None of the DDGS lots contained 3-ADON or NIV. Plants in OH had the highest levels of DON overall (mean of 9.51 μg g−1), and plants in NY, MI, IN, NE, and WI had mean DON levels >1 and <4 μg g−1. Twenty six percent (36/141) of the DDGS lots contained 1.0 to 5.0 μg g−1 DON, 2% (3/141) contained >5.0 and <10.0 μg g−1 DON, and 3% (4/141) contained >10.0 μg g−1 DON. All DDGS lots contaminated with unacceptable levels of DON evaded detection prior to their commercial distribution and were likely sold as feed products.Fuel ethanol co-products known as distillers’ dried grains with solubles (DDGS) are a significant source of energy, protein, and phosphorous in animal feed. Fuel ethanol production may concentrate mycotoxins present in corn into DDGS. One hundred and forty one corn DDGS lots collected in 2011 from 78 ethanol plants located in 12 states were screened for the mycotoxins deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), nivalenol (NIV), and zearalenone (ZON). DON ranged from <0.50 to 14.62 μg g Zea mays L.) as the primary feedstock [Fuel ethanol co-products known as distillers’ dried grains with solubles (DDGS) contain high levels of protein, fiber, minerals and vitamins ,2, are aeedstock . Fuel eteedstock , and addeedstock  is expeceedstock . Fuel eteedstock , and witFusarium  produce dangerous mycotoxins that may contaminate corn destined for fuel ethanol production and the resulting DDGS. These mycotoxins include deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV), and zearalenone (ZON) [−1 DON, 5 μg g−1 for grains and grain by-products intended for swine, and 10 μg g−1 in grains and grain by-products directed for cattle [−1 (1 country) to 1 μg g−1 (8 countries) [et al. [Several known fungal plant pathogens in the genus ne (ZON) . Consumpne (ZON) . 15-ADONne (ZON) , and havne (ZON) . NIV is ne (ZON) . The U.Sr cattle . Currentr cattle . There iuntries) . Cheli e [et al.  reviewedThe Food and Agriculture Organization estimates that over one quarter of the world’s crops are affected by mycotoxins every year , with anFusarium mycotoxins. We screened lots of DDGS for five mycotoxins that would likely be associated with corn diseases caused by Fusarium. Consequently, we did not conduct a complete screen of all potential mycotoxins  in corn DDGS; such an exhaustive effort was beyond the scope of our study. The specific objective of our work was to screen 141 DDGS lots collected in 2011 from 78 ethanol plants located in 12 states in the U.S. for the mycotoxins DON, 15-ADON, 3-ADON, NIV, and ZON. Little is known about the current and future impacts of mycotoxins in DDGS, and commercially viable methods to reduce mycotoxin contamination in DDGS are presently unavailable.Corn DDGS may be contaminated with trichothecenes, fumonisins, aflatoxins, and zearalenone ,22,23,24−1 DON, 2% (3/141) of the lots contained greater than 5.0 μg g−1 but less than 10.0 μg g−1 DON, and 3% (4/141) of lots contained 10.0 or more μg g−1 DON  of the lots contained greater than 5.0 μg g−1 but less than 10.0 μg g−1 15-ADON , 5% (7/141) were above advisory levels set for swine (>5 μg g−1), and 3% (4/141) were above levels considered safe for cattle consumption (>10.0 μg g−1) (−1 (−1 ZON (DON levels ranged from <0.50 to 14.62 μg g2 μg g−1 . None of5 μg g−1 . The mea and ZON . Our stu μg g−1) . ZON was μg g−1) , but foug−1) (−1  with oneg−1)  and ZON,  and mean ZON concentrations were positively correlated with mean 15-ADON concentrations . Zhang and Caupert [Fusarium grown on maize meal [Fusarium graminearum, DON and ZON both increased over a 9-week sampling period [Fusarium strains (including variability in the production of DON and ZON) infecting field crops [Mean DON concentrations for DDGS lots were positively correlated with mean concentrations of 15-ADON   had the highest levels of DON overall (mean of 9.51 μg g−1) . Ethanol4 μg g−1 . There w4 μg g−1 . Most of4 μg g−1 . The hig−1 (multiple lots) to 1.97 μg g−1  (All three repetitions (independent samples from DDGS lots) were significantly correlated for all repetition pairs for DON , 15-ADON , and ZON . This is consistent with previous studies that have determined mycotoxin levels in grains ,36,37, s μg g−1) . PreviouOne hundred and forty one corn DDGS lots from 78 ethanol plants in 12 states in the U.S. were sent to the Schmale Lab at Virginia Tech for mycotoxin analysis by Rob Musser from Nutriquest, Mason City, IA, USA. The DDGS lots were collected by operators at the ethanol plants or pulled as quality control samples at unloading at the feedmill. Multiple sub-samples were collected from a pile of DDGS or from the bottom hopper of a truck during unloading. The samples were then received by a third party analytical lab and distributed to members of the project team for analysis. Information for each of the DDGS lots is listed in v/v) acetonitrile in DI water) in a capped 50 mL polypropylene tube, and the solvent-subsample mixture was then placed on a shaker at 200 rpm for 1 h at room temperature. A portion of the solvent was passed through a clean-up column composed of a 1.5 g mixture of C18 (40 μm particle size)  and aluminum oxide   at a 1:3 ratio. The resulting eluent was then passed over a second clean-up column (same as above). A 2 mL aliquot of the eluent was transferred to a glass test tube and evaporated to dryness using a nitrogen evaporator set at 55 °C. Dried samples were then derivatized at 65 °C for 5 min with a 100 μL mixture of N-trimethylsilylimidazole (TMSI), trimethylchlorosilane (TMCS), N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA), and Pyridine at a 1:1:1:1 ratio [−1 mirex, followed by 1.0 mL of water to quench the reaction. Samples were vortexed until clear and 100 μL of the isooctane-mirex supernatant was removed and transferred to chromatography vials for gas chromatography-mass spectrometry (GC-MS) analysis. For quality assurance, a corn DDGS sample previously tested by the Mostrom Lab  was included as a check with expected concentrations of DON (8.0 μg g−1), 15-ADON (1.3 μg g−1), and ZON (1.0 μg g−1). Quality assurance samples were considered acceptable if the quantified DON concentration did not deviate more than 20% of the expected concentration.Mycotoxin extractions were performed on 5 g of each DDGS sample. Each 5 g sample was combined with 20 mL extraction solvent  at concentrations of 0.10, 0.25, 0.5, 1.0, 2.50, 5.0, 10.0, and 15.0 μg ml−1. Mycotoxin values below 2.5 μg g−1 were quantified using a standard curve ranging from 0.1 to 2.5 μg mL−1. Mycotoxin values above 2.5 μg g−1 were quantified using a standard curve from 0.1 to 15.0 μg mL−1. The limits of quantification (LOQ) for the analyses were 0.5 μg g−1 for DON, 0.5 μg g−1 for 3-ADON, 0.3 μg g−1 for 15-ADON, 0.3 μg g−1 NIV, and 0.3 μg g−1 for ZON  mode. A 1 μL volume of each sample or known standard was injected by an autosampler in splitless mode onto an HP-5MS column (0.25 mm inner-diameter by 0.25 μm film thickness by 30 m length) for detection and quantification of the mycotoxins DON, 15-ADON, 3-ADON, NIV, and ZON. The inlet temperature was set at 250°C, with a column flow rate of 1.2 mL/min. The initial column temperature was held at 150 °C for 1 min, increased to 280 °C at a rate of 30 °C/min, and held constant for 4.8 min. This was followed by a post run of 325 °C for 2.5 min. Mirex  was used as an internal standard at 0.5 μg mL for ZON .−1 DON when assayed in triplicate as the data set in the calculations of the blank. The standard deviation of these 18 data points was then used in the LOD and LOQ calculations. LOD was calculated as 3 times the standard deviation of the blank and LOQ was calculated as 6 times the standard deviation of the blank. All calculations used raw response values of the quantitating target ions. All calculations were performed for each of the four mycotoxins assayed in this study. Once the response values for LOD and LOQ were calculated, the corresponding concentration in μg g−1 was defined. The mean value of the raw responses for each level of nine calibration curves was determined. The LOD and LOQ concentration levels (μg g−1) were defined as the calibration levels whose mean response values was greater than and closest to the calculated LOD and LOQ responses. To determine the percent recovery of each of the mycotoxins analyzed, appropriate volumes of reference material in acetonitrile were added to a dry DDGS blank sample (11-N15735). The reference material, equivalent to 1, 5 and 10 μg g−1, was added in triplicate to 5 g of sample and allowed to completely dry with air flow in fume hood. Spiked samples were then assayed as previously described.Six DDGS samples were selected to determine the limit of detection (LOD) and limit of quantitation (LOQ) following established guidelines ,40,41. TA least squares fit model in JMP PRO  was used to analyze the association between concentrations DON and 15-ADON, DON and ZON, and 15-ADON and ZON. Only values greater than the LOQ were used in the analyses. A match pair analysis in JMP PRO was used to determine the correlation between each of the three sample replicates from all DDGS lots.One hundred and forty one corn DDGS lots collected in 2011 from 78 ethanol plants located in 12 states were screened for the mycotoxins deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), nivalenol (NIV), and zearalenone (ZON).−1, 15-ADON ranged from <0.10 to 7.55 μg g−1, and ZON ranged from <0.10 to 2.12 μg g−1. None of the DDGS lots contained 3-ADON or NIV.DON ranged from <0.50 to 14.62 μg g−1 DON. However, 26% (36/141) of the DDGS lots contained 1.0 to 5.0 μg g−1 DON, 2% (3/141) contained >5.0 and <10.0 μg g−1 DON, and 3% (4/141) contained >10.0 μg g−1 DON.The majority of the DDGS lots analyzed in the present study contained less than 1 μg gDDGS lots contaminated with unacceptable levels of DON evaded detection prior to their commercial distribution and were likely sold as feed products.−1), and plants in NY, MI, IN, NE, and WI had mean DON levels >1 and <4 μg g−1. These data suggest that corn grain and/or DDGS from these states should be tested in the future to examine the potential for mycotoxin contamination.Plants in OH had the highest levels of DON overall  and zearalenone glucosides . DON sugar glucosides and ZON glucosides break down during digestion and may be released as DON and ZON in the animal gut [Unknown are the contribution of masked mycotoxins to the dangers of contaminated DDGS. Masked mycotoxins include DON glucosides (3-β-imal gut ,43. Futuet al. [The continued excitement for the inclusion of DDGS in animal feeds and human foods, coupled with the concern for mycotoxin contamination in DDGS, underscores the need for new research aimed at detecting and mitigating mycotoxins in DDGS. Work by Khatibi et al.  highligh


Ophiopogon japonicus from Cixi city of Zhejiang and Santai county of Sichuan were quantified using HPLC-ELSD method and compared. Ophiopogonin B and ophiopogonin D′ content in tubers of HMD were higher than those in radix of CMD, whereas ophiopogonin D in HMD was about twice lower than that of CMD. Three ophiopogonins were all detected in fibrous roots of both HMD and CMD. Their averaged content in fibrous roots of CMD was about twice higher than that in tubers of CMD. Ophiopogonin D in fibrous roots of CMD was about five times higher than that of HMD. Our biochemical assay revealed that fibrous roots and tubers of CMD can be of an important saponin source, especially for ophiopogonin D.Zhejiang and Sichuan are regarded as two genuine producing areas of Ophiopogonis radix in China. To study the difference in the quality of Ophiopogonis radix from these two places, the contents of three reported bioactive saponins, that is, ophiopogonins B, D, and D′, in tubers and fibrous roots of Ophiopogon japonicus (Thunb) Ker-Gawl.  belongs to the family Liliaceae. Ophiopogonis radices, dried tubers of maidong, are routinely used in traditional Chinese medicine to nourish lung and body fluids, benefit the stomach, and remove stresses [stresses , 2. Maidstresses , 4. Howestresses . With th Ophiopogon japonicus, including steroidal saponins, polysaccharides, homoisoflavonoids, and amino acids [A lot of chemical compositions have been isolated from radix or fibrous roots ofno acids , 2, 6–9.no acids . HMD conno acids  found thno acids –15. Exceno acids , ophiopoMaidong tubers and fibrous roots were collected from Santai county, Sichuan province, and Cixi city, Zhejiang province of China . The samTwo grams of powder was extracted under reflux with 100 mL methanol for 1 hour and filtered with cheesecloth. The extract was evaporated to dryness in a rotary evaporator at 65°C under the reduced pressure and redissolved in 40 mL of water. The obtained solution was subsequently extracted with 20 mL of petroleum ether. The petroleum ether phase was discarded, and the fraction was then extracted three times with 90 mL of water-saturated butanol. The pooled butanol portions were evaporated to dryness at 65°C under the reduced pressure and dissolved in 2 mL methanol for the HPLC-ELSD analysis below.