the animal measurements, thereby validating the model. Next, we considered a predictive model in which three parameters, PVR, elastic modulus (EM), and arterial thickness, were varied singly from one simulation to the next to study their individual roles in PH progression. Finally, we used the model to predict the individual impacts of clinical (vasodilatory) and theoretical (compliance increasing) PH treatments on improving pulmonary hemodynamics. Our model (1) displayed excellent patient-specific agreement with measured global pulmonary parameters; (2) quantified relationships between PVR and mean pressure and PVS and pulse pressure, as well as studiying the right ventricular (RV) afterload, which could be measured as a hydraulic load calculated from spectral analysis of pulmonary artery pressure and flow waves; (3) qualitatively confirmed the derangement of vascular wall shear stress in progressive PH; and (4) established that decreasing proximal vascular stiffness through a theoretical treatment of reversing proximal vascular remodeling could decrease RV afterload.We develop a simple computational model based on measurements from a hypoxic neonatal calf model of pulmonary hypertension (PH) to investigate the interplay between vascular and ventricular measures in the setting of progressive PH. Model parameters were obtained directly from  Pulmonary hypertension (PH) is associated with a progressive increase of pulmonary vascular resistance (PVR) and sustained elevation of mean pulmonary artery pressure (mPAP), which together contribute to the right heart dysfunction. However, recent studies showed that mPAP apparently does not correlate with either the severity of symptoms or survival. Although many recent developments employing clinical and experimental measurements have investigated symptoms –4 to furClinical and traditional scientific studies of PH focus on changes in the distal circulation. For example, the distal pulmonary vascular bed can undergo remodeling resulting from growth of smooth-muscle cells, which narrow and restrict distal pulmonary vascular geometry. Additionally lumen obliteration, medial hypertrophy, and adventitial proliferation contribute to the distal diameter decrease and thickness increase in PH –7. In seAlthough vascular distal remodeling and distal vessel vasculopathy are generally considered the only major contributors to PH progression –15, recein vivo study. Here, we propose a modeling procedure to simulate PH progression by dividing it into 4 stages. At each stage we only consider system changes due to varying a single pulmonary vascular parameter, from PVR, elastic modulus (EM), and arterial thickness (h), starting with the control condition. We evaluate the role of these three key parameters in modulating input impedance and hemodynamics using a two-dimensional (2D) pulmonary vascular computational model based on in vivo and ex vivo measurements obtained from normal and chronically hypoxic neonatal calves [Z1) by comparing animal measurements to 17 calf specific computational models. We then perform predictive studies to assess the effects of PVR, EM, and h on the arterial pressure, WSS, impedance and RV power. Finally, the impacts of theoretical and available PH treatments are examined by reversing these parameters, thereby investigating the added effect of targeting proximal vascular remodeling for treatment in PH. We believe this simple but validated model offers important insight of PH progression and might encourage new treatment targets.Progression of pulmonary hypertension involves continuously changing vascular hemodynamics and remodeling of the proximal and distal arteries. It is understandably difficult to investigate an individual parameter's effect on PH in an l calves , 29. We The ability to develop a unique and validated model for each subject and achieving reasonable solution times that allow for routine simulation are the two critical factors that motivate us to employ numerical techniques, and should enable simpler application of these techniques in both research and clinical environments. Although there have been a variety of numerical models developed by our group and others, ranging in complexity from fully three dimensional, exact geometry models to simple one dimensional bulk flow models, we believe neither fully satisfies these two critical factors. By developing a simple but still subject-specified model, an approach is proposed through which we obtain information needed to study PH progression yet perform validation with a larger number of subjects.D, thickness h, and a total length of 0.1 m. Three simulation types are performed in this paper: a validation study, a PH progression study, and a PH treatment study. For the first, parameters from seventeen calves (two groups: control and hypertensive) are used to develop subject-specific models and validation occurs through comparison of multiple model results to experimental measurements. The PH progression and treatment studies then utilize mean values from the control and hypertensive groups to assess the impacts of specific parameters on afterload. Our primary intent here is to examine how hemodynamics and RV power change in PH progression and during treatment. Our model consists of a single elastic tube, representing the larger upstream compliant vessels, which is modeled in two dimensional (2D) axisymmetric coordinates. 3. At model initiation, there is no flow in the domain as well as no transmural pressure. At the artery model lumen the nonslip condition is applied to the fluid velocity, and normal stress is continuous across the lumen. An axisymmetric boundary is defined at the centerline of the arterial wall, thereby reducing the mesh size, and in turn, the simulation time. The inlet flow rate is transient, with pulse shape obtained from experimental in vivo measurements, as explained below. The outlet boundary condition of the flow domain is specified as a resistance boundary condition (32), R = P/Q, with P and Q being given by as the measurements mPAP and CO, respectively.The blood is modeled as an incompressible, laminar, Newtonian fluid with a viscosity of 3.5 mPa·s and a density of 1050 kg/m3 and Poison's ratio 0.48; the elastic moduli of the models were obtained from ex vivo measurements and are discussed further below. The boundary condition at each end is specified as free radial motion and zero longitudinal motion; there is no load on the exterior surface, and only internal coupled fluid pressure/shear stress is applied on the interior surface (fluid structure interface).The pulmonary artery wall is considered as an incompressible isotropic linear elastic model with density 1200 kg/mThe CFD-ACE multiphysics package  and MATLAB  are used to perform the numerical simulations for both fluid and solid domains, including experimental data and results processing. A rectangular mesh is generated for the axisymmetric geometry using CFD-GEOM initially containing uniform fluid and solid elements. The mesh contains 4221 fluid nodes and 1005 solid nodes, with 201 nodes on the fluid-structure boundary. Time integration utilizes a first-order Euler approach with a time step of 2 ms. Details of the solution of the fluid and structural equations can be referred to our previous work –32. BothBecause the resistance boundary condition coupled with wall stiffness allows for fluid storage, the simulation needs to take several cardiac cycles to become periodic. Here, we run each model for 6 continuous cardiac cycles to insure that the simulations are convergent in the transient inlet condition. A serial computer system (AMD Opteron (tm) Processor 280 2.4 GHz, 2.0 G memory) performed the simulations; typical wall time for one model is about 5 hours for a typical simulation time of 3 seconds (6 cardiac cycles).We carried out mesh validation by comparing our baseline mesh to a 1.5 × refined mesh  and verified time convergence by halving the time step (1 ms). A total 6 cardiac cycles are simulated and the fluid pressure/velocity at the end of simulations are compared with and without these temporal and spatial refinements. At nine different locations, defined in the axial direction at the inlet, midpoint, and outlet; along the radial direction at the axis of symmetry, halfway between the axis and the wall; and at the arterial wall, the fluid pressure or velocity has less than 1% change with refined mesh and less than 1% difference with refined time step, respectively.Z); several of the experimental measurements were described in previous papers [All studies were performed after the approval by the appropriate Institutional Animal Care and Use Committees. Seventeen newborn male dairy calves (Holstein) weighing between 35 and 50 kg were used. Data obtained from intact calves include the MPA lumen diameter, thickness, and elastic modulus (PVS); the mean MPA pressure (mPAP) and cardiac output (CO), the MPA centerline velocity time history, and pulmonary vascular input impedance  were computed from pressure and PW Doppler [Ex vivo measurements were taken to obtain the MPA diameter, thickness, and elastic modulus [ex vivo elastic modulus [Our  history . A solid history . Finally Doppler . We note modulus . Extract modulus  over a w modulus . AdditioThe model geometry and elastic properties, including the diameter, thickness, and elastic modulus, are all obtained from measurements of calf MPAs . For valWe have previously used our inlet condition in clinical models . BrieflyFor the predictive studies, we average heart rates of all seventeen calves and use the same heart rate for both control and hypertensive models. Because the heart rate is affected more sensitively by daily activities , its effect on the PH will not be considered in this study . Additioex vivo methods [Based on our recent vascular stiffening research in this animal model , there a methods .μ(∂u/∂r)r=R and derived from velocity u by fitting the near-wall velocity data with a three-point polynomial function [μ is the viscosity of blood, r is radial vector, and R is the local instantaneous radius of the pulmonary arterial wall. The mean WSS is time-average WSS and the pulse WSS is defined as (WSSmax − WSSmin) in one fully developed cardiac cycle. The wall pressure is obtained at the middle point between inlet and outlet boundaries, the same location of WSS. The definitions of mPAP and PP are similar to those of mean and pulse WSS. Details regarding the calculation of impedance from pressure and flow waveforms may be found in our previous works [WM = P0 · Q0, where P0 and Q0 are the zero-frequency components of the pressure and flow moduli, respectively. The oscillatory power was calculated asn represents the harmonic of the fundamental frequency. Finally, total RV power was calculated asMATLAB codes were used  to calculate the WSS, input impedance, and ventricular powers. The WSS is function . Here, μus works , 28, 33.us works , 35. TheZ1 and mPAP computed in our seventeen subject-specific models were compared with the results from measurements with Student's t test. P values refer to the probability associated with the two-tailed Student's t test, and results were deemed statistically significant for P values less than 0.05. Linear regression and Bland-Altman methods [Z1 and mPAP between numerical results and measurements and thereby validate the computational modeling approach. Limits of agreement (LoA), defined as the interval expected to contain 95% of the differences, were calculated and are given by the mean difference plus or minus 1.96 standard deviations. 95% confidence intervals were taken around the LoA to evaluate their precision and consistency. MATLAB codes were written  to perform the t test, linear regressions, and Bland-Altman analyses.With the exception of heart rate, all data from experimental measurements are presented as mean ±  SD. For validation, the first harmonic impedance  methods , 38 are ex vivo elastic modulus), and exit conditions (PVR). The thickness, elastic modulus, PVR, and mPAP are higher in hypertensive than those in control conditions (P < 0.05). There was no statistically significant change in the lumen diameter and CO as a result of hypertension. Qualitatively comparing parameters that did change between the control and hypoxic conditions, PVR (208%), elastic modulus (82%) and mPAP (164%) change substantially more than thickness (17%).Z1) and computed mPAP were compared to experimental measurements. N results to experimental mPAPC measurements, and between the numerical and experimental impedance Z1. The regressions equations are y = 0.980x + 0.448, R2 = 0.999 (P < 0.05) for mPAP and y = 1.032x – 0.275, R2 = 0.941 (P < 0.05) for the impedance modulus Z1. Figures Z1 between numerical and (gold-standard) experimental results. The Bland-Altman analyses show a bias ± LoA of 0.46 ± 1.74 mm Hg for mPAP and 0.13 ± 1.12 mm Hg/(L/min) for the impedance modulus Z1. Clearly the bias and LoA for mPAP are small compared to the typical pressure value, while the bias and LoA are more significant for the impedance modulus.To validate the artery model, seventeen calf-specific simulations were performed, and the computed first harmonic modulus of input impedance (h)—on pulmonary hemodynamics and RV power during PH progression—are studied for both progression (increases in parameters) and treatment (reduction in parameters). First we consider the progression of PH into four stages: (1) control, (2) hyper PVR (PVR: 4.91 → 15.1 mm Hg/(L/min)), (3) hyper EM (EM: 97.1 → 177 kPa), and finally (4) hyper h (thickness: 3.38 → 3.95 mm), which are described in The simulated effect of changes in our key parameters, namely, PVR, EM, and proximal thickness  on pulmonary hemodynamics in PH progression and treatment were examined in a 2D numerical model. The model was first validated using specified data from calves in control and hypertensive conditions. In simulated disease progression, the model suggests that PVR, elastic modulus, and thickness have quantitatively different effects on pulmonary pressures, RV afterload, and power. The combined changes are shown to increase the mean and pulsatile component of hemodynamics while decreasing WSS, which supports the idea of WSS derangement in the PH condition. In simulated disease treatment, the model suggests that targeting proximal vascular remodeling, and in turn stiffness, may aid in recovery by further reducing RV afterload and power requirements. Finally, the relatively small impacts that stiffness has on RV parameters suggest that, given its newly found prominence in clinical outcomes prediction, it may affect RV function through yet undetermined pathways.


