Studies have shown an increasing number of type 2 diabetes (T2D) patients with concomitant obesity and hyperlipidemia syndromes, resulting from relevant metabolic disorders. However, there are few medications and therapies, which can thoroughly address these issues. Therefore, the current study evaluated the efficacy and safety of using JTTZ, a Chinese herbal formula, to treat T2D with obesity and hyperlipidemia.  A total of 450 participants with T2D  ≥ 1.7 mmol/L) were randomly assigned, in equal proportions, to two groups in this multicenter randomized, positive-controlled, open-label trial. One group received JTTZ formula, and the other received metformin (MET) for 12 consecutive weeks. The primary efficacy outcomes were changes in HbA1c, TG, weight, and waist circumference. Adverse reactions and hypoglycemia were monitored. β. In addition, symptoms were significantly relieved in participants in the JTTZ group compared to those in the MET group. One case of hypoglycemia was reported in the MET group. No severe adverse events were reported in either group.  HbA1c decreased by 0.75 ± 1.32% and 0.71 ± 1.2% in the JTTZ and MET groups, respectively, after 12 weeks of treatment. TG levels in the JTTZ and MET groups were reduced by 0.64 ± 2.37 mmol/L and 0.37 ± 2.18 mmol/L, respectively. Weight was decreased by 2.47 ± 2.71 kg in the JTTZ group and by 2.03 ± 2.36 kg in the MET group. JTTZ also appeared to alleviate insulin resistance and increase HOMA-β cell function for T2D patients with obesity and hyperlipidemia. The JTTZ formula has shown that it could potentially be developed as an alternative medicine for patients with T2D, particularly those who cannot tolerate metformin or other hypoglycemic drugs. This trial was registered with Clinicaltrials.gov NCT01471275. The JTTZ formula led to safe and significant improvements in the blood glucose, blood lipids, and weight levels; relieved symptoms; and enhanced  Type 2 diabetes (T2D) is a complex progressive metabolic disorder, which poses a significant threat to human health and accounts for more than 91% of all diabetes cases. In 2015, it was estimated that 415 million adults suffered from diabetes mellitus, representing 9.1% of the global adult population. China has the largest number of diabetes patients worldwide—up to 109 million . T2D patTreatments for T2D are aimed at balancing the antihyperglycemic efficacy, risk of inducing hypoglycemia, risk of weight gain, tolerability, and other adverse effects. When diabetes is associated with obesity and hyperlipidemia, it is recommended that the algorithm for the comprehensive management of T2D be used . HoweverTreatise on Febrile Diseases  and used for stomach heat syndrome in ancient China. We have used JTTZ formula in our clinical application for thousands of patients, which was satisfying in lowering blood glucose, blood lipids, and weight levels. In the current study, the preliminary efficacy and safety of the JTTZ formula were evaluated for the treatment of T2D with obesity and hyperlipidemia and were also compared to those of the first-line antidiabetic oral agent, metformin.In previous studies, Chinese herbal medicines have been shown to effectively reduce blood glucose –12. HoweNCT01471275.The research was a multicenter randomized, positive-controlled, open-label clinical trial, which included a 4-week screening period and a 12-week treatment period. The study was approved by the Ethics Committee of Guang'anmen Hospital of the China Academy of Chinese Medical Sciences (number eighty-sixth 2011) and was conducted in accordance with the principles of the Declaration of Helsinki. Participants were recruited by nine branch centers, and all participants signed informed consents before the study. The registration number is Patients were diagnosed with T2D via fasting plasma glucose (FPG) tests and 75 g oral glucose tolerance tests (OGTT). Hyperlipidemia and obesity were diagnosed via measurements of triglyceride level (TG) and waist circumference (WC).