μL of the standard mixture were separately taken and diluted in methanol for HPLC-ELSD analysis to make their corresponding calibration curves. Calibration curves were constructed by plotting the logarithm of the HPLC-ELSD peak areas versus the logarithm concentration of each standard.HPLC grade acetonitrile was purchased from JT Baker Inc. (USA). Standards of ophiopogonin B (≥98%) and ophiopogonin D′ (≥98%) were obtained from Sichuan Weikeqi Biotechnology Co., Ltd., China. The standard ophiopogonin D (≥98%) was purchased from Shanghai Yilin Biotechnology Co., Ltd., China. The concentrations of ophiopogonins B, D, and D′ in the mixed stock standard solution were, separately, 0.082 mg, 0.111 mg, and 0.095 mg in 1 mL of methanol. 2, 4, 6, 8, 10, 15, and 20 μm), Waters 2424 evaporative light-scattering detector, and 2707 autosampler (USA). A linear gradient mobile phase containing acetonitrile (A) and water (B) was applied as follows: 45% A to 55% A for 30 min, 55% A to 45% A for 5 min, and 45% A held for another 5 min. The flow rate was 1.0 mL/min. The other ELSD settings included the following: drift tube temperature, 70°C; nitrogen flow rate, 2.4 mL/min; and heating power level of the sprayer, 60%. Three replicates of each sample were measured.Ten microliters of extracts and standards were run at 25°C on a Waters 600E system equipped with an Agela Venusil ASB C18 column , available P and K, total N, and organic matter content, which were assayed by the central laboratory of Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences, Beijing. Soil pH was measured in 1 : 2.5 soil (gram)/water (mL) solution by a pH meter. Soil electric conductivity was measured in a 1 : 5 soil/water extract using a conductivity meter. Available P and K, total N, and organic matter content were determined by molybdenum antimony blue colorimetry, flame photometry, Kjeldahl nitrogen method, and dichromate oxidation, respectively. The standard procedures for each method were followed during the extraction and quantification.λ 200–210 nm was generally affected by polysaccharides and interfering peaks, making UV readings unstable and irreproducible. HPLC-ELSD method has successfully determined the content of ophiopogonins D and D′ in maidong [Saponins have no UV absorption due to the absence of chromophores in their structures. Also, their UV absorption at  maidong . So we ay = 1.022x + 12.96, R2 = 0.9991; ophiopogonin D, y = 1.032x + 13.47, R2 = 0.9995; and ophiopogonin D′, y = 1.398x + 13.12, R2 = 0.9986. For the precision estimation, a sample extract was assayed successively for 6 times. The precisions calculated for ophiopogonin B, ophiopogonin D, and ophiopogonin D′ were 0.91%, 0.59%, and 0.87%, respectively. This indicated that our instrument was in good precision condition. Repeatability was obtained via parallel preparation of six sample extracts from the same batch. RSD for ophiopogonin B, ophiopogonin D, and ophiopogonin D′ were 1.00%, 1.32%, and 1.11%, respectively, which indicated that our HPLC method was reproducible.The linear regression equations obtained were ophiopogonin B, To check the stability of extracts, the same sample extract was assayed separately at 0, 2, 4, 8, 12, and 24 h after extraction. The values of relative standard deviation (RSD) for ophiopogonin B, ophiopogonin D, and ophiopogonin D′ were 1.22%, 1.52%, and 1.31%, respectively, indicating that our sample extract remained stable at least till 24 hours.To test the sample recovery, the standard solutions with known concentrations were added to 2 grams of maidong powder at the beginning of extraction. The recovery rates for standards of ophiopogonin B, ophiopogonin D, and ophiopogonin D′ were 98.20%, 103.80%, and 99.30%, respectively.μg/g to 139.04 μg/g, with an average of 114.22 μg/g, and, in CMD, from 70.16 μg/g to 132.05 μg/g, with an average of 107.71 μg/g. However, the amount of individual saponins between HMD and CMD was significantly different. The content of ophiopogonins B and D′ in tubers of HMD was significantly higher than that in CMD. For example, ophiopogonin B in tubers of HMD was between 26.25 μg/g and 57.03 μg/g, with an average of 41.90 μg/g; however, the content of ophiopogonin B in CMD was only from 7.10 μg/g to 27.7 μg/g, with an average of 13.98 μg/g. The content of ophiopogonin D in tubers of CMD was about twice higher than that in HMD, which was consistent with Yu et al.'s result [The content of three steroidal saponins in tubers of HMD and CMD was shown in μg/g to 368.65 μg/g, with an average of 241.06 μg/g. The total amount of three saponins in fibrous roots of HMD varied from 59.75 μg/g to 143.46 μg/g, with an average of 114.50 μg/g. Wu et al. found that the total saponin content in fibrous roots of CMD was higher than that in tubers of HMD and CMD [Our HPLC-ELSD analysis revealed that fibrous roots of both CMD and HMD contained ophiopogonins B, D, and D′ . The tot and CMD . So was The difference in saponin content between HMD and CMD, together with other bioactive compounds such as homoisoflavonoids and polysaccharides, was determined by a combination of cultivation habits, environmental conditions, and genetic difference between HMD and CMD. For example, Chuan maidong has to be harvested yearly, due to high temperatures and rainfall in the summer which make most tubers turn black and decay , 20; Han Ophiopogon japonicus from Zhejiang and Sichuan were quantified and compared. Ophiopogonin B and ophiopogonin D′ content in tubers of HMD were higher than those in radix of CMD, whereas ophiopogonin D in HMD was about twice lower than that of CMD. Three ophiopogonins were all detected in fibrous roots of both HMD and CMD. Ophiopogonin D in fibrous roots of CMD was much higher than that of HMD. Our biochemical assay revealed that fibrous roots and tubers of CMD can be of an important saponin source, especially for ophiopogonin D. Our results also provided the information on the quality control for future cultivation of maidong in new places and breeding new varieties containing desired or higher amount of bioactive compounds.Ophiopogonins B, D, and D′ in tubers and fibrous roots of


Middle-censoring is considered as a modern general scheme of censoring. In this paper, we study the analysis of middle-censored data with Burr-XII distribution which is considered one of the most popular and flexible distributions for modeling stochastic events and lifetime for many products.The parameters are estimated by the maximum likelihood method and the Bayes estimation under gamma prior and by applying the Lindley’s approximation.A simulation study is carried out to compare the performances of the two estimates. Both estimators behave almost similarly and verified the consistency property. A real medical data set is considered for illustration. Exact value of Ti is observable only if Ti ∉ , otherwise the actual value is not observable, but we observe the interval .Jammalamadaka and Mangalam  proposedIyer et al.  claimed Middle-censoring may arise in several situations as presented by Jammalamadaka and Mangalam . In any Iyer et al.  applied T1, …, Tn are i.i.d Burr-XII  random variables. Let Zi = Ri − Li, i = 1, …, n to be another random variable defines the length of the censoring interval with exponential distribution with mean γ-1, where the left-censoring point for each individual Li is assumed to be also an exponential random variable with mean λ-1. Moreover, the In this paper, we analyze the Burr-XII lifetime data when they are middle-censored. Assume that n randomly selected units from Burr-XII  population, where a and b are both unknown, are put on test under middle-censoring scheme. To write up the likelihood function, assume that there are n1 > 0 uncensored observations and n2 > 0 censored observations. Then, without loss of generality, by re-ordering the observed data into the uncensored and censored observations. Therefore, we have the following data n1 + n2 = n. Thus, the likelihood function of the observed data is given by:Suppose that c is a normalizing constant depending on λ and γ. The estimation of λ and γ is not of interest, thus it is left as a constant. The log-likelihood function is given bywhere a and b denoted by âM and The maximum likelihood estimation (MLE) of anda and b cannot be solved explicitly. Therefore, the solutions could be obtained by using Newton–Raphson method, or numerically by using the solve systems of nonlinear equations “nleqslv” package in R.It is obvious that the MLE of a and b are given by the elements of the inverse of the Fisher information matrixThe asymptotic variance-covariance of the MLE for parameters The approximate asymptotic variance-covariance matrix for the MLE will be considered because the exact mathematical expression for the above expectation is very difficult to obtain. Therefore, the approximate asymptotic variance-covariance matrix is given bywithandwhere following re-parameterization is used for the simplicity of expression presentations anda and b can be computed as follows α is the level of significance.Since the MLE is asymptotically normal, thus the approximate confidence intervals for the parameters a, b) with middle-censoring. Since a and b are both unknown, we will assume that the parameter b has an exponential distribution with mean 1/ β and the prior density of b is given by π1(b) = βeβb−  for b, β > 0, while the parameter a given the parameter b has a gamma prior distribution with shape parameter θ and scale parameter b. The conditional density function of a given b for b, θ > 0 is given by:This section considers the Bayesian formulation of the problem of estimating the scale and shape parameters of lifetime data from Burr-XII , Ûs is the posterior expectation given asUnder the Squared Error Loss (SEL) function, There is no closed form of the ratio of the two integrals in . Lindleyp + s = 3. Furthermore, εij are the elements of the inverse of the matrix having elements {−Lij}, where L is the log likelihood of the joint prior and where φ1, φ2) =  andNow by applying the Lindley’s approximation into our case where  under the SEL function, given by Lindley’s method in (3.5), turn out to be:The Bayes estimator of the function whereU = a thenIf U = b thenIf This section presents the numerical results for evaluating the performance of the two estimation methods for different sample size and censoring schemes. The author wrote R- subroutine to conduct the simulation study and it is available upon request.n =10, 30, 50, 70 and 100 with five combination of the censoring schemes  =, , ,  and . For all considered cases and without loss of generality, the random samples with desired sizes are generated from the Burr-XII distribution with parameters a = 1 and b = 1 are middle-censored according to (1.1). The MLE based on the iterative procedure given in  are obtained and reported in Table For each combination of sample size and censoring scheme the process is repeated 1000 times and the average estimates, the mean squared error (MSE) within brackets and the average censoring percentage . There is an inverse relationship between the average lengths of the confidence interval and sample size.Results in Table For the illustrative purpose, we consider a real data set which was generated from a clinical trial describing a relief time (in hours) for 50 arthritic patients as given in Wingo  who showThe arthritic data were artificially middle-censored by considering that the left end was an exponential random variable with mean 0.3 and the width was exponential with mean 0.3. Then the data were rearranged and given below:Data set: 0.29, 0.29, 0.34, 0.34, 0.35, 0.36, 0.36, 0.36, 0.44, 0.44,0.46, 0.46, 0.49, 0.49, 0.50, 0.50, 0.54, 0.55, 0.55, 0.55, 0.56, 0.57, 0.58, 0.59, 0.59, 0.60, 0.60, 0.61, 0.61, 0.62, 0.68, 0.70, 0.70, 0.71, 0.71, 0.71, 0.72, 0.73, 0.75, 0.75, 0.80, 0.81, 0.82, 0.84, 0.84, 0.87, , , , .n1 = 46 and n2 = 4 with censoring percentage 8.69%. The MLE of scale and shape parameters are â =7.423 and â and a and b are 7.628 and 4.157, respectively.There are four middle-censored observations are listed at the end of the data set, where The analysis of Burr XII distribution with middle-censoring was considered, where the parameter estimates were obtained by the maximum likelihood based on iterative procedures and Bayesian methods using the Lindley’s approximation. Both estimators behave almost similarly and verified the consistency property. Several related open problems would be interesting to be considered such as exploring the middle-censoring of Burr-XII model of covariates.