This study was designated to investigate whether increased extravascular lung water index (EVLWI) may correlate multiple organ dysfunction syndrome (MODS) and mortality in sepsis.We designed a prospective cohort study in an intensive care unit of a tertiary care hospital. Sixty-seven patients with severe sepsis were included. Data were used to determine an association between EVLWI and the development of MODS and mortality. These connections were determined by the multiple logistic regression, plotting the receiver operating characteristic (ROC) curve and by Spearman test.p<0.0001) than in those without (median (IQR), 12.4 (7.9–16.3) ml/kg, n = 29) and day 3 (median (IQR), 17.8 (11.2–22.8) ml/kg, n = 29, p = 0.004) than in those without (median (IQR), 12.4 (8.0–16.3) ml/kg, n = 29). EVLWI was used as an independent predictor of the development of MODS  during ICU stay. The area under the ROC curve showed that EVLWI levels could predict MODS (0.866) and mortality (0.881) during ICU stay. Meanwhile, the higher of SOFA score, the more EVLWI was found on day 1  and day 3 .EVLWI levels were higher in MODS patients on day 1 (median (IQR), 18(12.8–23.9) ml/kg, n = 38, Increased EVLWI levels correlates development of MODS and mortality during the patients' ICU stay. Further more, the potential of novel treatment in severe sepsis with lung injury may develop. The incidence of sepsis has been increasing because of the advancing age of the general population, a greater number of invasive procedures, and more immunosuppressive therapies Endothelial activation and damage occur early during sepsis in vivo. EVLWI determined with the single transpulmonary thermodilution technique correlates with that determined with the thermal-dye dilution The increased capillary permeability manifests in the lungs as altered alveolar–capillary barrier function and is characterized by accumulation of extravascular lung water index (EVLWI). Among the methods measuring EVLWI, the thermal-dye dilution method is considered to be the “gold standard” of EVLWI measurements This study was designed to investigate whether EVLWI is an independent factor for MODS in patients with severe sepsis.A total of 67 patients were enrolled in this study. There were 38 (56.7%) patients with MODS on day 1. In the cohort, 56 patients required mechanical ventilation. The characters of patients with and without MODS on day 1 and 3 were listed in p value less than 0.05 , SOFA score  and APACHE II score  remained significant predictors of MODS development during ICU stay after controlling for other variables. BMI, prior 24 hrs fluid balance, vasopressore use, PaO2/FiO2 ratio, lung injury score, PEEP and compliance of the lung failed to maintain their prognostic value for the MODS development during ICU stay in the adjusted analysis.Univariate analyses were primarily used for the selection of variables, based on a han 0.05 . The selp = 0.001) than in those without . Similarly, fatalities had increased levels of EVLWIs  compared with survivors . In 21 patients with sepsis from non-pulmonary origin , EVLWIs were higher in MODS patients  than in those without . Similarly, fatalities had increased levels of EVLWIs  compared with survivors .Comparisons between areas under the ROC curve for EVLWI, SOFA score, APACHE II scores, lung injury score, and prior 24 hrs fluid balance on day 1 of severe sepsis in prediction of clinical outcomes during ICU stay are listed in Sensitivities, specificities, and predictive values of elevated EVLWI (≥10 ml/kg) for the development of MODS and mortality during ICU stay are shown in This study has demonstrated that EVLWI and APACHE II scores are independent factors for the development of MODS in patients with severe sepsis. Increased EVLWI was associated with MODS on the day of the patients' ICU admission and subsequent development of organ failure during their ICU stay. With the cut-off value of 10 ml/kg, EVLWI offered good diagnostic sensitivity, specificity, and negative predictive value for MODS and mortality in those patients.The APACHE II score and SOFA score have already been used to evaluate the illness severity of ICU patients A recent study reported that 27% of severe sepsis patients who fulfilled the clinical consensus conference criteria for ARDS had never displayed raised EVLWI. In contrast, 57% of severe sepsis patients without clinical ARDS have increased EVLWI With a cut-off value of 10, this study has demonstrated that patients with EVLWI >10 ml/kg on day 1 had a higher chance of MODS on day 1 of ICU admission and subsequent development of MODS during their ICU stay. Patients with increased day 1 EVLWI had higher chances of subsequent dysfunction of each organ system and are more prone to ICU mortality when compared with those without. The area under the ROC curve confirms that day 1 EVLWI levels are superior to APACHE II and lung injury score in predicting the development of MODS and mortality during patients' ICU stay. In addition, day 1 EVLWI offered good sensitivity, specificity, and negative predictive values for MODS and ICU mortality.There are some limitations in current study. First, Frederic Michard reported the limitations of dilution methods may lead to an underestimation of EVLW in large pulmonary vascular obstruction, focal lung injury, and lung resection, but dilution methods remains an easy and clinically acceptable estimation of EVLW in most critically ill patients, including those with acute respiratory distress syndrome (ARDS) In conclusion, EVLWI is an independent factor for the development of MODS in patients with severe sepsis. Elevated EVLWI levels on day 1 of severe sepsis may serve as a predictor for the development of MODS and mortality during the patients' ICU stay. Further more, the potential of novel treatment in severe sepsis with lung injury may develop. By current studies, someone can develop to design further researches to discover possible mechanism of sepsis with lung injury and develop novel treatment. However, further investigation is required to confirm these findings and to determine the utility of EVLWI as an early prognostic marker in patients with severe sepsis.This study was conducted from April to December 2008 in a 37-bed medical intensive care unit (MICU). With approval of Institutional Review Board, Chang Gung Medical Foundation (IRB no.: 97-0374B) and written informed consent from all patients, we prospectively recruited 50 male and 17 female patients  on the day of MICU admission and within 24 hrs of the diagnosis of severe sepsis. All enrolled patients were recruited consecutively and met the criteria of sepsis with at least one organ failure. Patients were followed up until death or transferring out of ICU. Patients with the following criteria were excluded: pregnancy, age less than 18 years old, and uncontrolled malignancy. Severe sepsis was defined by the consensus committee of the American College of Chest Physicians and Society of Critical Care Medicine MODS was defined as more than 2 organs dysfunction (>3 organs) according to the Consensus Committee of American College of Chest Physicians and Society of Critical Care Medicine The physiological parameters, including the previous 24-hour net input/output of fluid balance, and shock status were assessed on patient enrollment on day 1 and day 3. Shock was defined as systolic blood pressure <90 mm Hg or mean arterial pressure <60 mm Hg and vasopressor necessarily. The serum albumin level, white blood cell counts, platelet counts, arterial oxygen tension [PaO2]/fractional inspired oxygen [FiO2] ratio, lung injury score, and chest X-ray score were also collected on day 1 and day 3. Ventilator parameters collected were tidal volume indexed to predicted body weight, positive end expiratory pressure (PEEP), compliance of the lung, plateau pressure, mean airway pressure, and minute volume on day 1 and day 3.The extravascular lung water (EVLW) measurement was based on the transpulmonary thermodilution method. The EVLW was measured everyday for 3 days but organ dysfunction was assessed on the day 1 and day 3 after ICU admission. The detail was well reported in our previous study Parameters were indexed to total body surface area or to predicted body weight in order to facilitate comparisons. We selected 10 ml/kg as a cut-off value by ROC curve method analysis, which was highly sensitive (85%) and specific (75%) in predicting development of MODS. Therefore, elevated EVLI was defined as a value exceeding 10 ml/kg on the day of undergoing PiCCO system monitoring.p value less than 0.05. The selected variables were entered into a multinominal logistic regression analysis to identify the net effects of each individual factor. The potential problem of colinearity among several variables was evaluated using the Spearman correlation coefficient before running the analysis. Odds ratios (OR) and their 95% confidence intervals (CI) were computed by logistic regression model analysis to clarify the impact of several potentially independent prognostic factors. We plotted receiver operating characteristic (ROC) curves for day 1 EVLWI, SOFA score, APACHE II scores, lung injury scores, and prior 24 hrs fluid balance for prediction of development of MODS and mortality during ICU stay; the respective areas under the curves were calculated. The relationship between EVLWI and SOFA score was assessed with Spearman test. A p-value<0.05 was considered statistically significant. All analyses were conducted using SPSS software  and Prism 4 for Windows .All data are expressed as median (IQR (interquartile range)) or number (%). Since most continuous variables were skewed, nonparametric approaches were used in the study. Quantitative variables between two groups were compared using the Mann-Whitney test for continuous and ordinal variables and the chi-square test for nominal variables. The univariate analyses above were primarily used for the selection of variables, based on a