(1) Informed consent was signed. (2) WC was ≥90 cm in males and ≥80 cm in females. (3) Patients were newly diagnosed with T2D, according to the 1999 World Health Organization criteria  ≥ 11.1 mmol/L), after a screening period (4-week diet and exercise therapy), and had not previously received pharmacological treatment. (4) Patients had a glycosylated hemoglobin (HbA1c) ≥ 7.0% and an FPG < 13.9 mmol/L. (5) TG levels were ≥1.7 mmol/L but <5.65 mmol/L. (6) Patients were between 18 and 70 years of age.The exclusion criteria are as follows: (1) patients who had used insulin therapy or previously undergone continuous treatment for diabetes lasting 3 months or longer, including Chinese and western medicine, physical therapy, psychological therapy, and health food; (2) patients who received hypoglycemic or hypolipidemic medication within the previous month; (3) patients with diabetic complications  or patients with diabetes accompanied by other serious primary diseases; (4) a systolic blood pressure (SBP) ≥ 160 mmHg or diastolic blood pressure (DBP) ≥ 100 mmHg; (5) patients that had diabetic ketosis, diabetic ketoacidosis, or severe infections within the previous month; (6) patients with psychiatric disease; (7) patients who were pregnant, who were planning to become pregnant, or who were breast-feeding; (8) patients who were allergic to the JTTZ formula or any of its major components or who had allergic constitutions; (9) patients involved in other clinical trials currently or within the previous month; (10) alcohol abuse and/or use of psychoactive substances or drug abuse and dependency within the past 5 years; (11) patients who were less likely to complete the study, according to the researcher's judgment, such as those being lost to follow-up due to frequent changes in work or living environments; (12) fluctuating dosage and category of antihypertensive drugs; (13) patients taking diet pills or health food that had an effect on body weight; (14) patients with impaired liver and renal function  and aspartate aminotransferase (AST) 2 times greater than the upper limit of normal and serum creatinine (Cr) greater than the upper limit of normal); and (15) patients that had asymptomatic hypoglycemia.The dose selection of metformin follows the “small starting dose” principle  and the (1) Participants experienced specific physiological changes or serious diabetic complications . (2) Participants had poor compliance, with test medication use less than 80% or more than 120% of the prescribed dose. (3) Participants had violated the protocol . (4) Participants withdrew from the study voluntarily. Participants had the right to withdraw from the trial voluntarily, according to the informed consent. Reasons for withdrawal were noted as much as possible, and the efficacy and any adverse reactions were recorded.Aloe vera), Huanglian (Coptis chinensis), Zhimu (Rhizoma Anemarrhenae), Hongqu (red yeast rice), Kugua (Momordica charantia), Danshen , Wuweizi (Schisandra chinensis), and Ganjiang (dried ginger). The dose was given in the form of boil-free granules. Herbs were boiled, filtered, concentrated into a cream, and dried into granules. The Jiangyin Tianjiang Pharmaceutical Co. Ltd.  provided all the herbs, and herbs were quality controlled in accordance with the Pharmacopoeia of the People's Republic of China (2010). The manufacturer was not involved in the design or analysis of this study. The granules were packed into individual bags. Participants in the JTTZ group took the granules orally 2 times per day, at mealtimes. The method of taking the medication is just like drinking an instant coffee: put the granules into a cup, and then pour some hot water and stir well.The JTTZ formula was composed of 8 herbs, including Luhui . Participants in the control group took 0.25 g metformin tablets orally 3 times per day. Participants in both groups underwent treatment for 12 weeks.μL test sample was injected into the column and eluted at a constant flow rate of 2 mL/min. Water with 0.02% phosphoric acid (mobile phase A) and acetonitrile (mobile phase B) was used. Composition was determined at a wavelength of 237 nm. The JTTZ formula powder was dissolved in methanol. All solutions were filtered through 0.45 μm nylon membrane Millex syringe filters before use. Chemical structures of the eight major compounds  were identified in the finished dose. Representative chromatograms of the JTTZ formula and the single granule and chemical structures of the major compounds are shown in The chemical composition of the JTTZ formula was analyzed using high-performance liquid chromatography (HPLC). The separation was carried out on a Merck Chromolith RP-18e column (4.6 × 100 mm inner diameter). For HPLC analysis, a 5 β cell function index (HOMA-β), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), body mass index (BMI), and hip circumferences (HC); and symptom scores of