Coronary artery disease (CAD) is one of the most important issues in modern medicine due to its high mortality and prevalence. An early detection and prevention can reduce morbidity and mortality. Arterial stiffness is a potent and independent predictor of CAD. We aimed to investigate the arterial stiffness in CAD patients undergoing the coronary angiography. Also, we investigated a possible correlation between arterial stiffness and in-stent restenosis (ISR), an important limitation of percutaneous coronary intervention (PCI).The study included 160 patients undergoing coronary angiography, treated either with PCI or with CABG. The pulse wave velocity (PWV) and augmentation index (AIx) were measured by the Arteriograph.PWV in the CAD group (12.24 ± 2.78 m/s) was significantly higher compared to the control group (8.27 ± 1.89 m/s). PWV in ISR and left main (LM) stenosis group (14.03 ± 3.15 and 13.89 ± 2.95 m/s) was significantly higher compared to the control and CAD groups. Peripheral and central AIx were significantly higher in CAD group (1.38 ± 30.63 % and 38.35 ± 15.52 %) than in control group (−11.35 ± 26.74 % and 26.91 ± 10.62 %). Patients with LM stenosis have significantly higher values of peripheral and central AIx (23.37 ± 23.77 % and 49.71 ± 12.02 %) than the CAD and ISR group.The study confirmed a positive correlation between arterial stiffness measures, PWV and AIx, and CAD. Also, this study showed the correlation between PWV and ISR which can help to select more appropriate stent.The online version of this article (doi:10.1186/s12872-016-0305-4) contains supplementary material, which is available to authorized users. Coronary artery disease (CAD) is one of the most important issues in modern medicine due to high prevalence and mortality. Both, the prevention and treatment of CAD in recent decades have been improved, however, CAD remains the first inductor of morbidity and mortality in humans . An earlTreatment of CAD could involve pharmacotherapy, percutaneous coronary interventions (PCI), and surgical revascularization. All methods are associated with some limitations and disadvantages. In the past few decades the development of PCI, atherectomy, with bare-metal (BMS) and drug eluting stents (DES) has made a significant progress in the management of CAD . The mosFactors that contribute to ISR are not fully understood. Cassese et al. showed that diabetes mellitus, multiple lesions, small vessels, multiple stents, smaller final stent lumen diameter and stent design are predictors for ISR , 13. SomIn this study we aimed to investigate the correlation between CAD assessed by coronary angiography graded by SYNTAX score and arterial stiffness measured by Arteriograph, a simple, inexpensive, reproducible and investigator-independent method. Also, we investigated the correlation between ISR and aortic PWV and Aix.st April 2014 to 1st October 2014. The control group consisted of 59 sex and age-matched healthy people. All subjects in the control group underwent the clinical examination, ECG, stress test, echocardiography, measuring of arterial stiffness and laboratory testing. All those subjects had a clear medical history. The CAD group excluded subjects with atrial fibrillation, acute coronary syndrome, and with significant valvular disease. All CAD patients had previous concordant non-invasive findings for CAD and experienced angina pectoris. They received an appropriate treatment  in accordance with the guidelines for CAD [The study included 160 patients with CAD who underwent an elective coronary angiography in the Department of Invasive Cardiology of Clinical Hospital Mostar from 1 for CAD . Out of All patients underwent routine coronary angiography using the Judkins technique on digitized coronary angiography equipment . Coronary angiograms were computerized and assessed by two experienced angiographers who were blinded to the results of arterial stiffness measurements. A significant CAD was defined as at least 50 % or more stenosis. ISR was defined as >50 % diameter stenosis at the stent site.The SYNTX score is a semi-quantitative angiographic tool to determine the extent of CAD . The algArterial stiffness was measured by an oscillometric non-invasive device Arteriograph . The device detects and processes oscillations on the upper-arm positioned cuff by a special high fidelity sensor during a complete occlusion of brachial artery. Simultaneously with the parameters of arterial stiffness (PWV (m/s) an AIx (%), the device also recorded systolic and diastolic blood pressures and heart rate .Twenty-four hours prior to the examination subjects were asked to refrain from exercise, fruits, vegetables, dietary supplements, tea, alcoholic beverages, and caffeine containing foods. Also, 12 hours before measurement of arterial stiffness patients did not take any drugs. All experiments were carried out in a quiet, temperature controlled room maintained around 24 °C and were started at 8 a.m. The subjects had rested quietly for 15 min in the supine position before the measurement.P <0.05), Bonferroni test was used for the post hoc analysis.Data are expressed as mean ± SD. Statistical analyses were performed using GraphPad Instat and GraphPad Prism . All variables were normalized before data statistical analyses on age, heart rate and blood pressure. One-way ANOVA test was used to evaluate changes in AIx and PWV. When statistical significance was reached by ANOVA  stenosis and ISR groups.The general characteristics of control, CAD, ISR and LM stenosis groups are shown in Table P <0.05). Also, PWV of an ISR and LM stenosis group (14.03 ± 3.15 and 13.89 ± 2.95 m/s) was significantly higher than in the control and CAD group (P <0.05)  was significantly higher compared to the control group (8.27 ± 1.89 m/s) (P <0.05). Also, central AIx of an LM stenosis group was significantly higher than in the CAD and ISR group (P <0.05)  was significantly higher compared to the control group (26.91 ± 10.62 %) (P <0.05). Also, peripheral AIx of an LM stenosis group was significantly higher than in the CAD and ISR group (P <0.05)  was significantly higher compared to the control group (−11.35 ± 26.74 %) (P <0.05). Among patients treated with PCI and patients who underwent CABG we did not find a difference in the parameters of arterial stiffness   compared to the PCI and ISR group (17.63 ± 9.77 and 19.33 ± 6.9) (In this study we showed a positive correlation between arterial stiffness parameters and CAD proved with coronary angiography. Also, our study showed the correlation between PWV and ISR.Some previous studies showed the correlation between arterial stiffness and CAD –22. In tFurthermore, we investigated a correlation between ISR and arterial stiffness. The correlation between PWV and ISR was found, but not between AIx and ISR. Arterial stiffness is strongly dependent on the balance of two major proteins, elastin and collagen . NormallOur results confirm the importance of measuring arterial elasticity in patients with CAD predisposition. Also, we have found that an elevated PWV is associated with ISR suggesting its measuring before the procedure which contributes for a better selection of the stent implant. This study confirms the value of Arteriograph in cardiovascular risk assessment as a broadly applicable method for screening the general population.ACE, angiotensin-converting-enzyme; AIx, augmentation index; BMS, bare-metal stent; CABG, coronary artery bypass graft; CAD, coronary artery disease; DES, drug eluting stent; ISR, in-stent restenosis; LAD, left anterior descending artery; LM, left main; PCI, percutaneous coronary intervention; PWV, pulse wave velocity


Piper nigrum [Piperaceae], commonly known as black pepper is used as medicine fairly throughout the greater part of India and as a spice globally.in vitro cytotoxic [antiproliferative] activity and in silico method.To isolate piperine and evaluate P.nigrum. Piperine was characterized by UV,IR, 1H-NMR, 13C-NMR and Mass spectrum. Standardization of piperine was done also by HPTLC fingerprinting. In vitro cytotoxic activity was done using HeLa cell lines by MTT assay at different concentrations ranging from 20 to 100 μg/ml in triplicate and in silico docking studies using enzyme EGFR tyrosine kinase.Piperine was isolated from the fruits of 50 value was found to be 61.94 ± 0.054 μg/ml in in vitro cytotoxic activity in HeLa Cell lines. Piperine was subjected to molecular docking studies for the inhibition of the enzyme EGFR tyrosine kinase, which is one of the targets for inhibition of cancer cells. It has shown −7.6 kJ mol−1 binding and 7.06 kJ mol−1 docking energy with two hydrogen bonds.Fingerprinting of isolated piperine were done by HPTLC method. The ICin vitro cytotoxic activity and in silico studies.piperine has shown to possess  Cancer is one of the highest impacting diseases worldwide with significant morbidity and mortality rates. The current known therapies are based on radio and chemotherapies and although in many cases, the patients have their health re-established, the treatment is very painful since their immunological system is severely compromised, because these procedures are not cells selective  and the results were expressed as mean of percentage inhibition. A graph of the concentration versus percentage growth inhibition was plotted, and the concentration at which 50 % cell death occurred was considered as the IC50 value. Before adding MTT, bright field images  were taken for visualizing the cell death.The MTT assay was carried out as described previously to measure cell viability  structural database. This file was then opened in SPDB viewer edited by removing the heteroatoms, adding C terminal oxygen. The active pockets on target protein molecule were found out using CASTp server  was downloaded from PDB : The signal at δ 1.55–1.71 , 3.52 , 3.63 , 5.98 , 6.44 , 6.76 , 6.77 , 6.89 , 6.98 , 7.40 . The δ values were comparable with that of reported 1H NMR piperine : the signals at δ 24.66, 25.66,26.67, 43.25, 46.93, 101.24, 105.71,108.46,120.15,122.41,125.41,131.07,138.14,142.39,148.11,148.21 and 165.43. The δ values were comparable with that of reported 13C NMR piperine  corresponding to piperine is visible in both the test and standard solution tracks under UV at 254 nm,366 nm and after derivatization.The HPTLC fingerprinting confirmed the presence of Piperine Figs. , 3 and 450 value was found to be 61.94 ± 0.054 μg/ml. Microscopy images representing the cell death caused by the compounds are as seen in Fig. The MTT values obtained demonstrated that piperine has cytotoxic effect as the ICThe tyrosine kinase receptors have multidomain extracellular Ligands for specific Ligand, a signal pass transmembrane hydrophobic helix and tyrosine kinase domain. The receptor tyrosine kinases are not only cell surfaces transmembrane receptors, but are also enzymes having kinase activity (Bari et al. −1 with two hydrogen bonds formed.Piperine was taken and docked to get the best conformer. Results were compared for the binding energy, docking energy and number of hydrogen bonds formed. According to the docking results Table , it has  in vitro studies the molecule emerged to be active against the cell line used in inhibiting the cell growth.Molecular docking with EGFR tyrosine kinase domain revealed that, our compound has inhibitory capability and thereby exhibiting interactions with one or the other amino acids in the active pockets as shown in Fig.  in vitro cytotoxic activity and in silico studies. The IC50 value was found to be 61.94 ± 0.054 μg/ml and in silico studies, it has more number of hydrogen bonds with minimum binding and docking energy and may be considered as inhibitor of EGFR tyrosine kinase. More experiments are required to understand the exact mechanism by which the cells are affected. It is important to correlate the structure of these compounds with their biological effect, which will be valuable to propose new lead compounds with better cytotoxic potential.Piperine has shown to possess