Boswellia carterii have been used in traditional medicine for many years for management different gastrointestinal disorders. In this study, we wish to report urease inhibitory activity of four isolated compound of boswellic acid derivative.Boswellia carterii and identified by NMR and Mass spectroscopic analysis , compared with thiourea as a standard inhibitor (IC50 = 21.1 ± 0.3 μM).It was found that compound The inhibition potency is probably due to the formation of appropriate hydrogen bonds and hydrophobic interactions between the investigated compounds and urease enzyme active site and confirms its traditional usage. Boswellia carterii is widely distributed in the Indian subcontinent and Africa. The gum exudates of Boswellia spp. comprise of several pentacyclic triterpenoid acids, which are known as boswellic acids (BA’s) and reported to have different biological activities. Their ability to induce apoptosis choose them as potent compounds in treatment of colon, prostate and breast cancers [Inhibition of enzymes by natural products which are proven to be the source of therapeutic agents, have increasingly attracted the interests of scientists as a valuable strategy in drug discovery.  cancers -7 and a  cancers -11. Also cancers .B. carterii has urease inhibition activity [In addition, a number of extracted organic acids of this plant show other biological activities such as inhibition of peptic ulcer formation, probably by increasing the gastric mucosal resistance which its exact molecular mechanism is still not clear. In the recent report, it was shown that methanolic extract of activity ,14.Helicobacter pylori infection is the main cause of gastritis, peptic ulcer disease and gastric cancers [Enzyme inhibition has already led to the discovery of the wide variety of useful drugs in the treatment of several diseases. It is found that  cancers ,16.H. pylori producing abundant amounts of ammonia , make the growth and survival of bacteria possible, by increasing the pH of environment [H. pylori has become resistant to many antibiotics, thus introducing new agents, like natural urease inhibitors, is of special importance. In addition, urease activity not only participates in the formation of kidney stones [Urease of ironment . Therefoy stones , but alsy stones .Throughout our work to search for natural urease inhibitor’s compounds from medicinal plants, we now report the isolation of four derivatives of boswellic acids to test their inhibitory activities through Jack bean urease. We employed Jack bean urease instead of bacteria’s one because it was previously found out that the mechanism and kinetics of inhibition for bacteria urease and Jack bean urease are comparable .Molecular docking and simulation studies improve the reliability, accuracy of biological test, and show possible interactions between molecules and their target receptors. So the extracted compounds were subjected to molecular docking for better recognition of their interaction with urease.Jack bean urease , thiourea and all other chemicals used were of analytical grade . All solutions were prepared in MilliQ  water.Boswellia spp.An earlier report claimed that acetyl-keto-β-boswellic acid could not be separated from a mixture of acetyl-β-boswellic acid and acetyl keto-β-boswellic acid by any of the chemical methods, such as ketal formation  or semi-B. carterii. A total of 10 g of oleo-gum resin was grounded. Of this, extraction performed in 100 ml methanol three times. After filtration, the extract was concentrated and solvent was removed. KOH 2% was added to separate the acidic fraction and extraction was performed by ethyl acetate five times. In the next step, the aqueous phase was acidified with HCl 2%, and previous step was repeated. Then the organic phase washed with distilled water to obtain fractions containing BA’s compounds. Liquid chromatography was used to obtain related derivatives in organic fraction. The organic layers were dried, filtered and concentrated under vacuum on a rotary evaporator. Liquid chromatography of 5 g of sample which dissolve in chloroform is performed on 100 g of silica gel 60 (0.063 - 0.2 mm). Solvent partitioning of the crude extracts was used to divide the extract into eight fractions with different polarities. The fractions were combined based on similarity of the chemical composition determined by TLC analysis.In this research, solvent-solvent extraction, thin-layer (TLC) and column chromatography were performed to isolate the main active constituent of 1H and 13C-NMR spectra were recorded on a Bruker Avance 300-MHz NMR spectrophotometer, with internal standard tetramethylsilane (TMS). The structures were further confirmed by mass-spectral analysis and comparing with reported data for all the compounds.Crude fractions 1-5 were concentrated under vacuum on the rotary evaporator. Fractions 6-7 were combined based on similarity of their TLC profile and subjected to further column chromatography and eluted with a methanol/chloroform gradient solvent mixture  to give fractions K, L, M and O. pure compound was identified using spectroscopic analysis which their structures are shown in Figure bc, where c is the concentration of solution (mol/L), b the Length of the UV cell, we can calculate the concentration of initially urease solution in this way. After appropriate dilution, the concentration of enzyme solution adjusted 2 mg/ml.For urease inhibition assays after addition of phosphate buffer pH = 6.8, sonication was performed for 3 min, followed by centrifugation and evaluating absorbance of upper solution in λ = 278 nm which is attributed to the urease. By using following equation A = λThe assay mixture, containing 50 μl (2 mg/ml) of Jack-bean urease and 100 μl of different concentration of test compounds, which were dissolved in ethanol 20% previously, was added to 850 μl of 25mM urea and pre-incubated for 0.5 h in water bath at 37°C. The urease reaction was stopped after 30 min incubation by following procedure.Urease activity was determined by measuring ammonia production using the indophenol method as described by Weatherburn . After pThe absorbance was measured at 625 nm. All experiments were performed in triplicate in a final volume of 1 ml, and thiourea was used as a standard urease inhibitors. Percentage inhibitions were calculated using the formula (100 – (OD sample / OD control) × 100).50) was determined by monitoring the inhibition effect of various concentrations of compounds in the assay. The IC50 values were then calculated using GraphPad Prism 5 software.The concentration that provokes an inhibition halfway between the minimum and maximum response of each compound  n of 2°A .In the present study, non-standard protein residues (KCX and CME) and the metal ions were included in the binding site specification. The docking calculations adopted in this work yield favorable orientations of ligands in a receptor. The graphical user interface ADT was employed to set up the enzymes: all hydrogen calculated and nonpolar hydrogens were merged to carbons. For determination of pharmacophore, Ligandscout 3.03 program was used .6 energy evaluations were performed. After clustering analysis, conformation with the most favorable binding energy was selected.Four potent inhibitors have sketched by Marvin sketch applet . Then adding polar hydrogens and rotatable bonds were done with Openbabel and AutodockTools, respectively ,25. DockB. carterii which has been numbered as medicinal plant and had been used in stomach and colon disorders [Plants are the important source of new drug developments. Current study presents an investigation on urease inhibition of four isolated compounds from isorders ,27, on t50 values were depicted in Table The aim of the present study is to confirm the possible anti-ulcer activity of the olibanum through inhibition of urease enzyme to support for its use as gastrointestinal agents in traditional medicine. Isolated compounds Figure  were scr4 was the only weakly active (IC50 = 85.23 ± 0.06 μM) and compound 1 (IC50 = 6.27 ± 0.03 μM) was found to be the most potent compound, compared to the standard inhibitor . In this series, compounds 2 and 3  are exhibited remarkable inhibition toward urease.Out of four BA’s tested for their urease inhibitory activity, compound 1 is depicted in Figure It could be seen that the dehydro-substituted compound has stronger activity against urease than the hydroxy or keto-substituted ones. To investigate the binding effects between extracted BA’s and the Jack bean urease the molecular docking study was performed. The 3D binding model of compound 1. Moreover, the compound may form hydrophobic interactions with Ala 150, Leu 457, Try 474 and Try 475 of urease  compared with other extracted compounds, which may also explain the excellent inhibitory activity of it. As a comparison, a molecular docking study on the other compounds was also carried out, as it shown in Figures 1, 2 and 4 with 60.5, 56.2 and 39.1%, respectively, compared to the structurally related compound 3 (37.4%) may either be due to the presence of methyl group and hydrophobic interaction of them with adjacent amino acids. This interaction becomes lower in the case of compound 3 because of steric factors. This phenomenon is depicted clearly in Figure As it has shown, the enhanced percentage of urease inhibitory activity of compounds 3, due to hydrogen bonding between oxygen of carboxyl group and Arg 368 and Gln 364, conformational locking happened, which possibly make the approach of the compound difficult towards the enzyme’s binding site and results in a sharp decline in inhibitory potential as compared to compound 4 (Figure As it has shown in Figure 1, two kinds of hydrogen bonds in compound 2 and one kind in compound 4 with respective amino acids in the hydrophobic pocket can explain their percent of inhibition activity order against urease (Table 3, despite the existence of four hydrogen bond’s percent of inhibition decrease dramatically. This observation can be explained by existing steric hindrance, which prevents the compound to interact with the hydrophobic site appropriately.Besides, presence of three kinds of hydrogen bonds formed by the carboxyl group of compound B. carterii has been tested against urease, which may confirm its potential traditional use for stomach problems. Based on the above study, we can preliminarily conclude that the substituted C ring of BA’s may result to decreased inhibition concentration compared with the reference inhibitor thiourea. In addition, hydrophobic interactions between methyl group of compounds and near residues play an important role in the inhibition. This observation can be applied to the development of new antiulcer agents.In this research isolated compounds from There are no other conflicts of interest related to this publication.All authors contributed to the concept and design, making and analysis of data, drafting, revising and final approval. MA is responsible for the study registration, financial and administrative support. SG, FN & ZBO are responsible for biological assays. RB, SaG & BY were involved for docking studies. RH & ShR were responsible for preparation of extract and isolation of compounds 1-4. SG, RB& MA were participated in data assembly and analysis, interpretation and manuscript writing. All authors read and approved the final manuscript.