the participants. Measurements of FPG, weight, WC, and HC were done every 4 weeks; other assessments were performed before and after the 12-week treatment phase.The primary efficacy outcomes were changes in HbA1c, TG, weights, and WC. Secondary efficacy outcomes were the target achievement rates for HbA1c and TG; changes in FPG, 2 h PG, insulin resistance index (HOMA-IR), 2) was calculated according to height and weight. Plasma insulin level was measured, and the homeostatic model assessment was performed to quantify HOMA-IR and HOMA-β.Evaluation of 2 h PG was done using a standard 75 g OGTT. Body mass index . Secondary symptoms were other symptoms of gastrointestinal excess heat syndrome. Tongue and pulse presentations were divided into 2 categories (0 being no and 2 being yes), and the rest of the symptoms were divided into 4 levels and were quantified as scores 0, 1, 2, and 3 (3 being the most severe).Vital signs; routine blood, urine, and stool samples; electrocardiograms; and liver and renal function  were all used to evaluate safety. Safety indexes were tested every 4 weeks. Hypoglycemia is divided into severe hypoglycemia and nonsevere hypoglycemia. Severe hypoglycemia refers to a disturbance of consciousness that requires help from others. Neurological symptoms are significantly improved or disappeared after hypoglycemia has been corrected. Nonsevere hypoglycemia refers to diabetic patients with blood glucose levels ≤ 3.9 mmol/L after taking medication, which may have hypoglycemia symptoms, but no disturbance of consciousness . AdverseAn Interactive Web Response System designed by the Clinical Evaluation Center of China Academy of Chinese Medical Sciences was used in this study to produce a random code for the trial group and control group in equal proportions. Each participant was assigned a random number, and the drugs were distributed according to the number. Case report forms were obtained at the end of the experiment, and the data were analyzed by an independent third-party statistical agency.t-test or Wilcoxon rank sum test was used to analyze differences between two groups. In each group, changes between baseline (before treatment) and posttreatment (end of the study) were analyzed using a comparative t-test; the primary endpoints were compared using a covariance analysis model with the baseline as the covariate. Enumeration data was described using frequency. Changes, which occurred during the period before and after the treatment, were compared using the CMH χ2 test; the incidences of adverse events between the two groups were compared by the CMH χ2 test or Fisher exact probability test. All statistical tests were two-sided. A significance level of 5% was used, and 95% confidence intervals were calculated.Data was entered using the EpiData3.1 software . Data was analyzed using the SPSS 20.0 software . Measurement data is described as mean ± standard deviation. A n = 225) and the control group (metformin (MET); n = 225). In the JTTZ group, 216 subjects completed the study and 215 were included in the PPS analysis. In the MET group, 212 subjects completed the study and 199 were included in the PPS analysis , 450 subjects were enrolled and randomly divided into the trial group , with a 0.75 ± 1.32 reduction (95% CI = 0.58–0.93) in the JTTZ group and a 0.71 ± 1.2 reduction (95% CI = 0.54–0.87) in the MET group.At week 12, the HbA1c levels (%) in the JTTZ and MET groups were 7.51 ± 1.44 and 7.57 ± 1.42, respectively. HbA1c level was significantly lower in both groups, compared to that at baseline (P < 0.001) in the JTTZ group and by 0.37 ± 2.18 (P < 0.05) in the MET group, compared to that at baseline. The 95% CI was 0.32–0.96 in the JTTZ group and 0.06–0.67 in the MET group  in the JTTZ group and MET groups were 2.79 ± 1.86 and 2.82 ± 1.91, respectively. The level was significantly reduced by 0.64 ± 2.37 .The HbA1c value in 45.1% (97/215) of the subjects in the JTTZ group and 42.7% (85/199) in the MET group achieved the target, using HbA1c ≤ 7.0% as the target achievement. There was no statistical difference between the two groups (CMH P < 0.001).The FPG levels (mmol/L) in the JTTZ and MET groups were 8.24 ± 2.35 and 8.21 ± 2.23, respectively, at week 12. The level was significantly reduced by 1.4 ± 2.4 in the JTTZ group and by 1.33 ± 2.33 in the MET group compared to that at baseline (P < 0.001) (The 2 h PG levels (mmol/L) in the JTTZ and MET groups were 14.52 ± 4.73 and 14.83 ± 4.32, respectively, at week 12. The level was significantly reduced by 2.42 ± 4.53 in the JTTZ group and by 2.18 ± 4.48 in the MET group (< 0.001) .