Background. Shaoyao-Gancao Decoction (SGD), a well-known traditional Chinese medicine prescription, has been widely used to treat adenomyosis, dysmenorrhea, abdominal pain, and inflammation in Asia. However, the mechanism underlying the effectiveness of SGD in the treatment of adenomyosis still remains elusive. The present study aimed to investigate the bioactivity of SGD and its underlying molecular mechanisms using cultured human adenomyosis-derived cells. Methods. Human adenomyosis-derived cells were treated with SGD and its major constituents (paeoniflorin and liquiritin) in vitro. Effects of SGD, paeoniflorin, and liquiritin on cell proliferation and apoptosis were examined by MTT assay and flow cytometry analyses. The effects of SGD, paeoniflorin, and liquiritin on the production of PGE2 and PGFα2 were assayed using ELISA. ER-α and OTR mRNA expression levels were also evaluated by real-time qRT-PCR. Results. SGD, paeoniflorin, and liquiritin inhibited proliferation and induced apoptosis of human adenomyosis-derived cells in a dose-dependent manner. SGD and paeoniflorin significantly reduced the PGE2 and PGFα2 production. Furthermore, they remarkably decreased the mRNA levels of ER-α and OTR. Conclusions. The results of this study provide possible mechanisms for the bioactivity of SGD for treating adenomyosis and contribute to the ethnopharmacological knowledge about this prescription. Adenomyosis, also known as endometriosis interna, is a common gynecological disorder defined as the benign invasion of endometrial glands and stroma into the myometrium . The repShanghan lun in 210 CE. The herbal prescription, which is made up of two herbs , is commonly used in the treatment of gynecological disorders in China, including dysmenorrheal [Shaoyao-Gancao Decoction , a well-known traditional Chinese medicine prescription, was sourced from the Chinese Medical Classics text—norrheal , menorrhnorrheal , and infnorrheal , 14. Simnorrheal . In addinorrheal  and antinorrheal  effects.norrheal .2 (PGE2) and prostaglandin Fα2 (PGFα2) production and estrogen receptor-α (ER-α) mRNA and oxytocin receptor (OTR) mRNA expression were also investigated.However, little is known about the potential mechanisms of the antiadenomyosis effect of SGD. In this study, we examined the effects of SGD and its major constituents (paeoniflorin and liquiritin) on the proliferation and apoptosis of human adenomyosis-derived cells. In addition, their effects on prostaglandin E2 enzyme-linked immunosorbent assay (ELISA) kit was obtained from R&D Systems ; PGFα2 ELISA kit was purchased from ENZO Life Sciences.Acetonitrile ; Paeoniflorin and liquiritin were obtained from The National Institute for the Control of Pharmaceutical and Biological Products ; 3--2, 5-diphenyltetrazolium bromide (MTT), Apoptosis Detection Kit , sodium dodecyl (lauryl) sulphate-hydrochloride (SDS-HCl), and Trizol were purchased from Invitrogen; Dulbecco's modified Eagle's medium (DMEM), Collagenase I, 0.25% trypsin, fetal bovine serum (FBS), and phosphate buffered saline (PBS) were obtained from Gibco; M-MLV RTase cDNA Synthesis Kit and SYBR Premix Ex Taq were obtained from Takara ; PGEPaeoniae Radix and Glycyrrhizae Radix were purchased from Caizhilin Materials Company of Guangzhou and authenticated by professor Zi-Ren Su in the School of Pharmacy of Guangzhou University of Chinese Medicine.According to the preparation method of SGD by Shen et al. with slight modifications , extract Paeoniae Radix (0.2 mL), Glycyrrhizae Radix (0.08 mL), and SGD (0.28 mL) with a concentration equivalent to 1 g/mL crude herb materials were dried and then extracted with methanol-water  under ultrasonication for 30 min. The solutions were filtered and then determined for UFLC analysis.Ultra-fast liquid chromatography (UFLC) profiles of two single herbs and SGD were performed as the following procedures: extract solutions of18 column . The elution gradient was carried out with binary solvent system consisting of 0.5% acetic acid in H2O (solvent A) and MeCN (solvent B). A linear gradient profile with the following proportions (v/v) of solvent B was applied: gradient profile 0 to 10 min and 5% of B, 10 to 20 min and 5% to 15% of B, 20 to 30 min and 15% to 30% of B, 30 to 40 min and 30% to 50% of B, and 40 to 45 min and 50% to 70% of B. The flow rate was controlled with LC 20AD at 0.4 mL/min, injection volume 5 μL, and the column temperature was maintained at 28°C. The effluents from the column were detected at absorbencies ranging from 200 to 400 nm using a photodiode-array detector. The three-dimensional data collection and processing for peak analysis were accomplished with CLASS-LC10 System Analysis Software .UFLC equipment was controlled with LC-20AD pump  using a YMC-UltraHT Pro CAdenomyosis specimens were obtained at the time of laparoscopy or laparotomy performed for adenomyosis treatment from eight Chinese women aged 20–45 years. All patients had regular menstrual cycles. No patients were undergoing hormone therapy prior to surgery. All biopsy specimens were collected at the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine between November 2009 and August 2010 with the permission of the local ethics committee, and written informed consent was obtained from each patient.5 cell/mL in DMEM supplemented with 10% FBS, then transferred to a tissue culture dish, and incubated at 37°C. In order to accumulate enough cells for further study, cultured cells of individual patients were pooled and cryopreserved. The lower passage number (three to six) of cells was used for experiments to avoid changes in phenotype and gene expression.The biopsy specimens were immediately placed in ice-cold DMEM for transport to the laboratory. The tissues were then dissociated as previously described with some modifications . The bio4 cells per well and cultivated in 100 μL of the DMEM containing 10% FBS. After a 3-day preincubation period, culture media were replaced with 100 μL DMEM and then incubated at 37°C for 24 h. The supernatants were removed, and 100 μL of fresh DMEM containing various concentrations of SGD (0.1–500 mg/mL), paeoniflorin (0.0001–10 mg/mL), liquiritin (0.0001–10 mg/mL), and mifepristone (0.005–100 μM as positive control) was added. Cells were incubated at 37°C for 24 h; then 10 μL MTT (5 mg/mL) was added, and the cells were incubated at 37°C for 4 h. After the medium and MTT were removed, 100 μL of SDS-HCL was added to each well and then placed on a plate shaker for 10 min at room temperature. For each well, absorbance at 570 nm was measured using a microtiter plate ELISA reader. The cell survival rate was calculated as follows: percent survival =   × 100.Isolated cells were first placed in 96-well microtiter plates at a density of 5 × 10μM as positive control) for 24 hours, cells were collected and washed with PBS. Cells were harvested by incubating with 0.25% trypsin for 3–6 min, and then DMEM containing 10% FBS was added. Cells were centrifuged at 1000 rpm for 5 min and then washed 3 times with PBS, followed by being resuspended in 500 μL of binding buffer containing 5 μL annexin V-FITC and 5 μL PI and then incubated for 5 min in the dark at room temperature. Following this, the cells were analyzed by flow cytometry. The percentage of cells undergoing apoptosis was determined by three independent experiments.The flow cytometric analysis of annexin V-FITC and propidium iodide (PI) stained cells was performed using the Apoptosis Detection Kit . Annexin2 and PGFα2 in the culture supernatant were determined by ELISA kits according to the manufacturer's protocol.Human adenomyosis-derived cells were pretreated with SGD (10 and 20 mg/mL), paeoniflorin (0.25 and 0.5 mg/mL), and liquiritin (0.25 and 0.5 mg/mL) for 24 hours. The concentrations of PGEα and OTR were determined by qRT-PCR analysis. Cells preincubated with or without SGD (10 and 20 mg/mL), paeoniflorin (0.25 and 0.5 mg/mL), and liquiritin (0.25 and 0.5 mg/mL) for 24 hours, and then total RNA was isolated using Trizol Reagent according to the manufacturer's protocol. Total RNA was reverse-transcribed using a cDNA synthesis kit. qRT-PCR was performed on the Thermal Cycler Dice Real Time System using SYBR Premix Ex Taq. The forward and reverse primers were 5′-GTCTCGTCTGGCGCTCCAT-3′ and 5′-CCCTGGTTCCTGTCCAAGAG-3′ for ERα mRNA; 5′-AGGAAGCCTCGGCCTTCAT-3′ and 5′-GAGGTGGCCCGTGAACAG-3′ for OTR mRNA; 5′-GCATGGGTCAGAAGGATTCCT-3′ and 5′-TCGTCCCAGTTGGTGACGAT-3′ for β-actin mRNA. The relative expression of ER-α and OTR mRNA was normalized to the amount of β-actin mRNA levels.The mRNA levels of ER-P < 0.05 was considered statistically significant.For all groups data are presented as the mean plus or minus standard deviation (SD). Statistical significance within a parameter was evaluated by one-way analysis of variance (ANOVA) using the SPSS. Value of UFLC profiles of two single herbs and SGD were shown in Inverted microscope was selected for the identification of human adenomyosis-derived cells. Examination of these cells in culture showed monolayers of cells, displaying a fibroblast-like, stellate, or spindle-like morphology, as reported by Suzuki-Kakisaka et al. .