Brazilian Amerindians have experienced a drastic population decrease in the past 500 years. Indeed, many native groups from eastern Brazil have vanished. However, their mitochondrial mtDNA haplotypes, still persist in Brazilians, at least 50 million of whom carry Amerindian mitochondrial lineages. Our objective was to test whether, by analyzing extant rural populations from regions anciently occupied by specific Amerindian groups, we could identify potentially authentic mitochondrial lineages, a strategy we have named 'homopatric targeting'.We studied 173 individuals from Queixadinha, a small village located in a territory previously occupied by the now extinct Botocudo Amerindian nation. Pedigree analysis revealed 74 unrelated matrilineages, which were screened for Amerindian mtDNA lineages by restriction fragment length polymorphism. A cosmopolitan control group was composed of 100 individuals from surrounding cities. All Amerindian lineages identified had their hypervariable segment HVSI sequenced, yielding 13 Amerindian haplotypes in Queixadinha, nine of which were not present in available databanks or in the literature. Among these haplotypes, there was a significant excess of haplogroup C (70%) and absence of haplogroup A lineages, which were the most common in the control group. The novelty of the haplotypes and the excess of the C haplogroup suggested that we might indeed have identified Botocudo lineages. To validate our strategy, we studied teeth extracted from 14 ancient skulls of Botocudo Amerindians from the collection of the National Museum of Rio de Janeiro. We recovered mtDNA sequences from all the teeth, identifying only six different haplotypes (a low haplotypic diversity of 0.8352 ± 0.0617), one of which was present among the lineages observed in the extant individuals studied.These findings validate the technique of homopatric targeting as a useful new strategy to study the peopling and colonization of the New World, especially when direct analysis of genetic material is not possible. When Europeans arrived in Brazil in 1500, they found more than two million Amerindians .The Descent of Man. The names this tribe used themselves were 'Gren' or 'Kren'; the name 'Botocudos'  was given to them by the Portuguese because they inserted into their lower lips and earlobes wooden disks similar to the corks of wine casks (botoques) used in Portugal. The Botocudos belonged to the Macro-Je linguistic group, and inhabited adjacent regions in the states of Minas Gerais, Bahia and Espírito Santo, in southeast Brazil [One of the best known eastern Brazilian Amerindian nations was the Botocudos, a hunting-gatherer group that is mentioned in Darwin's t Brazil . We do nIn 1808, the Portuguese royal court moved to Brazil, fleeing from the Napoleonic invasion of the Iberian Peninsula. Soon afterwards, Prince Regent João, acting on reports about the Botocudo savagery and their refusal to subject to European rule, declared war on their nation, a policy that eventually led to their virtual extinction . Nowadayet al. [In 2000, Alves-Silva et al.  reportedhomos) meaning 'the same' and πατρίδα (patrida) meaning 'fatherland'.We reasoned that the chances of success would be much improved if we studied extant populations that have always lived in small regions once inhabited by specific Amerindian nations. We have called this strategy 'homopatric targeting', a neologism made up of the Greek roots ομοιος  in the Vale do Jequitinhonha region of the state of Minas Gerais in Brazil, occupied before the 19th Century by the Botocudo Amerindian nation. Three-generation pedigrees were obtained, and individuals who belonged to the same maternal lineages were removed from the study. Thus, of the original 173 samples, we included 74 matrilineally unrelated individuals in the study. We investigated their mitochondrial ancestry by standard restriction fragment length polymorphism (RFLP) for haplogroups A, B, C, D, × and M, as described previously -10. In tLikewise, of the 100 cosmopolitan unrelated samples from northeastern Minas Gerais (MGNE), we identified 24 (24%) as belonging to Amerindian haplogroups: nine of haplogroup A (37.5%), seven of haplogroup B (29.2%) and four each of haplogroups C and D (16.7% each) (data not shown). Again, no matrilineage belonged to haplogroup M or Amerindian X.et al. [2 = 15.8; P < 0.001).The frequencies of 27.0% of Amerindian lineages in the rural population (QUEIX) and 24.0% in the cosmopolitan population (MGNE) are commensurate with the findings of Alves-Silva et al.  in Braziet al. . The difIn the 20 mtDNA HVSI sequences obtained from the QUEIX samples, we identified 13 different haplotypes, the sequence of which (318 bp from 16045 to 16362) is shown in Table The B, C and D founding haplogroups ,11 were The haplotype diversity of the Amerindian QUEIX samples was 0.9263 ± 0.0431, lower than that of the cosmopolitan Amerindian lineages in MGNE, which was 0.9746 ± 0.020, similar to that of the Amerindian lineages of the white population of Brazil (0.9780 ± 0.0083) . AdditioWe could not find any instance of haplogroup B lineages MG18, MG22, MG 23 or MG24 in available databases or in the literature  by themselves cannot generate a precise haplogroup classification, owing to the constant occurrence of recurring mutations, we established a strategy for confirmation of the Amerindian origin of the samples selected as possible genetic signatures for the extinct indigenous populations previously inhabiting the region. Studies on complete mtDNA sequences have allowed the identification of polymorphisms present at coding regions that can differentiate between the ancestral Asiatic haplogroups and the Amerindian descendents ,16,17. TWe analysed teeth extracted from 14 skulls in the collection of the Museu Nacional do Rio de Janeiro, which are thought to be remains of Botocudo Amerindians Table . By sequThe 14 HVSI sequences recovered from the ancient teeth code for six haplotypes defined by 15 different polymorphic sites. Gene diversity was calculated as only 0.8352 ± 0.0617 (even lower than the QUEIX samples), and nucleotide diversity was estimated at 0.014687 ± 0.008591.A haplotype network of the sequences found in the QUEIX and the Botocudo teeth samples is shown in Figure The founding haplotype of the Native American haplogroup C was present in three skulls, being coded as Bot01  is characterized by the transition in nt16129 (G→A) in addition to the aforementioned specific markers for haplogroup C. This lineage is shared with individuals of other South American populations, such as the Guahibo from Venezuela , and theHaplotype Bot03 Table  was seenThe Brazilian population is the product of genetic admixture between three ancestral groups: native Amerindians, European colonizers and African slaves. We have previously demonstrated that the vast majority of the chromosome Y lineages found in the contemporaneous Brazilian population, independent of their geographical region, is of European origin (98%) . By contExtrapolating from this to the current population of Brazil, which is approximately 190 million inhabitants, we would expect the existence of roughly 60 million Brazilians carrying Amerindian mtDNA. If we could study this DNA and ascertain the Amerindian group from which the individuals originated, it might be possible to reconstitute the mtDNA haplotype profile of many extinct original native populations. Our strategy, which we propose to call homopatric targeting, is to concentrate the searches in extant populations that have always lived in small regions once inhabited by specific Amerindian nations. In this way, we could explore the mtDNA lineages of population groups that no longer exist.The Botocudos present interesting anthropometric craniometric characteristics that distinguish them from the vast majority of other Amerindians, and suggest that they might conceivably be related to the Paleoindians from the Lagoa Santa region in Brazil . We studWe found 13 different Amerindian mtDNA haplotypes in samples from Queixadinha, nine of which could not be found in available databases or in the literature after extensive searches  found in Botocudos. Likewise, we suggest that the absence of Amerindian haplogroup A in both the Queixadinha and the Botocudo samples is related, probably also because of genetic drift. By contrast, the absence of Amerindian D lineages in the Botocudo sample gene pool was not unexpected, because this haplogroup represents only 10% of the Amerindian matrilineages in QUEIX.The analysis of the distribution of the ancient Botocudos HVSI haplotypes among 5,133 Amerindian haplotypes (beyond the public mtDNA sequences databases) showed that, except for one individual from Zona da Mata, the Bot04 haplotype is exclusively present in the QUEIX population. This result is sufficient to validate our 'homopatric targeting' strategy.In conclusion, our success in using the present-day population to retrieve the genetic lineages of peoples who are now extinct opens up an important pathway towards the reconstitution of our history, especially in cases in which direct analysis of the specific genetic material has become impossible.Consent was obtained from all participants, and all DNA analyses were performed anonymously. The study was approved by the local ethics committees of all the institutions involved in sample collection.The population studied has been described previously , and incHaeIII; haplogroup C, -13259, HincII; haplogroup D, -5176, AluI and haplogroup X, -1715, DdeI. Haplogroup B was identified using the 9 bp polymorphic deletion . All PCR amplifications and digestions were carried out according to previously described protocols [Five amplified segments in the mtDNA coding region were analyzed by RFLP tests to type haplogroup-specific sites as follows: haplogroup A, + 663, The nucleotide sequences of the HVSI and II of the control region of the mitochondrial DNA were determined for all individuals who had been typed as belonging to the Amerindian haplogroups A, B, C, D and X, and for those that possibly belonged to haplogroup M. The method used was direct sequencing from PCR products; both strands of each sample were sequenced and analyzed separately. Subsequently, the sequences were compared with the reference sequence of human mitochondrial DNA ,29, and The initial amplifications via PCR were performed using two pairs of specific primers Table  The PCR Two sequencing methods were used, using two different sequencers. For the reactions using the Automated laser fluorescence sequencer , amplified segments were purified  and around 300-400 ng of sample were used in the sequencing reactions, with a commercial fluorescence label kit . In sequencing reactions, fluorescein-labeled primers were use , around 40 to 50 ng of PCR product were mixed with 10 μM of primer  and 4 