χ2 = 0.035, P = 0.852).The TG value in 27.4% (59/215) of the subjects in the JTTZ group and 27.1% (54/199) of the subjects in the MET group achieved the target, when using TG ≤ 1.7 mmol/L as the target achievement. There was no statistical difference between the two groups (CMH P < 0.001), compared to that at baseline.The TC levels (mmol/L) in the JTTZ and MET groups were 5.06 ± 1.00 and 5.21 ± 1.00, respectively, at week 12. The level was significantly reduced by 0.39 ± 0.99 in the JTTZ group and by 0.36 ± 1.16 in the MET group (P < 0.001). The reduced levels of LDL-C showed no obvious differences between the two groups (The LDL-C levels (mmol/L) in the JTTZ and MET groups were 2.92 ± 0.73 and 2.98 ± 0.76, respectively, at week 12. Compared with the baseline, both groups had a very significant difference, respectively (o groups .P < 0.001). The JTTZ group decreased more than did the MET group in each project; however, there was no obvious difference between the changes ( changes .P < 0.01) and by 0.16 ± 0.82 (P < 0.05) in the MET group, compared to that at baseline.At week 12, the HOMA-IR levels in the JTTZ and MET groups were 1.39 ± 0.68 and 1.35 ± 0.67, respectively. The level was significantly reduced by 0.19 ± 0.91 in the JTTZ group . The increased levels of HOMA-β showed no obvious differences between the two groups (The HOMA-o groups .P < 0.001). The 95% CI was 4.67–5.80 in the JTTZ group and 1.64–2.79 in the MET group. The level decreased significantly more in the JTTZ group than in the MET group (P < 0.001).Participant symptom score level in the JTTZ and MET groups were 5.80 ± 4.05 and 8.57 ± 4.40, respectively, at week 12. The level was reduced by 5.23 ± 4.20 in the JTTZ group and by 2.21 ± 4.11 in the MET group, compared to that at baseline (P < 0.05). Comparison of symptom scores and symptom disappearance rates is shown in After the 12-week treatment, the following symptoms had improved more in the JTTZ group than in the MET group: thoracoabdominal distension, constipation, bitter mouth, halitosis, increased eating with rapid hunger, red tongue and yellow tongue coating, and powerful and rapid pulse (P = 0.490). There were two cases of transient slight ALT and AST elevation in the both groups. One case of nonsevere hypoglycemia was reported in the MET group. No abnormal ECGs, renal function tests (serum creatinine), or routine blood tests were observed during the trial.No severe adverse events were reported during the trial. Thirteen nonsevere adverse events (6.0%) were reported in the JTTZ group, and nine nonsevere adverse events (4.5%) were reported in the MET group (2 due to the risk of lactic acidosis [Metformin is a very effective antihyperglycemic and is recommended as a first-line choice for the treatment of T2D . In addiacidosis , 25. Furacidosis , 27.β levels and decreases in HOMA-IR levels. The TG levels were significantly reduced by 0.64 mmol/L in the JTTZ group and by 0.37 mmol/L in the MET group. Weight levels in the JTTZ and MET groups were decreased by 2.47 kg and 2.03 kg, respectively. Although there was no significant difference between the changes, using the JTTZ formula provided a greater reduction than using metformin in progression. Furthermore, in contrast to metformin, the JTTZ formula significantly improved patient symptoms. One case of hypoglycemia was reported in the MET group, and both groups had no severe adverse events.In this current study, after 12-week treatment, significant reductions in blood glucose, blood lipid, and weight levels were observed in both the JTTZ formula group and the metformin group. The HbA1c level was decreased by 0.75% in the JTTZ group, which had no obvious difference when compared to the MET group with a 0.71% reduction. Compared to baseline, both groups had significant improvements in HOMA-Aloe vera gel extract resulted in a reduction in fasting blood glucose, hepatic transaminases, plasma and tissue cholesterol, triglycerides, free fatty acids, and phospholipids in diabetic rats after 21 days of treatment [Salvia miltiorrhiza has cardiovascular protective effects, and Schisandra chinensis has antioxidant and hepatoprotective effects [Previous studies demonstrated that many active ingredients in the traditional Chinese medicine formula, JTTZ, can also effectively manage diabetes, obesity, and dyslipidemia. Some researchers have shown that mangiferin in Rhizoma Anemarrhenae has a positive effect on weight loss, lowering blood sugar and fat, decreasing arteriosclerosis, and improving cardiovascular complications –30. Berbreatment . Moreove effects , 34.