μM) for 24 h. The results showed that SGD, paeoniflorin, liquiritin, and mifepristone inhibited the survival of human adenomyosis-derived cells in a dose-dependent manner. The IC50 value of SGD, paeoniflorin, liquiritin, and mifepristone was 25.06 mg/mL, 1.03 mg/mL, 1.62 mg/mL, and 10.61 μM, respectively.The inhibitory effect of SGD, paeoniflorin, and liquiritin on the growth of human adenomyosis-derived cells was evaluated using MTT assay. The effects on human adenomyosis-derived cells are shown in μM), human adenomyosis-derived cells were analyzed by flow cytometry. P < 0.05), 23.5 ± 3.2% (P < 0.01), 23.0 ± 2.6% (P < 0.01), 28.3 ± 2.6% (P < 0.01), 10.7 ± 1.8%, 13.2 ± 2.2% (P < 0.05), and 18.5 ± 4.2% (P < 0.05) in the presence of SGD (10 and 20 mg/mL), paeoniflorin (0.25 and 0.5 mg/mL), liquiritin (0.25 and 0.5 mg/mL), and mifepristone (10 μM), respectively. These results demonstrated that SGD (10 and 20 mg/mL), paeoniflorin (0.25 and 0.5 mg/mL), liquiritin (0.5 mg/mL), and mifepristone (10 μM) could significantly induce the apoptosis of human adenomyosis-derived cells.Simultaneous staining with annexin V-FITC and PI distinguished between healthy, early apoptotic, late apoptotic, and necrotic cells . After 22 and PGFα2, were examined. As shown in 2  and PGFα2 , while PGE2 and PGFα2 in control group were 1592.63 ± 51.35 and 2553.58 ± 25.69 pg/106 cells, respectively. These results suggested that the decrease in production of PGE2 and PGFα2 might be a potential mechanism in relieving adenomyosis-associated dysmenorrhea using SGD. Meanwhile, the activity of SGD in decreasing PGE2 and PGFα2 production might be partly ascribable to paeoniflorin.To investigate the potential mechanism underlying the effectiveness of SGD in the treatment of adenomyosis-associated dysmenorrhea, the levels of two well-established pain mediators, namely, PGEα mRNA and OTR mRNA were investigated by qRT-PCR. As shown in α , in comparison to control group (0.353722 ± 0.060531). In addition, SGD (10 and 20 mg/mL), paeoniflorin (0.25 and 0.5 mg/mL), and liquiritin (0.25 and 0.5 mg/mL) obviously decreased the mRNA levels of OTR  in a dose-dependent manner, in comparison to control group (0.0693 ± 0.0164).The effects of SGD, paeoniflorin, and liquiritin on the expression of ER-Traditional Chinese medicines have attracted interest and acceptance in many countries with the merits of few side effects, affordability, and local availability . HoweverCell proliferation has been shown to have multiple effects in embryonic development and pattern formation, including cell growth, morphogenesis, and gene expression . HoweverApoptosis, a process of programmed cell death, is essential for embryonic development, proper development and functioning of the immune system, and the maintenance of tissue homeostasis . Accumul2 and PGFα2, are bioactive lipids derived from arachidonic acid and play an important role in the etiology of dysmenorrhoea [2 and PGFα2 were higher in the menstrual fluid of women with dysmenorrhoea than in women with painless periods [2 and PGFα2 could upregulate cyclooxygenase-2 (COX-2), which is an essential enzyme for the synthesis of PGE2 and PGFα2, causing dysmenorrhea in adenomyosis [2 and PGFα2 synthesis has become the main treatment of dysmenorrhoea [2 and PGFα2 was performed. Results showed that treatment with SGD dose-dependently and significantly decreased the production of PGE2 and PGFα2. A similar result has been reported by Imai et al. that incubation of human endometrial cells with SGD induced a significant decrease in PG levels [2 and PGFα2 in the culture medium of human myometrial biopsies were significantly decreased by the addition of SGD [2 and PGFα2 production.Dysmenorrhea, defined as painful menstruation involving low abdominal pains, is a major clinical symptom of adenomyosis , 36. Pronorrhoea , 38. In  periods . The incnomyosis , 40. Thenorrhoea . In the G levels . In addin of SGD . The aboα and ER-β, have been shown to be partly responsible for the cell proliferation, apoptosis, differentiation, and development [α is the principal ER expressed in the endometrium, and it is considered to be crucial in the development of endometrioid endometrial carcinoma [α is associated with the onset and growth of adenomyosis [α can help us understand the mechanism underlying the effectiveness of SGD in the treatment of adenomyosis. By qRT-PCR analysis, we found that SGD treatment remarkably inhibited the expression of ER-α mRNA.Adenomyosis is most frequent in women of reproductive age and regress after menopause, which is well regarded as an estrogen-dependent condition . The estelopment . ER-α isarcinoma . In addiarcinoma , and ER-nomyosis . ConsequAlthough the cause for OTR overexpression in adenomyosis is currently unclear, it has been shown that OTR is involved in adenomyosis-associated dysmenorrhea and may be potential therapeutic target in treating the symptom and perhaps chronic pelvic pain in women with adenomyosis , 49. Mea Paeoniae Radix. Liquiritin is a kind of flavonoids that is extracted from Glycyrrhizae Radix. Furthermore, in order to reveal the active constituents and their contribution to the effect of SGD, we also examined the antiadenomyosis effects of its 2 major ingredients, paeoniflorin and liquiritin, on human adenomyosis-derived cells. Results showed that both paeoniflorin and liquiritin significantly reduced proliferation, accelerated apoptosis, and suppressed ER-α mRNA and OTR mRNA expression in human adenomyosis-derived cells. Paeoniflorin can also decrease production of PGE2 and PGFα2. These findings suggested that the antiadenomyosis effects of SGD on human adenomyosis-derived cells would be partially achieved by these 2 components. In previous studies, it was reported that paeoniflorin possessed analgesic [In addition, it is widely accepted that the combined action of multiple constituents in traditional Chinese medicine is considered to be crucial for its therapeutic effects . Yin et nalgesic , anti-innalgesic , immunornalgesic , cognitinalgesic , neuromunalgesic , 61, andnalgesic  activitinalgesic , and supnalgesic . Liquirinalgesic , could inalgesic , and is nalgesic . These e2, PGFα2. Meanwhile, SGD, paeoniflorin, and liquiritin significantly suppressed the expression of ER-α mRNA and OTR mRNA in human adenomyosis-derived cells. The results of the present study suggest that SGD and its major constituents (paeoniflorin and liquiritin) possess active antiadenomyosis activities.In conclusion, our study showed that SGD and its major constituents (paeoniflorin and liquiritin) displayed a differential ability to inhibit proliferation and induce apoptosis of human adenomyosis-derived cells. SGD and paeoniflorin effectively decreased the production of PGE


Janibacter sp. As revealed by high performance liquid chromatography (HPLC) analysis, FAS23 strain was found to be efficient for PCP removal in the presence of 1% of glucose. The conditions of growth and PCP removal by FAS23 strain were found to be optimal in neutral pH and at a temperature of 30°C. Moreover, this strain was found to be halotolerant at a range of 1–10% of NaCl and able to degrade PCP at a concentration up to 300 mg/L, while the addition of nonionic surfactant (Tween 80) enhanced the PCP removal capacity.Many pentachlorophenol- (PCP-) contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, and high salt concentrations. PCP-degrading microorganisms, adapted to grow and prosper in these environments, play an important role in the biological treatment of polluted extreme habitats. A PCP-degrading bacterium was isolated and characterized from arid and saline soil in southern Tunisia and was enriched in mineral salts medium supplemented with PCP as source of carbon and energy. Based on 16S rRNA coding gene sequence analysis, the strain FAS23 was identified as  Mycobacterium chlorophenolicum [ Alcaligenes sp. [ Rhodococcus chlorophenolicus [ Flavobacterium [ Novosphingobium lentum [ Sphingomonas chlorophenolica [ Bacillus [ Pseudomonas [ Acinetobacter [ Arthrobacter [ Kocuria [ Janibacter which is recognized by Martin et al. [ J. limosus [ J. terrae [ J. melonis [ J. corallicola [ J. anophelis [p-dioxin [ Janibacter members was described. PCP and other POP compounds shared many physical properties, which limited biodegradation processes, and one of these properties was their lower solubility and therefore low bioavailability to the degrading bacteria. Nevertheless, the use of surfactants such as Tween 80 has the potential to increase the biodegradation rates of hydrophobic organic compounds by increasing the total aqueous solubility of these pesticides [ Janibacter sp., a halotolerant actinobacterium member isolated from arid and saline land in southern Tunisia.The polyorganochlorophenolic (POP) compounds have been extensively used as wide spectrum biocides in industry and agriculture . The toxenolicum , Alcaligenes sp. , Rhodocoenolicus , Flavobaacterium , Novosphm lentum  and Sphihenolica , BacilluBacillus , Pseudomudomonas , and Acitobacter , as welltobacter , most prtobacter . Besidestobacter . These mtobacter . Howeverrobacter  and Kocu Kocuria , were ren et al.  belongs  limosus , J. terr. terrae , J. melo melonis , J. coraallicola , and J. nophelis . Interesnophelis , monochlp-dioxin , dibenzop-dioxin , anthracp-dioxin , dibenzop-dioxin , and polp-dioxin . Howeversticides . In thisPCP (MW 266.34 and >99% purity) and acetonitrile (HPLC grade) were purchased from Sigma Aldrich (USA). All other inorganic chemicals used to prepare the different media are commercially available with highest purity and are used without further purification.2HPO4, and 2 mM KH2PO4) and stirred vigorously for 30 min. The soil suspension was diluted and 0.1 mL sample was spread on the surface of yeast extract-mannitol medium (YEM). YEM medium contained the following components at the specified concentrations (in g/L): mannitol, 5; yeast extract, 0.5; MgSO4·7H2O, 0.2; NaCl, 0.1; K2HPO4, 0.5; Na gluconate, 5; agar, 15; pH = 6, 8. After sterilization for 20 min at 120°C, 1 mL of 16.6% CaCl2 solution was added to 1 liter of YEM medium (1 : 1000). The plates were then incubated at 30°C for 7 days. Pure cultures of the isolates were obtained by streaking a single colony on the same medium.The sediment samples were collected in March 2011 from arid and saline ecosystems belonging to the site “Sebkha El Naouel” with GPS coordinates: N 34°26′951′′ E 09°54′102′′ altitude 150 ft/46 m, in southern Tunisia. Bacterial isolation was performed as described by Rösch et al.  with somμL containing 1 μL of the template DNA; 0.5 μM of each primer; 0.5 μM of deoxynucleotide triphosphate (dNTP); 2.5 μL 10X PCR buffer for Taq polymerase; MgCl2 1.5 mM; 1 UI of Taq polymerase. The amplification cycle was as follows: denaturation step at 94°C for 3 min, followed by 35 cycles  plus one additional cycle at 72°C for 7 min as a final elongation step. The 16S rDNA PCR amplicons were purified with Exonuclease-I and Shrimp Alkaline Phosphatase  following the manufacturer's standard protocol. Sequence analyses of the purified DNAs were performed using a Big Dye Terminator cycle sequencing kit V3.1 (Applied Biosystems) and an Applied Biosystems 3130XL Capillary DNA Sequencer machine. Sequence similarities were found by BLAST analysis [http://www.ncbi.nlm.nih.gov/) and the Ribosomal Database Project (RDP). Phylogenetic analyses of the 16S rRNA gene sequences were conducted with Molecular Evolutionary Genetics Analysis (MEGA) software, version 5 [For DNA extraction, the FAS23 strain was grown in tryptic soy broth (TSB) containing (in g/L) casein peptone, 17; soya peptone, 3; glucose, 2.5; sodium chloride, 5; dipotassium hydrogen phosphate, 4. DNA extraction was performed using CTAB/NaCl method as described by Wilson  and modianalysis  using thersion 5 . Trees wersion 5 . The seq6 CFU/mL of the strain FAS23. The MSM contained the following components at the specified concentrations (in g/L): KH2PO4, 0.8; Na2HP4, 0.8; MgSO4·7H2O, 0.2; CaCl2·2H2O, 0.01; NH4Cl, 0.5, plus 1 mL of trace metal solution which includes (in mg/L) FeSO4·7H2O, 5; ZnSO4·7H2O, 4; MnSO4·4H2O, 0.2; NiCl·6H2O, 0.1; H3BO3, 0.15; CoCl2·6H2O, 0.5; ZnCl2 0.25; and EDTA, 2.5. PCP was added to the medium after autoclaving [μm filters [μL were applied to C18 reverse phase column  at a flow rate of 1 mL min−1. The retained molecules were eluted over 35 min using the following gradient: 1% (v/v) phosphoric acid in water for 4 min, followed by an increase to 100% (v/v) acetonitrile within 21 min which was kept constant for 5 min and then decreased back to initial concentration and kept constant for another 5 min. PCP was quantified using external standards method. Percent removal was estimated using the following formula: removal (%) = area − area/area [The kinetics of the PCP removal under different conditions were conducted in 500 mL flasks, sealed with cotton stoppers, containing 100 mL of mineral salt medium (MSM) adjusted to pH 6.9, supplemented with 1% glucose and inoculated with 1% of 10oclaving . When ne filters . Samplesrea/area .P value of less than 0.05 was considered statistically