μl of reagent from a , thus making a total volume of 10 μl. The mixture underwent PCR, and the sequencing products were purified.et al. [To avoid the presence of phantom mutations generated artificially in the sequencing process, which might cause erroneous evolutionary interpretations, we followed the strategies described by Bandelt et al. . In addiBased on the results obtained from the phylogenetic analyses of the Amerindian sequences, some haplotypes were selected as possible genetic signatures of indigenous populations of the region. To verify the presence of recently described polymorphisms encountered in the coding region of mtDNA that can better distinguish between Asian and Amerindian haplogroups ,16,17, wet al. [In total, 13 polymorphisms were analyzed: three from haplogroup A, six from B, two from C and two from D, -irradiated water. Each tooth was ground with a mortar and pestle until a fine-grained powder was obtained. Samples (500 mg) of the powder were transferred into sterile 15 ml tubes, and the DNA was extracted as described previously . BrieflyThe mtDNA analyses were performed by DNA sequencing of HVSI of the control region and specific sites on the coding region (RFLP) of mtDNA. For these analyses, two primer pairs were designed to amplify overlapping fragments that divided the region in two smaller amplicons: fragment 1 (nucleotides 15989 to16251) and fragment 2 (16190 to 16410). For some samples, it was necessary to divide fragment 2 into two smaller amplicons (16190 to16322 and 16268 to 16410)  in a buffer (20 mM Tris-HCl pH8.4 and 50 mM KCl). The cycling parameters were: initial denaturation at 94°C for 4 min, followed by 44 cycles at 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds, with a final extension step at 72°C for 10 minutes. To confirm amplification, the PCR products (3 μl) were separated in 6% polyacrylamide gels (PAGE), stained with silver salts and precipitated with lysophosphatidic acid (LPA) and polyethylene glycol (PEG)8000.PCR conditions were the same for all reactions. A sample (3 μl) of the aliquot containing the ancient DNA (not quantified) was amplified in 20 μl reaction volume containing 0.2 mM of each dNTP, 0.3 μM of each primer, 2.5 mM MgClet al. [Sequencing reactions were performed on each strand, using the same primers as for PCR amplification. The sequencing reaction products were cleaned up and then run on an automated sequencer , using the same conditions as described above for the modern samples. Sequencing was performed in both directions (forward and reverse). The sequence files generated were analyzed together with the respective chromatograms using Bioedit v.7.0.9 software. . To monitor for the possible presence of phantom mutations generated artificially in the sequencing process, we followed the strategies described by Bandelt et al. .Haelll; haplogroup B, 9 bp deletion (COII/tRNALys); haplogroup C, + 13262, Alul and haplogroup D, - 5176, Alul. The PCR conditions were as described above. The PCR amplification product, (3 μl) was digested for 2 hours at 37°C with 1 U of the appropriated restriction enzyme. The digestion products were separated in 6% PAGE gels, which were stained with silver for identification of the presence or absence of the restriction sites that characterize haplogroups A, C or D and of the 9 bp deletion that defines haplogroup B.Four primer pairs  for 30 minutes before all experiments, and cleaned with a high concentration of sodium hypochlorite. All apparel  were disposable. Laboratory equipment  were sterilized by a long exposure to UV (254 nm). All metallic material and laboratory glassware were sterilized in an oven at 200°C for at least 6 hours. Preparation of ground tooth powder was performed in a separated room from the buffer preparation, DNA extraction procedure and PCR assays. To detect possible contamination by exogenous modern DNA, extraction and amplification blanks were used as negative controls, and all personnel involved, either directly or indirectly in the work were genetically typed (HVSI) and their profiles compared with the results obtained from the ancient teeth samples.http://empop.org, Ambase http://www.lghm.ufpa.br/ambase, mtDB http://www.genpat.uu.se/mtDB/, mitosearch http://www.mitosearch.org, hvrbase++ http://www.hvrbase.org), and with the FBI mtDNA population database http://www2.fbi.gov/hq/lab/fsc/backissu/april2002/miller1.htm. Our database search results were up to date as of July 2010.We manually compared our sequences with 5,133 HVSI mtDNA sequences from North, Central and South America (see Additional file To compare and understand the relationship between the sequences found in both Queixadinha and Botocudo populations, we also performed a median-joining network analysis using the software Network 4.502 .2 tests. The Raymond and Rousset test of population differentiation and estimates of haplotype and nucleotide diversity were calculated with the program Arlequin version 2.000 [The program CLUMP  was usedon 2.000 .The authors declare that they have no competing interests.VFG and FP carried out molecular genetic studies, participated in the data analysis, and drafted the manuscript. HGD carried out molecular genetic studies. CRC and HPS identified the skulls in the museum collection, and extracted and provided the teeth for DNA analyses. SDJP conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.Supplementary Table 1. Exact test of population differentiationClick here for fileSupplementary Table 2. Native Americans populations to which Queixadinha and Botocudos sequences were compared.Click here for fileSupplementary Table 3. Results of database searches for the 13 haplotypes found in Queixadinha on July 2010.Click here for fileSupplementary Table 4. Minisequencing results of selected Amerindian haplotypes.Click here for fileSupplementary Table 5. Sequencing primers used to amplify Botocudo DNA samples in this study, with annealing temperatures.Click here for fileSupplementary Table 6. Restriction fragment length polymorphism primers used to ancient DNA samples in this study, with annealing temperatures.Click here for file


A former Republican congressman from South Carolina has started an institution to promote conservative approaches to mitigating climate change and achieving energy security for the United States. According to Bob Inglis, whose Energy and Enterprise Initiative is housed within George Mason University, market distortions lead Americans to use far more fossil fuels than they would if they paid the full cost of that fuel. If fuel producers are held fully accountable for the health, productivity, and environmental costs of their products, he says, free enterprise will naturally give rise to the cleanest, most sustainable fuels.A committee of the National Research Council recently estimated that burning fossil fuels for electricity, heat, and vehicle operation cost the United States more than $120 billion in 2005. These estimates were based on damages to human health, grain crop and timber yields, building materials, and recreation by criteria air pollutants including particulate matter, sulfur dioxide, and nitrogen oxides.The majority of this cost was attributed to premature deaths resulting from exposure to these pollutants. The estimates omitted a number of factors that could further increase costs, including damages to ecosystem services, environmental damages from coal mining, and military costs of protecting fuel imports.Alexander Bozmoski, director of strategy and operations for the Energy and Enterprise Initiative, says society bears those costs not through payments at the gasoline pump or on their power bill, but instead through greater health insurance premiums, lost income, and increased taxes for defense spending.In 2009 the United States provided $5.3 billion in subsidies for fossil fuel producers and $4.9 billion in subsidies for fossil fuel consumers. Of the total $10.2 billion in producer and consumer subsidies, more than half went toward natural gas, one-third went toward petroleum, and about one-tenth went toward coal. These figures do not include costs for fossil energy research and development or for maintenance of the Strategic Petroleum Reserve or the Northeast Home Heating Oil Reserve, which were established as buffers against major supply disruptions.Hidden Costs of Energy: Unpriced Consequences of Energy Production and Use,According to the National Research Council report $62 billion in damage was attributed to 406 coal-fired electric power plants. Damages averaged $156 million per plant and 3.2¢ per kilowatt-hour of electricity produced.$740 million in damage was attributed to 498 natural gas electric power plants. Damages averaged $1.49 million per plant and 0.16¢ per kilowatt-hour of electricity produced.$1.4 billion in damage was attributed to burning natural gas for heating . Median estimated damages were about 11¢ per thousand cubic feet of heated space.$56 billion in damage was attributed to motor vehicles. Damages averaged 1.2–1.7¢ per mile traveled depending on vehicle technology and fuel type.Fossil fuel subsidies come in a variety of forms. Some of the largest subsidies on the production side include write-offs for properties used to process qualified petroleum fuels and costs associated with exploration and development for oil and gas wells. Consumer subsidies include the Low-Income Energy Assistance Program to help needy residents pay their energy bills, fuel tax exemptions for farmers (one of the biggest single subsidies at nearly $1 billion in 2009), and a tax credit for investing in power generation projects that use “clean coal” technologies.Guardian, Fatih Birol, the chief economist at the International Energy Agency, was quoted as saying, “Energy is significantly underpriced in many parts of the world, leading to wasteful consumption, price volatility, and fuel smuggling. It’s also undermining the competitiveness of renewables.” The United States is not alone in subsidizing these fuels; worldwide, fossil fuel subsidies amounted to $409 billion in 2010.According to Birol, phasing out subsidies could, in the course of discouraging waste and encouraging development of clean fuels, reduce emissions by about half the amount necessary to prevent warming beyond 2°C over preindustrial levels.Inglis says the initiative’s primary modus operandi will be educational: to provide information to conservatives, particularly young conservatives, by speaking at colleges and universities, convening forums, and sponsoring policy papers. The initiative website is clear that its founders “don’t subscribe to apocalyptic visions of climate change.” They do believe, however, that “the best science available indicates that America faces substantial risks in a changing climate [and] that conservative energy policy should mitigate those risks.”