β cell function, and relieved symptoms. It may be used as an alternative medicine for those with T2D and concomitant obesity and hyperlipidemia, which is supposed to be paid more attention in the treatments of type 2 diabetes, particularly in patients who cannot tolerate metformin or other hypoglycemic drugs. The limitation of this study was that the 12-week duration of the trial was relatively short. Maybe longer-term observation and mechanism research will demonstrate the formula effects more clearly.A comprehensive management of glycemic control and cardiovascular risk factors such as lipids and weight is crucial , 36. Thi


Aedes albopictus and Aedes triseriatus larvae in a controlled laboratory dose-response experiment. In the field, zinc concentrations were as high as 7.26 mg/L in a single tire and averaged as high as 2.39 (SE ± 1.17) mg/L among tires at a single site. Aedes albopictus  and A. triseriatus  were the most widespread mosquito species, co-occurred in over half  of all tires, and A. triseriatus was only collected without A. albopictus in one tire. Aedes triseriatus was more strongly negatively associated with zinc concentration than A. albopictus, and another common mosquito, C. pipiens, which was found in 17 tires. In the laboratory experiment, A. albopictus per capita rate of population change (λ′) was over 1.0, indicating positive population growth, from 0–8.9 mg/L zinc concentration , but steeply declined to zero from 44.50–89.00 mg/L zinc . In contrast, A. triseriatus λ′ declined at the lower concentration of 0.05 mg/L zinc (100 mg/L tire leachate), and was zero at 0.45, 8.90, 44.50, and 89.00 mg/L zinc . These results indicate that tire leachate can have severe negative effects on populations of container-utilizing mosquitoes at concentrations commonly found in the field. Superior tolerance to tire leachate of A. albopictus compared to A. triseriatus, and possibly other native mosquito species, may have facilitated the replacement of these native species as A. albopictus has invaded North America and other regions around the world.Discarded vehicle tire casings are an important artificial habitat for the developmental stages of numerous vector mosquitoes. Discarded vehicle tires degrade under ultraviolet light and leach numerous soluble metals  and organic substances  that could affect mosquito larvae that inhabit the tire casing. This study examined the relationship between soluble zinc, a common marker of tire leachate, on mosquito densities in tire habitats in the field, and tested the effects of tire leachate on the survival and development of newly hatched  The distributions and abundances of adult mosquitoes are strongly affected by processes occurring at the developmental  life stages . Larval Discarded vehicles are an important artificial habitat for the developmental stages of numerous vector mosquitoes . Rubber Aedes albopictus, the Asian tiger mosquito, was introduced to the continental United States in the mid-1980s through shipments of eggs and larvae in used tires , which is a composite index based on individual fitness parameters: survivorship, female development time, and female wing length (as a fecundity surrogate).In this study, we will examine the relationship between tire leachate and densities of mosquitoes in discarded tires in the field, and test the effects of tire leachate on the survival and development of material , but a rmaterial . Therefomaterial . In our material . In mosqMosquitoes were sampled from 42 tires among five sites (6–14 tires per site) in College Park  and Baltimore , Maryland, USA. All sampling was conducted from July to August 2011, which is the period of peak mosquito activity in the area . WeatherTire leachate was prepared by extracting ground tire material, minus 30 mesh (less than 0.59 mm) 99.51% dry content, with deionized water (DI) in either Teflon or Teflon coated polypropylene bottles at a solid to liquid ratio of 1:10 . A blank, containing only DI water was also prepared. The samples were shaken for a one-week period on an orbital table shaker at approximately 100 rpm at room temperature (19–22 °C). At the conclusion of the one-week