significant (test at P < 0.05). Three replicates were prepared for each treatment.Data were subjected to analysis of variance using SPSS software (version 14.0) and the mean differences were compared by Student-Newman-Keuls comparison test. A  Janibacter sp.  on bacterial growth of Janibacter sp. strain and PCP removal was studied in MSM. The result showed that the growth of the strain was possible only after the addition of glucose  on the growth and PCP removal was assessed . At bothVariation of PCP amount in the medium showed that the growth of the strain, as well as PCP removal, decreased with the increase of PCP concentration . At low The strain was tested for its ability to remove PCP (20 mg/L) at different NaCl concentrations . The best rate of growth and PCP removal was recorded at 1% of NaCl (more than 92% after 144 h of incubation). The growth and thus the capacity of PCP removal were inhibited when the concentration of sodium chloride was increased . At 3% NIn this study, the nonionic surfactant Tween 80 was found to enhance the growth and PCP-biodegradation process . Interes Janibacter sp., with respect to morphological and biochemical tests and 16S rRNA gene sequence. Despite their known high potential in recalcitrant compounds biodegradation [ Janibacter, described in this study, are reported for the first time for their ability to degrade PCP. In biodegradation process, glucose is commonly used as an additional source of carbon and energy and is the most metabolizable sugar which supported a maximum growth [The strain FAS23 isolated from saline sediment collected from Tunisian arid ecosystems was identified as an actinobacterium belonging to the genusradation , 33, bacm growth . In thism growth  and Singm growth  who repom growth . This efm growth . Arthrobacter and different Pseudomonas species, the neutral pH was found to be optimal for PCP degradation. However, for other bacterial species, such as Sphingomonas chlorophenolica, PCP degradation was more important at pH 9.2 [In the present study, PCP removal is affected by pH variation. As it was reported by Premalatha and Rajakumar , Wolski t pH 9.2 .Temperature is another important environmental factor that can influence the rate of pollutants degradation . The opt Janibacter sp. decreases when PCP concentration was increased. Furthermore, we found that the removal ability by Janibacter sp. has reached 40% after 144 h of incubation, when PCP concentration of 300 mg/L was used. These results are in accordance with those of Chandra et al. [ Bacillus cereus strain and may suggest that these bacteria may tolerate and remove high concentrations of PCP if we increase the incubation time. On the contrary, Kao et al. [The effect of different concentrations of PCP on growth of the strain proved that the PCP removal was more efficient at low concentrations (20 mg/L). This result was coherent with data of Webb et al.  reportina et al.  for Bacio et al.  reported Janibacter sp. was able to remove PCP even with salinity fluctuations (less than 10%). These results were in accordance with those of Gayathri and Vasudevan [ Janibacter sp. strain has an inherent flexibility to adapt to salinity fluctuations.As for bioremediation, the strain should possess not only the high removal efficiency for the target compounds but also the strong abilities of adapting some conditions such as pH, temperature, and salinity fluctuations. In this study, it was shown thatasudevan  suggesti Janibacter sp. strain.The use of surfactants has the potential to increase the biodegradation rate of hydrophobic organic compounds in contaminated environments. Nonionic surfactants are usually used in the bioavailability studies due to their relatively low toxicity compared to ionic surfactants . The enhJanibacter sp.) was isolated from saline soil of arid land and investigated for its physiological characteristics. Janibacter was able to remove high concentration of PCP and to tolerate fluctuation of NaCl. This removal potential was moreover accelerated by the addition of Tween 80. This study suggested that strain Janibacter sp. could be widely used for PCP bioremediation of polluted arid/extreme environments.In this study, a novel efficient PCP-degrading actinobacterium (


Our thermometer anchored at nearly-zero distance from the ER, i.e. the heat source, allowed the detection of the heat as it readily dissipated, and revealed the dynamics of heat production in real time at a subcellular level.The dynamics of cellular heat production and propagation remains elusive at a subcellular level. Here we report the first small molecule fluorescent thermometer selectively targeting the endoplasmic reticulum (ER thermo yellow), with the highest sensitivity reported so far (3.9%/°C). Unlike nanoparticle thermometers, ER thermo yellow stains the target organelle evenly without the commonly encountered problem of aggregation, and successfully demonstrates the ability to monitor intracellular temperature gradients generated by external heat sources in various cell types. We further confirm the ability of ER thermo yellow to monitor heat production by intracellular Ca Genetically-encoded calcium indicators expressed in the ER revealed that [Ca2+] in the ER changes only by a few hundred micromolars even with the addition of ionomycin (1 μM), which causes calcium influx from the extracellular medium202+] . This means that the ΔT distribution (SD = 0.4°C) of ER thermo yellow can be attributed to the intrinsic measurement error, and that the temperature measurement was accurate with a SD of 0.4°C from whole cell analysis. It is therefore apparent that heat production did not significantly vary from cell to cell. At the single cell level, heterogeneous heat production is likely to occur even within the cell shown in F-test showed ER thermo yellow (−6.1 ± 3.1%) and ER tracker (−0.4 ± 3.8%) to have equal variance . The difference in ΔT at the single cell level  is not attributed to the heterogeneous change in temperature caused by Ca2+ shock but the intrinsic measurement error  in fixed HeLa cells . This de2+ shock occurs throughout the whole cell but rather at specific organelles. The three-dimensional heat conduction equation can be described as follows: R denotes the radius of the sphere, q the heat per second (W), k thermal conductivity (W/m/K) and ΔT temperature difference. Given that the cell has a 10 µm sphere of water (0.6 W/m/K), a q of around 100 µW is needed to maintain a temperature rise of a whole cell by 1°C. However, this does not agree with the data obtained from the microcalorimetry studies in the field of thermal biology33q) in the order of pico to nanowatts, although it should be taken into account that these experiments are performed using cell suspensions that may therefore be a mixture of dead and live cells. Our thermometer is placed inside the compartmentalized interior of a cell, at a ‘nearly zero' distance from the heat source, which enables the accurate measurement of local and small changes in heat production. Until now, Yang et al.T induced by Ca2+ shock to be around 1.8°C inside the cell, which is in accordance with our results. The nanothermometers of Yang et al. also revealed that ΔT was heterogeneous with a broad distribution. They discussed that this heterogeneity was due to variation in distances between thermometers and heating spots. In contrast, ER thermo yellow localizes inside the ER, i.e. where the organelle heat source resides, at a distance from the heat source that is nearly zero. As judged from data variation from spot to spot within a single cell, we did not detect heterogeneity in ΔT  of 1500 dyes were diluted to 2.5 µM in HEPES buffer solution  containing 0.5% DMSO and were loaded on 96 well plates. The fluorescence intensity was measured using a Spectra Max Gemini XSF plate reader. Before measurement, the plate loaded with samples was kept at 32°C or 37°C for 1 hr until the temperature reached the stable state. The heating and cooling process from 32°C to 37°C was repeated twice. Detailed methods for screening for temperature sensitive dyes is described in the 3 saturated solution (50 µL) and chloroacetylchloride 20 µL were added to the reaction mixture. The crude compound was purified by column chromatography and the final product was 15 mg (81%). 1H-NMR (CD3OD + CDCl3) δ 7.67, 7.66, 7.62, 7.42, 7.30, 6.98, 6.76, 6.72, 6.34, 4.13, 1.56, 13C-NMR (CD3OD + CDCl3) δ 158.34, 154.45, 145.65, 142.04, 140.94, 138.07, 134.93, 133.90, 130.40, 130.39, 129.75, 126.22, 124.07, 123.39, 121.49, 120.14, 119.56, 119.44, 119.05, 116.53, 116.04, 53.30, 42.78, 29.53, 15.25; ESI-MS m/z (M + H) calc'd: 576.12, found 576.0, and 556.0(M - F).Aminophenyl bodipy  and 3-threefluoromethoxy-6-hydroxy-benzaldehyde  were dissolved with acetonitrile (5 mL) in a 20 mL vial, and 3 eq. of pyrrolidine and acetic acid were then added to the reaction mixture. The reaction vial was heated to 60°C using a heating block reactor for 3 hrs. After completion of the reaction, NaHCO2 for 30 minutes. After incubation, washing with fresh pre-warmed medium was carried out once. Regarding fixed HeLa cells, after staining with ER thermo yellow, they were treated with 4% formaldehyde solution for 30 min at room temperature and washed three times with 20 mM HEPES buffer solution . For co-staining experiments, the final concentration of ER tracker Green, DAPI, and Fluo 4 were 500 nM, 1 µM, and 1 µM, respectively. All microscopic experiments were carried out at a temperature of 37°C, maintained by a circulation chamber. Microscopic images using ER tracker Green (or Fluo 4), DAPI, and ER thermo yellow (or ER tracker red) were obtained with FITC (488 nm laser), DAPI, Texas red (543 nm laser) filter sets. The optical setup was built around the inverted microscope , and the facility to create local and transient temperature gradients was largely the same as previously reported2+ concentration was carried out with a confocal laser microscope . In order to avoid crosstalk resulting from the strong fluorescence of Fluo 4 and ER thermo yellow, the emission channel for ER thermo yellow was restricted to the tail end, i.e., between 590 and 690 nm. Photobleaching could not be ignored in this setup and therefore was corrected by fitting to a single exponential curve  was added into 2 mL of pre-warmed culture medium and then incubated at 37°C, 5% COal curve . A stock2. The C2C12 myoblast cell line was obtained from American Type Culture Collection . Undifferentiated myoblasts were grown in Dulbecco's modified eagle medium (DMEM) supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), and 10% fetal bovine serum (FBS). When myoblast culture reached 100% confluence, myoblasts were stimulated to differentiate by replacing the medium with DMEM supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), and 2% heat-inactivated horse serum. Differentiation was allowed to continue for 5 days to obtain differentiated myotubes, with media replacement every 2 days. NIH3T3 and Chang cells were cultured according to methods similar as those used with HeLa cells. Brown adipose tissue was collected from mice dissection. The animal experiment procedures were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC).HeLa cells were cultured in Dulbecco's modified eagle's medium (DMEM)  supplemented with fetal bovine serum (10%) and penicillin–streptomycin. Cells were grown in a 3.5 cm glass-based dish at 37°C in the presence of 5% COAll reactions were performed in oven-dried glassware under a positive pressure of nitrogen. Unless otherwise noted, starting materials and solvents were purchased from Aldrich and Acros organics and used without further purification. Analytical TLC was carried out on a Merck 60 F254 silica gel plate (0.25 mm layer thickness) and visualization was carried out with UV light. Column chromatography was performed on a Merck 60 silica gel (230–400 mesh). NMR spectra were recorded on a Bruker Avance 300 NMR spectrometer. Chemical shifts are reported as δ in units of parts per million (ppm) and coupling constants are reported as a J value in Hertz (Hz). The mass of all the compounds was determined by LC-MS of Agilent Technologies with an electrospray ionization source. Spectroscopic measurements were performed on a fluorometer and UV/VIS instrument, Synergy 4 from Bio-Tek and Gemini XS fluorescence plate reader, repsectively. The slit width was 1 nm for both excitation and emission. Relative quantum efficiencies were obtained by comparing the areas under the corrected emission spectrum. The following equation was used to calculate quantum yield: st is the reported quantum yield of the standard, I is the integrated emission spectrum, A is the absorbance at the excitation wavelength, and xη is the refractive index of the solvents used. The subscript x denotes unknown and st denotes standard. Rhodamine B was used as standard.Where ΦS.A., M.S. and Y.