Infection rates may be higher during pregnancy and lactation. Pteropus vampyrus and P. hypomelanus bats in Peninsular Malaysia. Neutralizing antibodies against NiV were detected at most locations surveyed. We observed a consistently higher NiV risk (odds ratio 3.9) and seroprevalence (32.8%) for P. vampyrus than P. hypomelanus (11.1%) bats. A 3-year longitudinal study of P. hypomelanus bats indicated nonseasonal temporal variation in seroprevalence, evidence for viral circulation within the study period, and an overall NiV seroprevalence of 9.8%. The seroprevalence fluctuated over the study duration between 1% and 20% and generally decreased during 2004–2006. Adult bats, particularly pregnant, with dependent pup and lactating bats, had a higher prevalence of NiV antibodies than juveniles. Antibodies in juveniles 6 months–2 years of age suggested viral circulation within the study period.We conducted cross-sectional and longitudinal studies to determine the distribution of and risk factors for seropositivity to Nipah virus (NiV) among  Pteropus) were the most likely reservoir host of the virus, and evidence of NiV in these bats had been consistently found in populations across southern Asia (–Nipah virus (NiV) disease emerged in Peninsular Malaysia during September 1998–April 1999 and resulted in 105 human deaths (≈40%) and compulsory culling of 1.1 million pigs (–Pteropus hypomelanus (variable flying fox) and P. vampyrus (large flying fox) bats, as reservoir hosts for NiV  and western (n = 4) coasts of the peninsula ; Terengganu ; Pahang ; and Johor (Benut and Kesang)  . We detePermission for the study was granted by PERHILITAN, and Institutional Animal Care and Use Committee approval was obtained from the Wildlife Trust Institutional Animal Care and Use Committee . A special permit to trap and humanely kill the bats was obtained from PERHILITAN. Bats were captured nonrandomly by using 2 methods: opportunistic sampling of hunted bats (hunters were not solicited or incentivized to hunt bats for this study) and mist nets.Hunted bats were sampled at the site of hunting, and bats were attributed to the location of the nearest known roost. Blood samples were collected by cardiac puncture, and the kidneys were harvested. The blood, urine, and kidney samples were processed as described , juvenile , or pup (dependent and attached to its dam)  between species were tested by using the Mann-Whitney U test. Comparison of the seroprevalence rates between the 2 pteropid species was performed by using the χP. hypomelanus bats in Pulau Tioman in which 50 bats from the same colony were captured by using mist nets and sampled approximately every 6–8 weeks during January 2004–October 2006. Monsoonal rains prevented access to the island of Pulau Tioman during December–February, which extended the interval between some sampling points. Captured bats were tagged by using a thumb band or implantable microchip  and unique identification numbers to ensure that sampling was not repeated on the same bat. The bats were anesthetized by using ketamine and xylazine  and reproductive maturity  and renamed the variable the sexual and reproductive maturity factor. The logistic regression analysis includes the sexual and reproductive maturity categories and sampling time for the bats. All hypothesis testing was 2-sided, with α = 0.05, and was performed by using SPSS version 19 .We investigated associations between serostatus (positive or negative) and each of the hypothesized risk factors  using the χPlasma-neutralizing antibodies against NiV were measured by using the serum neutralization test (SNT)  were seropositive. NiV antibodies were detected in 13 (13.8%) of 94 bats from islands off the east coast  of Peninsular Malaysia. Titers ranged from <8 to 256 (median <8); among seropositive bats, the median titer was 32. Samples from Pulau Pangkor (n = 24) were negative for NiV-neutralizing antibodies. All culture and PCR results were negative. Pups were not captured, but among juvenile and adult bats, 0% (0/31) and 11.1% (13/73), respectively, were seropositive . UnivariP.vampyrus and P. hypomelanus bats was significant  and P. vampyrus  bats was significant , and risk for a seropositive reaction to NiV was 3.9× higher for P. vampyrus bats than for P. hypomelanus bats.The antibody titer difference between hypomelanus bat samples had negative culture and PCR results. The overall NiV seroprevalence for P. hypomelanus bats from Pulau Tioman was 9.8%. Differences in seroprevalence between sampling times were significant (p<0.001). The highest seroprevalence rate was in 2004, which then waned in 2005 and 2006  was significantly higher than in juveniles , and adults were ≈4× more likely to be seropositive (odds ratio 3.9). Seroprevalence was not significantly different between male (11.1%) and female bats . However, when data for female bats were examined by their reproductive categories, a significant difference (p<0.001) was observed. Bats that were pregnant, carrying a pup, and lactating were 3.9×, 4.8×, and 3.0× more likely to be seropositive than adult female bats that did not show these features .Although our data were not sufficiently powered to detect significant differences among various sex and reproductive maturity categories (sample size was not large enough within each category level), when we controlled for the effect of time, the likelihood of seropositivity to NiV among pregnant, carrying, and nursing females remained higher than other female bats, and the highest risk was observed among those females carrying pups. Juvenile female bats were the least likely to have detectable NiV antibodies compared with other bats. Most seropositive cases appeared to cluster in 2004 and decreased toward the end of the study .P. hypomelanus bats to smaller (<20), more disparate cohorts that roosted higher in the trees and finally relocation to another site on Pulau Tioman, ≈6 km from the previous site. We believe roost size affected seroprevalence rates and thus reduced risks for seropositivity, as observed toward the end of the study.Anthropic disturbance to roosting sites during our study resulted in dispersal of ,P. vampyrus and P. hypomelanus bats were 32.8% and 11.1%, respectively, which differed from results of a study Yob et al.  and, although unpredictable, seroprevalence generally waned in the final 2 years (,–,,,,The longitudinal study of  2 years . We did ,,Sex was not a risk factor for NiV exposure, which is consistent with results of other studies (Pteropus spp. bats are known to carry their pups for <3 months (<4 months after the pup is independent. Pregnancy and lactation are the most metabolically demanding periods of mammalian life (Among mammals, P. hypomelanus bats reach sexual maturity at ≈12 months of age (P. vampyrus at 24 months of age (P. vampyrus bats demonstrated horizontal transmission of NiV from an adult to juvenile bats within the same colony after a brief shedding episode (P. hypomelanus bats with antibodies to NiV during the longitudinal study support the possibility that virus had been circulating in this population within the lifetime of the juvenile, assuming that the juvenile bats were old enough to have lost maternal antibodies.Our study has several limitations, including sampling bias, which results from the nonrandom sampling technique used. Sample numbers were often suboptimal because of extreme difficulty in catching pteropid bats. In addition, there are major challenges associated with interpretation of serologic data in wildlife populations. Although we detected differences in the prevalence of neutralizing antibodies between species and increased risk among bats in different reproductive categories, there is still little known about the timing of actual infection or the duration of NiV antibodies in bats. One technique used to overcome this difficulty was to examine age-stratified serologic data, which has been used in similar epidemiologic studies (Pteropus bats at almost all locations we studied in Malaysia, coupled with isolation of NiV from these bats by our group and another group, strengthened the theory that NiV is enzootic in both Pteropus bat species, and that these species serve as the natural reservoir for NiV in Malaysia. Longitudinal surveys of P. hypomelanus bats suggest size and colony density may cause lower seroprevalence, and female bats that were pregnant, carrying a pup, and lactating generally had higher rates of NiV exposure than males or nonpregnant adult females, which lend further support to the hypothesis that infection rates may be higher during periods of pregnancy and lactation. Further study of NiV infection and shedding rates in pteropid bats will help elucidate seasonal and intracolonial viral dynamics.Our finding of neutralizing antibodies to NiV in both species of


P = 0.0271). For each orchard, a 16S rRNA gene clone library was constructed from leaf samples. Proteobacteria dominated the bacterial communities at all orchards, accounting for 71 of 104 OTUs (determined at 97% sequence similarity) and 93% of all sequences. The genera Massilia, Methylobacterium, Pantoea, Pseudomonas, and Sphingomonas were shared across all sites. Shannon and Simpson’s diversity indices and Pielou’s evenness index were similar among orchards regardless of streptomycin use. Analysis of Similarity (ANOSIM) indicated that long-term streptomycin treatment did not account for the observed variability in community structure among orchards . Other variables, including time of summer, temperature and time at sampling, and relative distance of the orchards from each other, also had no significant effect on bacterial community structure. We conclude that factors other than streptomycin exposure drive both the proportion of streptomycin-resistant bacteria and phylogenetic makeup of bacterial communities in the apple phyllosphere in middle to late summer.We studied the effect of many years of streptomycin use in apple orchards on the proportion of phyllosphere bacteria resistant to streptomycin and bacterial community structure. Leaf samples were collected during early July through early September from four orchards that had been sprayed with streptomycin during spring of most years for at least 10 years and four orchards that had not been sprayed. The percentage of cultured phyllosphere bacteria resistant to streptomycin at non-sprayed orchards (mean of 65%) was greater than at sprayed orchards (mean of 50%) ( Erwinia amylovora, the fire blight pathogen, have emerged. In some cases strA or the gene pair strA-strB, which encode aminoglycoside phosphotransferases, are carried on transposon Tn53935393 have been described in bacteria inhabiting diverse ecological niches, including soil, plants, animals and humans http://www.ams.usda.gov/AMSv1.0/getfile?dDocName=STELPRDC5091714).The use of antibiotics in agriculture is controversial because of the possibility of selection for antibiotic resistant bacteria on farms and subsequent horizontal transfer of resistance genes to clinically important bacteria strA and strB was greater in treated soil. However, in a survey of soils from diverse habitats in Europe strA, strB, and genes encoding aminoglycoside nucleotidyltransferases was not greater in streptomycin-treated soil from an apple orchard in Germany compared to non-treated soil from the same location or soils from other polluted or pristine sources. Likewise, Duffy et al. The heightened regulations and general concerns surrounding the use of streptomycin and other antibiotics in plant agriculture have been an impetus behind studies addressing the impact of antibiotics on bacterial communities in cropping systems. Rodríguez et al. In the current study we test the hypothesis that the use of streptomycin in commercial apple orchards