period, the samples were filtered in a class 1,000 laminar flow clean bench through a sterile 0.2 µm filter, into a sterile 1,000 ml polycarbonate container. The filter membrane was surfactant-free Cellulose Acetate (CA); this membrane has no wetting agents that might affect sensitive cell culture lines and is cleaner than ordinary CA membranes for tissue culture use. The sample in the sterile container was bagged and refrigerated prior to usage. For conformity and comparison with earlier studies in the literature, this stock solution was designated as 100% tire leachate at a concentration of 100,000 mg/L. Analysis on this 100% solution using an ICP-OES indicated that it had a zinc concentration of 89 mg/L.A. albopictus and A. triseriatus, which were the two most widespread mosquito species from our field study (see Results), and for which we also had readily available individuals. Aedes albopictus and A. triseriatus eggs sourced from F1−2 colonies at University of Maryland were synchronously hatched in nutrient broth solutions in plastic trays. Within 24 h, 25 newly hatched larvae of each species were rinsed and added to 400 ml cups containing one of six concentrations of tire leachate using dilutions of the 100% stock solution and DI water. The six dilutions and their zinc concentrations (mg/L) were: 100.0% (89.000 mg/L), 50.0% (44.500 mg/L), 10.0% (8.900 mg/L), 1% (0.890 mg/L), 0.5% (0.445 mg/L), and 0.1% (0.089 mg/L). Each concentration × species combination had five replicates to yield 70 total experimental cups (7 concentrations × 2 species × 5 replicates). Cups were housed in an incubator at 25 °C and 16:8 h light-dark cycle. Each cup received 0.1 mg of bovine liver powder:lactalbumin (diluted 1:10). Pupae were removed from containers daily and placed into individual vials until adult emergence. Adults were sexed, identified, dried  and weighed, and females’ wings were measured. For each cup, proportion survivorship, median female development time, and median female mass were calculated. These fitness parameters were used to estimate the finite rate of population growth for each species λ′, , x is the mean time to eclosion (measured in days), Ax is the mean number of females eclosing on day x, wx is the mean body size on day x, and f(wx) is a function describing size dependent fecundity for each species, estimated from the mean wing length on day x, wx, of female mosquitoes  =  − 121.240 + 78.02wx, where wx is wing length (millimeters) (A. triseriatus was f(wx) = (1∕2)exp[4.5801 + 0.8926(lnwx)] − 1  . The funwx)] − 1 . D is thbopictus  and 12 dseriatus .A. albopictus, A. triseriatus, and C. pipiens (see Results) were analyzed using negative binomial regressions . Because a species’ abundance in a tire may be predicted by various physiochemical and biological container characteristics as well as surrounding landscape variables, we also included site, detritus amount, TN, TP, and the abundances of the other two mosquito species in an initial multi-factor model for each species. Final multi-factor models were selected using backward selection. If there was no significant loss of fit, as evaluated by AIC, we continued to eliminate the next least significant variable until all non-significant variables were removed or until there was significant loss of model fit . Correlations among predictor variables were tested using Pearson or Spearman rank correlations (SAS PROC CORR) and multicolinearity was checked by means of Variance Inflation Characteristics (VIF) (SAS PROC REG), with a VIF above 5 for a variable indicating a problem (λ′) failed to meet parametric assumptions despite transformations, and generalized models using variable distribution models  routinely failed to converge. Tire leachate concentration was treated as a categorical variable because preliminary data plots clearly showed that its relationship with each response variable was not linear. For all analyses, randomization ANOVA yielded conclusions identical to those of parametric ANOVA, probably because trends among treatment groups were clear (see Results). For brevity and accuracy, we report only the results from randomization ANOVA. Experimentwise α = 0.05 for all statistical analyses.Relationships between zinc concentrations and field abundances of each of the three collected mosquito species:  problem ; however problem  because Aedes albopictus, A. triseriatus and C. pipiens were collected from 88% (37/42), 55% (23/42), and 