-T.C. designed research. M.S. constructed the microscopic system with infrared laser and S.A. performed the microscopic experiments. S.-C.L. and D.Z. synthesized dyes and tested cell viability. S.A., M.S. and Y.-T.C. wrote this paper. Y.-T.C. organized this study.Supplementary informationSupplementary movie


Comarostaphylis arbutoides  from neotropical montane forests are rarely described. To date, only mycorrhizal associations with the fungal species Leccinum monticola, Leotia lubrica and Sebacina sp. are known from literature. The genus Cortinarius is one of the most species-rich ectomycorrhizal taxa with over 2000 assumed species. In this study, two sites in the Cordillera de Talamanca of Costa Rica were sampled, where Com. arbutoides is endemic and grows together with Quercus costaricensis. Using a combined method of rDNA sequence analysis and morphotyping, 33 sampled mycorrhizal systems of Cortinarius were assigned to the subgenera Dermocybe, Phlegmacium and Telamonia. Specific plant primers were used to identify the host plant. Here, we present the phylogenetic data of all found Cortinarii and describe four of the arbutoid mycorrhizal systems morphologically and anatomically.Arbutoid mycorrhizas of  Comarostaphylis arbutoides is a tropical woody plant of Central America, occurring in dry oak-pine and cloud forests, as well as in the páramo at an elevation of c. 2500–3430 m a.s.l. Together with Arbutus and Arctostaphylos, it belongs to the ericaceous subfamily Arbutoidea, which are known to form arbutoid mycorrhizas with ectomycorrhizal fungi  and internal transcribed spacer (ITS) sequencing as well as phylogenetic analysis. Plant primers were used to sequence the ITS region of the host plant from the same mycorrhizal system as used for fungal analysis. According to Agerer  several different o Agerer , we presQ. costaricensis mixed with solitary individuals of Com. arbutoides. At site II , Com. arbutoides itself is the dominating species, mixed with a few isolated Q. costaricensis. Members of the Araliaceae (Schefflera and Oreopanax); Cunoniaceae (Weinmannia); Ericaceae ; Poaceae (Chusquea); Primulaceae (Myrsine) and Winteraceae (Drimys) form the understory vegetation.Sampling was conducted in a secondary cloud forest around the Mountain Cerro de la Muerte (3491 m a.s.l.) in the Cordillera de Talamanca of Costa Rica, 54 km southeast of the capital city of San José. Site I  is dominated by Com. arbutoides were collected during the rainy seasons in October 2010 and 2011. For this, a soil corer  was used at distances of 50 and 100 cm from the trunk. At the University of Costa Rica, turgid and apparently healthy morphotypes were sorted out using a stereomicroscope. Systems with the same morphological features  were assigned to one morphotype. For further analyses, the morphotypes were preserved in 2 % glutaraldehyde with a 0.1 M sodium cacodylate buffer , KM457023-KM457055 (LSU), KF419121 (Com. arbutoides) and KM978077 (Quercus sp.).Genomic DNA was isolated from one unramified root tip per morphotype, using the DNeasy Plant Mini Kit  following the manufacturer’s recommendations. To identify the mycorrhizal fungi at both family and species level, PCR amplification and sequencing of the internal transcribed spacer (ITS) region and the ribosomal nuclear large subunit (LSU) were performed. Here, the primer combinations ITS1F/ITS4 (Gardes and Bruns http://technelysium.com.au). Identity of obtained sequence data was confirmed by BLASTn search against the NCBI database (http://www.ncbi.nlm.nih.gov/) and the database UNITE . For phylogenetic analysis at species level, the datasets of ITS sequences provided by Peintner et al.  at room temperature until further processing. Thereafter, six washes in 0.1 M sodium cacodylate buffer were performed. Samples were postfixed in 1 % osmium tetroxide in the same buffer for 1 h under light exclusion at room temperature. After six washes in double-distilled water, samples were dehydrated by immersion for 15 min in 25, 50, 70 and 95 % acetone and three times for 1 h in 100 % acetone, respectively. The mycorrhizal tips were embedded in Spurr’s plastic Spurr  and sectCortinarius after sequence comparison with BLASTn in the NCBI database and UNITE. In NCBI, best matches were mainly received with samples originally from North America, whereas comparison in UNITE almost exclusively resulted in European species  is obtained by KKM 144 with Cortinarius comarostaphylii from Costa Rica; in UNITE C. leucophanes from Finland is the closest match (99 %) for this sample. KKM 132 achieved the lowest identity match in both databases (89 as well as 88 %) with Cortinarius exlugubris from New Zealand and Cortinarius terpsichores from Sweden, respectively.A total of 399 root tips were analysed genetically, of which 33 were assigned to the genus ies Tab. . In NCBICortinarius, the 33 samples can be assigned to three different subgenera: Dermocybe, Phlegmacium and Telamonia.The Bayesian and RAxML phylogenies, generated by ITS sequences are concordant. Both trees show the same grouping structure, supported by mainly higher posterior probabilities (PP) in the Bayesian analysis and lower bootstraps (BS) in the RAxML analysis Fig. . Within Dermocybe subgenus (PP 1/BS 91), whereas 13 samples are assigned to subgroup “Dermocybe I” (PP 1/BS 100), and a further two samples  to subgroup “Dermocybe II” (PP 0.99/BS 73) , whereas the cluster with KKM 132 received a good PP (0.93), but a weak BS support (53). The remaining 16 cortinarioid samples are assigned to the subgenus Telamonia and are distributed amongst three very well-supported clusters (PP 1/BS 96 to 100) as shown in Fig. Around half of the analysed cortinarioid sequences belong to the genus PP /BS 91, wQuercus sp. was proven as host plant 1* Mycorrhizal system silvery, rapidly displaced by water when touched, then mantle transparent; epidermal cells generally visible through mantle along the whole mycorrhizal system; rhizomorphs frequent, with smooth margins: 22 Mycorrhizal system densely silvery; all mantle layers with open anastomoses; no colour reaction with KOH and NH4OH: KKM 298 (Telamonia)2* Mycorrhizal system slightly silvery; only inner mantle layer with open anastomoses; colour reaction with KOH and NH4OH: 33 Mycorrhizal system ochre to yellowish brown, very tip yellowish; rhizomorphs ochre to yellowish brown; anastomoses of emanating elements likewise closed by a clamp or open: KKM 255; KKM 359; KKM 388 (Dermocybe)3* Mycorrhizal system brown, very tip brownish to greyish; rhizomorphs ochre to reddish brown; anastomoses of emanating elements closed by a clamp or rarely open: KKM 149 (Dermocybe)Identification key for the cortinarioid mycorrhizas:Mycorrhizal systems irregularly pinnate to dichotomous, with 0–1 orders of ramification, systems abundant and dense, up to 6.8 mm long, strongly hydrophobic, of medium distance fringe exploration type. Main axes 0.3–0.5 mm diameter. Unramified ends sinuous to tortuous, not inflated, cylindric, up to 1.1 (2.1) mm long and 0.2–0.3 mm diameter; mantle ochre to brownish, very tip ochre to yellowish, older parts dark brown and mycorrhizas not carbonising. Surface of unramified ends densely stringy, not smooth, very tip transparent and epidermal cells visible through mantle, older parts of mantle not transparent and occasionally, very tip partly silvery due to enclosed air. Rhizomorphs abundant, up to 0.12 mm diameter, roundish to flat in cross-section, emanating from all parts of the mycorrhiza, connection oblique, distal rhizomorphs connected over a long distance with mantle surface, growing into soil or organic layers, ochre to brownish, repeatedly ramified into smaller filaments, with hairy or even fan-like margins; rhizomorphs appear very wiry to brittle and are frequently found in short broken fragments around the mycorrhizal systems. Cystidia lacking. Sclerotia not observed.Fig.  MycorrhiOuter mantle layers densely plectenchymatous, hyphae irregularly to somewhat star-like arranged, rarely ramified, some hyphae in bundles, without any special pattern  μm long, 1.6–4.6 (6.4) μm in diameter, cell walls (0.2) 0.3–0.4 μm thick, smooth and yellow; hyphae rarely septate, clampless and constricted at septa, septa as thick as cell walls. Inner mantle layers densely plectenchymatous, hyphae irregular in shape, hyphae (4) 8–27 (63) μm long, 1.8–6.8 (8.3) μm in diameter, cell walls 0.3–0.4 μm thick, smooth and yellow; septa not observed. Very tip like other parts of the mantle.Fig.  Mantle lRhizomorphs undifferentiated, hyphae loosely interwoven and of uniform diameter  μm diameter, cell walls 0.3 μm, and distal ends of hyphae simple; ramification backwards-oriented or acute, with one side branch at septum, one or two hyphal diameter below the septum; septa with clamps, constricted at septa, backwards-oriented clamps not observed and septa as thick as hyphal walls; anastomoses rare and open with a short bridge, bridge as thick as hyphae, cell walls of anastomoses as thick as remaining walls. Cystidia not found.Fig.  Lacking Fig.  Mantle p4, guaiac, KOH 10 %, lactic acid, Lugol’s solution, Melzer’s reagent, NH4OH conc., sulpho-vanillin, H2SO4.Cotton blue: hyphae blue or greenish; toluidine blue: hyphae blue to violet. No reaction was observed with: acetic acid, ethanol 70 %, Fe(II)SOQ. costaricensis, soil core exc., myc. isol. Katja Kühdorf; KKM 132, 12 October 2010; mycorrhiza deposited by B. Münzenberger .Costa Rica, province of San José, canton of Pérez Zeledón, at mountain Cerro de la Muerte, Reserva Forestal Los Santos ), in a secondary cloud forest with Mycorrhizal systems irregularly pinnate to dichotomous, with 0–4 orders of ramification, systems nest-like, forming hyphal mats, up to 8.4 mm long, strongly hydrophobic, of medium distance fringe exploration type. Main axes 0.3–0.4 mm diameter. Unramified ends bent or sinuous, not inflated, cylindric, up to 1.8 (3) mm long, 0.2–0.3 (0.4) mm diameter; mantle and very tip white and yellowish to ochre, older parts light orange, not carbonising. Surface of unramified ends densely stringy or forming rings, not smooth, fan-like cottony between side branches and main axis, densely silvery by enclosed air, rapidly displaced by water when touched, then mantle transparent and epidermal cells visible through mantle. Rhizomorphs frequent, up to 0.25 mm diameter; flat in cross-section, emanating from all parts of the mycorrhiza, connection oblique, distal rhizomorphs connected over a long distance with mantle surface, growing into soil or organic layers, white, repeatedly ramified into smaller filaments, with smooth margins. Cystidia lacking. Sclerotia not observed.Fig.  