alters bacterial community structure on apple leaves. We compared phyllosphere bacterial communities in four commercial apple orchards where streptomycin had been used for several years with communities from four orchards where streptomycin had not been used. Our goal was to assess the long-term effects of streptomycin use, rather than the transient disruption of communities that might be expected in the spring immediately after spraying. To this end, we sampled leaves in middle to late summer, constructed 16S rRNA clone libraries from phyllosphere bacteria, identified operational taxonomic units (OTUs) from the cloned sequences, and then used diversity indices to describe communities. Because certain bacterial taxa have previously been identified as likely reservoirs of antibiotic-resistance genes in the phyllosphere No specific permits were required for the described field studies. At each study site, the landowner granted us permission to collect apple leaves. The studies did not involve endangered or protected species.Apple leaves were collected in southern Wisconsin, USA, at eight commercial orchards during early July through early September, 2009 . At all At each orchard, 50 fully-expanded, healthy-appearing leaves from each of five or six arbitrarily selected trees  of the cultivar ‘Gala’ were collected at least 2 days after the last rainfall to minimize the effect of rain on bacterial community structure −1) to inhibit fungal growth, as well as 0.1× TSA amended with cycloheximide (100 µg ml−1) and streptomycin sulfate (50 µg ml−1) to select for streptomycin resistant bacteria. After incubation for 7 days at room temperature and with ambient light, bacterial colonies were counted and the proportion resistant to streptomycin was calculated. A two-tailed Student’s t-test was calculated on the mean percent streptomycin resistance between sprayed and non-sprayed orchards to determine the long-term effect of streptomycin use on bacterial populations. To examine the shorter term effect of streptomycin on the leaf bacterial community, colony counts from the 10 individual trees from the orchards that were sprayed in the sampling year  were combined into a single dataset, while colony counts from the 10 trees from orchards that were not sprayed in the sampling year but had a history of streptomycin exposure  were combined. A two-tailed Student’s t-test was then calculated, with N = 10 for sprayed in sampling year and N = 10 for not sprayed in sampling year.An aliquot of the combined leaf extracts from each tree was serially diluted and then plated onto 0.1× tryptic soy agar (TSA) amended with cycloheximide  and M13r(-27) primers according to Bräuer et al. Bacterial cells in leaf extracts from each tree were pelleted by centrifugation at 12,857×g at 4°C for 15 min. Pellets were then resuspended in residual supernatant and centrifuged at 16,870×g for 5 min in a microcentrifuge to further concentrate the bacteria. Genomic DNA was extracted from each individual cell pellet using the FastDNA spin kit . PCR amplification of 16S rRNA genes was performed in triplicate using primers 27f and 1492r as described in Bräuer et al. E. coli base positions 103 to 920 were also omitted from further study. The remaining sequences were aligned using the SILVA aligner (25) and imported into ARB The sequences were screened against chloroplast and other non-ribosomal DNA using BLASTn and the Ribosomal Database Project Classifier Mothur The 16S rRNA gene sequences obtained in this study were deposited into the NCBI GenBank database under accession numbers JQ046416 to JQ048217.P = 0.0271). Among sprayed orchards, the observation of higher CFU counts at orchards sprayed in 2009, the year of sampling (Ep and SR), versus those not sprayed in 2009 (BFF and LP), prompted an analysis of shorter-term effects of streptomycin on total CFU. The mean log10 CFU/g leaf tissue was 5.58 and 6.29 for sprayed and non-sprayed orchards, respectively. A two-tailed Student’s t-test indicated that the difference in total CFU was highly significant (P<0.001).At sprayed orchards 43% to 59% (mean of 50%) of the total culturable bacteria were resistant to streptomycin, whereas at non-sprayed orchards 57% to 72% (mean of 65%) of the total culturable bacteria were resistant to streptomycin . A two-tMassilia, Methylobacterium, Pantoea, Pseudomonas, and Sphingomonas, were shared across all sites , Pseudomonas (orchards SR and DC), or Massilia (BW)  and 37% (range 6 to 64%), respectively (Pseudomonas in sprayed and non-sprayed orchards was 23% (range 10 to 37%) and 17% (range 5 to 38%), respectively (Sphingomonas (P = 0.7470) or Pseudomonas (P = 0.5165) at orchards sprayed with streptomycin compared to non-sprayed orchards. A total of 3199 16S rRNA gene clone sequences were obtained for the eight-orchard study. After excluding non-ribosomal DNA (notably chloroplast DNA), low quality or short sequences, and chimeras, 1802 sequences remained for analyses, with a range of 169 to 335 sequences per orchard ; Fig. 1.ll sites . At indilia (BW) . Becauseectively . The perectively . Two-taiP = 0.293 for Shannon; P = 0.225 for Pielou’s), whereas the Simpson’s index indicated somewhat greater diversity (P = 0.083) for communities from sprayed orchards compared to non-sprayed orchards.Rarefaction curves indicated that more OTUs would have been obtained with additional sequencing . ShannonP = 0.655), nor an effect of not spraying streptomycin during the sampling year regardless of long-term treatment . Although sampling of the eight orchards extended from early July to early September, the effect of sampling time did not explain the variability in community structure . The time of day of sampling, which ranged from 0900 to 1100 hr, did not account for community structure , nor did the ambient temperature at the time of sampling, which ranged from 16°C to 29°C . For total log10 CFU, R = 0.625 and P = 0.030 . For log10 CFU on streptomycin-amended TSA, R = 0.056 and P = 0.594. Geographic proximity among the orchards also did not influence the community structure . The same conclusions  were obtained using Bray-Curtis distance, a non-phylogenetic metric of community similarity  represented 40.8% and 22.7%, respectively, of the total variation . ANOSIM milarity .Sphingomonas from the dataset did not reveal any effects of streptomycin. The bacterial communities at orchards Ep, DC, and SR clustered together, while orchards EL and GPS became more distant from LP and BFF on a PCoA plot  for the effect of spraying during the spring of the sampling year on bacterial communities in mid to late summer.Recalculating the weighted Unifrac after removing CoA plot . Closer er sites . Recalcuommunity . Orchardbacillus . Also, sorchards . If orchSphingomonas and Pseudomonas in non-exposed orchards, since these genera are known to have a high level of stable, intrinsic or acquired resistance to streptomycin regardless of exposure Sphingomonas and Pseudomonas based on their abundance in clone libraries. The validity of this approach is supported by the finding that the relative abundance of each of these genera was similar among cultured isolates and 16S rRNA gene clones derived from the same field samples Sphingomonas and Pseudomonas sequences in clone libraries was similar between the sprayed and non-sprayed orchards, making it unlikely that those genera accounted for the difference in the percentage of streptomycin-resistant colonies. We previously found a greater abundance of Actinomycetales, specifically the genera Curtobacterium and Frigoribacterium, in the apple phyllosphere by culturing on TSA than by cloning 16S rRNA genes The results indicate that the routine springtime use of streptomycin in apple orchards for the control of fire blight disease does not have long-term effects on the diversity or phylogenetic composition of bacterial communities in the phyllosphere in middle to late summer. However, the proportion of cultured bacteria resistant to streptomycin was lower in orchards with a history of streptomycin exposure than in non-exposed orchards. We hypothesized that this unexpected result might be explained by a chance higher abundance of Despite a lack of long-term effects of streptomycin on bacterial phyllosphere communities, where the antibiotic was applied during spring of the year of sampling, the total bacterial population, but not the proportion of bacteria resistant to streptomycin, was reduced in mid-summer. When site BW, an outlier on PCoA plots due to observable differences in its bacterial community composition, was treated as an outlier in ANOSIM, we found weak evidence that exposure to streptomycin during the year of sampling affected the phylogenetic composition of communities.Pantoea agglomerans (formerly Erwinia herbicola) has been cited as a likely reservoir of mobile streptomycin resistance genes in apple orchards Pantoea was abundant in one non-sprayed and two sprayed orchards, and therefore was not obviously affected by long-term streptomycin use. We had expected a greater abundance of Pantoea and the related enterobacterial genera Erwinia and Enterobacter across all orchards, since these genera are commonly isolated from apple tissues during spring Pantoea dominating in early summer and diminishing by fall Pantoea among orchards.The enterobacterium Previous studies concerning the use of antibiotics in crop production report the proportion of antibiotic resistant bacteria in leaf, flower, or soil samples relative to antibiotic exposure, and some provide information on the presence of resistance genes and/or potential resistance plasmids Our analyses were based on a modest number of sequences (1802 across eight orchards) relative to the thousands of sequences per sample that are routinely obtained through modern high-throughput technology. Indeed, rarefaction curves suggested that further sequencing would have revealed greater richness in our samples. While we cannot dismiss the potential ecological importance of rare taxa that might have been detected through deeper sequencing, the dominance in our dataset of a few taxa common to all sites makes it unlikely that deeper sequencing would have revealed significant differences in bacterial communities at sprayed and non-sprayed orchards, at least when analyzed by methods that take into account sequence abundance .We conclude that long-term streptomycin use does not increase the frequency of resistant bacteria or disrupt bacterial communities on apple leaves. Our findings contribute to a growing body of literature that indicates using streptomycin to control fire blight has low environmental impact Figure S1Weighted Unifrac PCoA plot of the orchard bacterial communities, excluding Sphingomonas species. The streptomycin treated sites were Ep, SR, BFF, and LP. The nontreated sites were DC, EL, GPS, and BW.(DOC)Click here for additional data file.Figure S2Weighted Unifrac PCoA plot of the orchard bacterial communities, excluding Sphingomonas and Pseudomonas species. The streptomycin treated sites were Ep, SR, BFF, and LP. The nontreated sites were DC, EL, GPS, and BW.(DOC)Click here for additional data file.Table S1Phylogenetic characterization and abundance of bacterial 16S rRNA gene sequences in libraries constructed from apple leaves differing in streptomycin treatment.(DOC)Click here for additional data file.Table S2ANOSIM and Mantel values from Bray-Curtis matrix of the bacterial communities at the 100%, 99%, and 97% similarity threshold. The Bonferroni corrected alpha is 0.0056.(DOC)Click here for additional data file.Table S3ANOSIM and Mantel values from weighted Unifrac matrix for the bacterial communities excluding Sphingomonas and Pseudomonas species. The Bonferroni corrected alpha is 0.0056.(DOC)Click here for additional data file.