MycorrhiOuter mantle layers densely plectenchymatous, hyphae irregularly to somewhat star-like arranged, occasionally ramified, without any special pattern, often with bundles of parallel hyphae  20–32 (50) μm long, (1.9) 2.3–4.3 (5.2) μm in diameter, cell walls 0.3 μm thick, irregularly inflated, smooth and colourless; hyphae with simple septa, occasionally with clamps, constricted at septa and septa as thick as cell walls and anastomoses open or closed, with short bridge, bridge thinner or as thick as hyphae. Inner mantle layers densely plectenchymatous, hyphae in bundles, forming ring-like structures, hyphae uneven in diameter, some hyphae epidermoid, sometimes ampullate at one side of septum and hyphae up to (9) 20–115 (200) μm long, 2.4–5.3 μm in diameter, cell walls 0.3 μm thick and colourless; hyphae with simple septa, rarely with clamp connection, constricted at septa, septa as thick as cell walls and anastomoses open, with short bridge, bridge thinner or as thick as hyphae, anastomoses closed, with long bridge, bridge bigger than hyphae. Very tip like other parts of the mantle.Fig.  Mantle lRhizomorphs undifferentiated, hyphae loosely interwoven and of uniform diameter  65–115 μm long, 2.4–4.8 μm diameter, cell walls 0.3 μm and distal ends of hyphae simple; ramification acute or approximately 90 °, with one side branch at septum and one or two hyphal diameter below the septum; septa with clamps, constricted at septa, backwards-oriented clamp observed only once, septa as thick as hyphal walls; anastomoses frequent, open with a short bridge, bridge thinner or as thick as hyphae and cell walls of anastomoses as thick as remaining walls. Cystidia not found.Fig.  Lacking Fig.  Mantle p4, guaiac, KOH 10 %, lactic acid, Lugol’s solution, Melzer’s reagent, NH4OH conc., sulpho-vanillin, H2SO4.Cotton blue: hyphae blue; toluidine blue: hyphae violet and cell content pink. No reaction was observed with: acetic acid, ethanol 70 %, Fe(II)SOQ. costaricensis, soil core exc., myc. isol. Katja Kühdorf; KKM 298, 4 October 2011; mycorrhiza deposited by B. Münzenberger .Costa Rica, province of San José, canton of Pérez Zeledón, at mountain Cerro de la Muerte, Estación Biológica de la Muerte ), in a secondary cloud forest with Mycorrhizal systems irregularly pinnate to dichotomous, with 0–2 orders of ramification, systems solitary or in small numbers to abundant and dense, up to 9.9 mm long, slightly hydrophobic, of medium distance fringe exploration type. Main axes 0.2–0.6 mm diameter. Unramified ends bent or sinuous, not inflated, cylindric, up to 1.5 (2.7) mm long, 0.2–0.3 mm diameter; mantle ochre to yellowish brown, very tip yellowish, older parts dark orange and mycorrhizas not carbonising. Surface of unramified ends loosely stringy to loosely cottony, not smooth, between side branches and main axis sometimes fan-like cottony, slightly silvery by enclosed air, rapidly displaced by water when touched, then mantle generally transparent; epidermal cells visible through mantle. Rhizomorphs frequent, up to 0.11 mm diameter; flat in cross-section, emanating from all parts of the mycorrhiza, connection oblique, distal rhizomorphs connected over a long distance with mantle surface, growing into soil or organic layers, ochre to yellowish brown, repeatedly ramified into smaller filaments, with smooth margins. Cystidia lacking. Sclerotia not observed.Fig.  MycorrhiOuter mantle layers loosely plectenchymatous, hyphae irregularly arranged, some hyphae in bundles, rarely ramified, without any special pattern  μm in diameter, cell walls 0.3 (0.4) μm thick, smooth and colourless; hyphae with simple septa, constricted at septa and septa as thick as cell walls; anastomoses open, with short bridge, bridge thinner or as thick as hyphae. Very tip like other parts of the mantle.Fig.  Mantle lRhizomorphs undifferentiated, hyphae loosely interwoven and of uniform diameter , with a short bridge or bridge almost lacking, bridge as thick as hyphae, cell walls of anastomoses as thick as remaining walls. Cystidia not found.Fig.  Lacking Fig.  Mantle p4OH conc.: rhizomorphs bright pink after a few minutes; toluidine blue: hyphae violet, cell content pink. No reaction was observed with: acetic acid, ethanol 70 %, Fe(II)SO4, guaiac, lactic acid, Lugol’s solution, Melzer’s reagent, sulpho-vanillin, H2SO4.Cotton blue: hyphae blue to patchy greenish; KOH 10 %: rhizomorphs bright pink; NHQ. costaricensis, soil core exc., myc. isol. Katja Kühdorf; KKM 255, 4 October 2011 and mycorrhiza deposited by B. Münzenberger . Further material studied Costa Rica, province of San José, canton of Pérez Zeledón, at mountain Cerro de la Muerte, Reserva Forestal Los Santos ), in a secondary cloud forest with Q. costaricensis, soil core exc., myc. isol. Katja Kühdorf; KKM 359 and KKM 388, 18 October 2011; mycorrhiza deposited by B. Münzenberger .Costa Rica, province of San José, canton of Pérez Zeledón, at mountain Cerro de la Muerte, Estación Biológica de la Muerte ), in a secondary cloud forest with Mycorrhizal systems irregularly pinnate to dichotomous, with 0–2 orders of ramification, systems abundant and dense, up to 11.3 mm long, slightly hydrophobic and of medium distance fringe exploration type. Main axes 0.3–0.5 mm diameter. Unramified ends sinuous to tortuous, not inflated, cylindric, up to 1.6 (2.5) mm long, 0.3 mm diameter; mantle brown, very tip brownish to greyish, older parts dark brown and mycorrhizas not carbonising. Surface of unramified ends loosely stringy to loosely cottony, not smooth, between side branches and main axis sometimes fan-like cottony, slightly silvery by enclosed air, rapidly displaced by water when touched, then mantle generally transparent and epidermal cells visible through mantle. Rhizomorphs frequent, up to 0.13 mm diameter, flat in cross-section, emanating from all parts of the mycorrhiza, connection oblique, distal rhizomorphs connected over a long distance with mantle surface, growing into soil or organic layers, ochre to reddish brown, repeatedly ramified into smaller filaments, with smooth margins. Cystidia lacking. Sclerotia not observed.Fig.  MycorrhiOuter mantle layers loosely plectenchymatous, hyphae irregularly arranged, rarely ramified, without any special pattern  μm diameter, cell walls 0.3 μm, and distal ends of hyphae simple; ramification acute or approximately 90 °, with one side branch at septum, one or two hyphal diameter below the septum, backwards-oriented ramification observed only once; septa with clamps, constricted at septa and septa as thick as hyphal walls; anastomoses frequent, closed by a clamp (contact clamp) or rarely open, with a short bridge or bridge almost lacking, bridge as thick as hyphae and cell walls of anastomoses as thick as remaining walls. Cystidia not found.Fig.  Lacking Fig.  Mantle p4OH conc.: rhizomorphs bright pink after a few minutes; toluidine blue: hyphae violet and cell content pink. No reaction was observed with: acetic acid, ethanol 70 %, Fe(II)SO4, guaiac, lactic acid, Lugol’s solution, Melzer’s reagent, sulpho-vanillin, H2SO4.Cotton blue: hyphae blue to patchy greenish; KOH 10 %: rhizomorphs bright pink; NHQ. costaricensis, soil core exc., myc. isol. Katja Kühdorf; KKM 149, 12 October 2010; mycorrhiza deposited by B. Münzenberger .Costa Rica, province of San José, canton of Pérez Zeledón, at mountain Cerro de la Muerte, Reserva Forestal Los Santos ), in a secondary cloud forest with Despite the efforts in investigating the biodiversity of Costa Rican Cortinarii, only Ammirati et al.  suppliedC. comarostaphylii. This species occurs in the Costa Rican páramo  hitherto described in DEEMY .  Palfner –2015.N. reticulosa” in showing similar plectenchymatous mantle structures. However, in contrast to KKM 132, “N. reticulosa” possess anastomoses in the outer mantle layer, a rough hyphal surface in the outer mantle layer as well as in the emanating elements, and simple septa as well as intrahyphal hyphae in the emanating hyphae.Anatomically, KKM 132 resembles “C. alnobetulae and “N. reticulosa”. However, both samples differ from KKM 132 in lacking reaction with cotton blue and toluidine blue, and a positive reaction with sulpho-vanillin, respectively. The likewise absent reaction with NH4OH of KKM 132 was not investigated in the other mycorrhizal samples of Phlegmacium.The phlegmacioid mycorrhiza of KKM 132 shows no reaction with KOH, an attribute in common with Telamonia are described as follows: Cortinarius armillatus  shows a brown to dark brown mantle with a reddish tint, as is also common with D. phoenicea. However, both described mycorrhizas show a distinctly transparent mantle clearly distinguishing them from the other Dermocybe species described in DEEMY so far.Currently, nine ECMs of the subgenus nnamomea , Dermocyntha Uhl , DermocyCuvelier , Dermocyanguinea , c, as winea Uhl . Due to Dermocybe species , as well as of the outer mantle layer (D. cinnamomeolutea) possess a rough surface. D. cinnamomeolutea and D. holoxantha have a matrix in the middle and inner mantle layers and, furthermore, none of these three species show a ring-like inner mantle layer as occurs in both, “Dermocybe I” and “Dermocybe II”. Anastomoses open or closed by a clamp, are very common in the emanating elements, whereas in “Dermocybe II”, open anastomoses are less frequently observed than in “Dermocybe I”. Within the described Dermocybe ECM only, D. phoenicea also feature these two types of anastomoses.Anatomically, the mycorrhizas of “Dermocybe I” and “Dermocybe II” do not differ much. Both types show a plectenchymatous mantle throughout undifferentiated rhizomorphs, infrequently with smooth margins (types A or B) and both form short anastomoses with contact clamps. These features are typical for s Agerer . Accordi4OH. A colour reaction with NH4OH was only tested in D. cinnamomea, where no reaction was observed. A reaction with KOH was investigated in seven of the nine Dermocybe species, where again, D. cinnamomea, as well as D. palustris showed a reaction, but which only affected the hyphal content or the mantle, respectively.The rhizomorphs of “Dermocybe I” and “Dermocybe II” show a bright pink colour reaction with KOH, as well as with NHQuercus sp., Com. arbutoides and Alnus acuminate. Even though oak species have an extensive distribution and are the most common ectomycorrhizal host plant for Cortinarius, they only occur in small numbers. Additionally, some Cortinarius species have very special habitat preferences (Liimatainen Cortinarius findings in this study primarily differ from Ammirati et al. (Dermocybe have not been reported yet for Costa Rica. Moreover, in contrast to Ammirati et al. (Cortinarius fruit bodies in this area.According to Ammirati et al. , Costa Ri et al. , and whyi et al. , who des