Follicular stimulating hormone (FSH) is a glycoprotein and widely used for the treatment of infertility; FSH action is mediated by FSH receptor (FSHR), SNPs of which determine the ovarian response. Two polymorphisms of the FSHR gene were identified, which caused a change of threonine (T) to alanine (A) at position 307 and asparagine (N) to serine(S) at position 680. Both polymorphic sites give rise to three discrete variants of the FSHR: TT, TA, and AA for position 307; NN, NS, and SS for position 680.2=0.84).450 Chinese women were recruited in an assisted reproductive technology program from October 2011 to March 2012. FSHR polymorphisms at the positions 307 and 680 were examined by PCR-RFLP. Serum FSH and estradiol level, FSH amount, ovarian response and pregnancy rate were recorded during treatment. The basal FSH levels were higher in AA  and SS  compared to other genotypes respectively; the days for ovulation induction was slightly longer in AA and SS. Women with AA and SS have higher rates of poor response compared to carriers of other genotypes (P<0.05). Furthermore, there is a nearly complete linkage between these two polymorphisms in Chinese women (D’=0.95, rIn Chinese women receiving ART, the subjects with AA and SS genotypes have higher basal FSH levels, and these genotypes are associated with an increased risk of poor response. Our data suggested that the personalized FSH therapy may be applied according to patient’s genetic background in clinical settings. The linkage suggested that the polymorphisms of Thr307Ala and Asn680Ser may be used as TAG-SNP markers for analysis of potential genotyping in ART. In assisted reproductive technology (ART) programs, the response of ovulating women to exogenous follicular stimulating hormone (FSH) is quite variable in individuals. Past clinical results indicated that it is difficult to prognosticate the ovarian response to intense gonadotropin stimulation. Poor ovarian response results in the insufficient stimulation and cycle cancellation . ReverseFSH and FSH receptor (FSHR) play the essential roles in the regulation of steroidogenesis and follicle proliferation in ovary stimulation ,6. The FThe gene of FSHR harbors more than 900 SNPs, two of which at positions of 307 and 680 are located in the exon 10 . These SIn the study of position 680 in women undergoing ovarian stimulation, the results demonstrated that carriers of SS variant had significantly higher basal FSH levels, and required higher doses of exogenous FSH for stimulation ,19. In aHere, we hypothesize that there is an association between FSHR polymorphisms (Thr307Ala and Asn680Ser) and ovarian response in Chinese women. To test this hypothesis, we employed the methods of Polymerase Chain Reaction-restriction polymorphism length fragment (PCR-RFLP) and direct sequencing to analyze the polymorphisms of FSHR gene at position 307 and 680 from 450 patients in Chinese population. The study demonstrated that the polymorphisms at positions 307 and 680 are associated with the ovarian response to FSH. Furthermore, the SNP at position 680 (Asn680Ser) was closely linked to the disequilibrium of Thr307Ala.in vitro fertilization (IVF) at the Reproductive Medicine Department of XiangYa Hospital from October 2011 to March 2012. The reason of infertility has been identified as male causes, tubal factor, or both. Male infertility refers to the inability of a couple to achieve a pregnancy after one year of unprotected sexual intercourse with a fertile female. Tubal infertility includes either tubal occlusion or adhesions. Mix factor infertility includes both male infertility and tubal infertility. Each patient received a full explanation of purpose of the study, and signed the consent form. All patients had body mass index of 16–35 kg/m2 with ages ranged from 20 to 46 years old.The study protocol was approved by the Chinese clinical trial register (ChiCTR-OCH-12002155) and the research ethics committee, xiangya hospital, China. All subjects were women born in southern China. Peripheral blood was obtained from 450 females who are infertile and received Ovulation was induced according to the standard protocol using gonadotrophin-releasing hormone agonist. Ovarian was exposed to an empirical dose of Urofollitropin , depending on serum estradiol levels and ultrasonography in order to reach an optimal ovarian response. Follicular development was monitored by transvaginal sonography. The monitoring started at 3rd or 4th day post stimulation and continued every other day. Sonography was performed daily when the leading follicle exceeded 17-18 mm in diameter. Ovulation was induced by 10,000 IU hCG when at least one follicle attained more than 20mm in diameter. Oocytes were collected by transvaginal ultrasoundguided aspiration 36h after hCG administration.  Basal levels of FSH, luteinizing hormone (LH) and estradiol on day 3 of the menstrual cycle were measured in one of the cycles before ovarian stimulation. In all subjects, the level of gonadotropins, number of follicles, number of retrieved oocytes, clinical pregnancy rate and the related parameters were recorded. The ovarian response was classified to poor (less than five), normal (between five and fourteen) and high (more than fourteen) based on the number of retrieved oocytes .. A volume of 1 ml whole blood was obtained from each subject, and was anticoagulated with EDTA. Genomic DNA was extracted from leukocytes in peripheral blood with the Wizard® Genomic DNA Purification Kit  according to the manufacturers’ manual. For testing FSHR polymorphisms, the PCR reactions were performed in an Eppendorf Personal Cycler Thermocycler  with 5ul DreamTaq TM Green PCR Master Mix , 20 pmols of each primer and 100 ng of DNA in a final volume of 10 μL.A-G transition. Then the PCR product of 364 bp was digested at 37 °C by Bsu36I restriction enzyme  overnight and assayed on 2.8% agarose gel stained with SYBR Safe DNA Gel Stain . DNA sequencing of PCR–RFLP products was used to confirm the results obtained by RFLP.The polymorphism of T307A variant was detected by the nested PCR–RFLP method. Firstly, a 657 bp fragment of FSHR gene was amplified from the genomic DNA by PCR, PCR was ran at an annealing temperature of 58°C with 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds. The primers used in the reaction are upstream 5’TCTGAGCTTCATCCAATTTGCA3’, downstream 5’GGGAAAGAGGGCAGCTGCAA3’. The amplified fragment after the first round of PCR (2 μL) was further amplified by secondary PCR using another set of primers . An endonuclease Bsu36I recognition site was created by the The N680S variant in the exon 10 of FSHR gene was amplified by PCR-RFLP using a set of primers . A 519 bp DNA fragment was amplified with 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 45 seconds. The PCR-RFLP fragment was digested by BsrI  and 2.8% agarose gel electrophoresis with SYBR Safe DNA Gel Stain. DNA sequencing of PCR–RFLP products was used to conﬁrm the results obtained by RFLP.The statistical tests were performed using the SPSS 19 . The observed genotype frequencies in patients compared with the expected frequencies under Hardy–Weinberg equilibrium were evaluated by the Chi-square test. ANOVA was used to analyze the differences in age, BMI and basal hormone levels. ANCOVA was used to analyze the differences in other clinical parameters between the study groups. Chi-square analysis was used to analyze the data of infertility etiology, ovarian response, OHSS and pregnancy rate. The haplotype analysis for FSHR was performed using PHASE 2.1. Linkage disequilibrium between SNPs was estimated using Haploview version 3.2 . P<0.05 We first established a method to analyze the genotypes of FSHR by RFLP assay. For T307A variant, three different patterns of restriction enzyme digestion were revealed . A. A 36No significant differences were observed between the genotype distribution of the polymorphism and that expected from Hardy–Weinberg equilibrium in patients for both SNPs. PCR-RFLP assay was used to analyze 450 samples from subjects undergoing ART program. As shown in 2=0.84). The results were discordant in only 31 patients out of all 450 research subjects. To further analyze the allelic distributions, we examined the frequencies of homozygous and heterozygous individuals for SNPs at 307 and 680 . A. The Clinical and endocrinologic parameters were analyzed based on the polymorphism at position 307  and 680 However, no statistically signiﬁcant difference was observed among three groups in these parameters: age, BMI, infertility etiology, basal estradiol and LH levels, amount of the FSH required for ovulation induction, FSH, estradiol ,LH levels after down regulation, estradiol, LH , progesterone levels, number of antral follicle and follicle (d≥14mm) on the day of hCG administration, preovulatory follicles, and number of retrieved oocytes  and 3 .The ultrasonographic parameters showed a potential association between FSHR polymorphisms and the ovarian response in subjects undergoing ovarian induction. The ovarian response of different FSHR gene polymorphisms for the residues 680 during ovulation stimulation was shown in OHSS occurred in 54 women in the study group of 450 subjects prospectively enrolled in the ART program. Chi-square test revealed that there was no signiﬁcant association between the occurrence of OHSS and the genotypes at either position 307 or 680: Thr307Ala (P=0.254), Asn680Ser (P=0.092). Also, the biochemical pregnancy rates and clinical pregnancy rates were not significantly different among three polymorphisms at positions 307 or 680.It has been previously reported that the response of ovaries to FSH stimulation is variable in ART program, and FSHR polymorphisms of Thr307Ala and Asn680Ser may be related to the response of the patients to FSH stimulation. One study suggested that the effects of the FSHR polymorphism are independent of ethnic background, in spite of lack of the data from Chinese woman population . To veriIt has been reported that subjects with homozygous Ser/Ser have higher basal levels of FSH, and require more exogenous FSH to induce ovulation ,18,19. BPrevious studies suggested that the same dose of FSH resulted in much lower estradiol levels in subjects with homozygous Ser/Ser than in Asn/Asn in German women . In addiIt has previously been reported that the Ser/Ser carriers may have an increased risk of poor outcome during COH . In the Although previous study reported that FSHR polymorphism at position 680 could be used to predict the severity of symptoms of OHSS patients, but could not predict the occurrence of OHSS . We did 2=0.84, n=450). These data, together with others, suggest a use of Thr307Ala and Asn680Ser in FSHR gene as TAG-SNPs for potential genotyping in ART.A possible linkage between Thr307Ala and Asn680Ser has been reported in the infertile women. A complete linkage has been reported between polymorphisms Thr307Ala and Asn680Ser of FSHR gene in Japanese women (n=522) , while oIn all, the results in the present study provided new additions to the understanding of the impact of FSHR SNPs on exogenous FSH induction. Personalized FSH therapy may be used in ovarian stimulation program according to the patient’s genetic background in clinical settings. In ART treatment, genetic testing may help to identify patient’s likely response when given FSH in relation with genetic polymorphisms, and provide tools for the determination of optimizing drug dose to magnify the clinical benefit of ART.