41% (17/42) of tires, and constituted 55% (2941/5383), 8% (415/5383), and 38% (2027/5383) of total immatures, respectively. Aedes albopictus and A. triseriatus co-occurred in individual tires the most frequently among all pairwise species combinations, being collected together in 22 tires. Aedes triseriatus was only collected without A. albopictus in one tire. Aedes albopictus and A. triseriatus co-occurred with C. pipiens in 17 and 15 tires, respectively. There was considerable variation among individual tires and sites in mosquito abundances and physiochemical characteristics of tires  was most strongly negatively associated with zinc concentration, followed by A. albopictus , and C. pipiens . Additionally, A. albopictus was associated positively with detritus amount ; A. triseriatus was positively associated with A. albopictus  and C. pipiens ; and C. pipiens varied with site (P = 0.0080) and was positively associated to A. triseriatus  and TN . TP was excluded from final models because it was always non-significant .of tires . Zinc coof tires . The throf tires . In finaλ′, P < 0.0001) and survival (P < 0.0001) both showed a significant interaction between species and tire leachate concentration. Aedes albopictus had similar λ′ than A. triseriatus in the control treatment (0 mg/L) but clearly divergent responses to increasing tire leachate concentration. Aedes albopioctus λ′ was consistently high and similar from 0–10,000 mg/L tire leachate but steeply declined to zero from 50,000 to 100,000 mg/L. In contrast, A. triseriatus λ′ declined at the lower concentration of 100 mg/L tire leachate, and was zero at 500, 10,000, and 100,000 mg/L  and mass  of both sexes, nor were any significant interactions of tire leachate with species  , and A. albopictus had faster female development than A. triseriatus (P < 0.0001) (P = 0.3000).Estimated finite rate of population increase (000 mg/L . Survivoleachate . There w 0.1000) . Aedes t 0.0001) . Developλ′ and survival of both the invasive mosquito A. albopictus and the native congener A. triseriatus, but that the invasive A. albopictus was clearly more tolerant. In field tires, A. triseriatus was more strongly negatively associated with zinc concentration compared to A. albopictus and another common co-occurring mosquito, C. pipiens. In our laboratory experiment, A. albopioctus λ′ was over 1.0, indicating positive population growth, and survival was high from 0–10,000 mg/L tire leachate, but both λ′ and survival steeply declined to zero from 10,000 to 100,000 mg/L. In contrast, A. triseriatus λ′ and survival declined at the lower concentration of 100 mg/L tire leachate, and was zero at 500, 10,000, and 100,000 mg/L.This study showed that tire leachate had negative effects on A. triseriatus after A. albopictus invasion by releasing compounds that are more toxic to A. albopictus. Instead, our data suggest that tire habitats may in fact support the spread of A. albopictus and the concomitant decline in A. triseriatus. Three of the five field sites that we sampled had tire water with mean zinc concentrations (1.35–2.39 mg/L) in excess of concentrations that caused a decline in A. triseriatus survival and λ′ (0 − 0.45 mg/L) in the laboratory experiment. This result, combined with the strong negative association between A. triseriatus and zinc concentrations in the field, suggests that A. triseriatus is likely to be negatively affected by tire leachate under many field conditions. Mean A. triseriatus λ′ was <1.0 across all tire leachate concentrations in our experiment, including in the control concentration that consisted only of DI water. This result suggests that our baseline experimental conditions were generally unfavorable to A. triseriatus, possibly because of food limitations. Nevertheless, the decline in A. triseriatus performance with even relatively small increases in leachate concentration was clear and not apparent in A. albopictus, which only showed declines in population performance in conditions higher than 8.9 mg zinc/L. Since its establishment in North America in the mid-1980s, A. albopictus has rapidly become the most common urban mosquito in many areas in the eastern United States. There are several explanations for this dominance; the most well documented being that A. albopictus is a superior competitor at the larval stage  and synthetic  toxins via a number of biochemical mechanisms, and among the most well documented are P450 monoxygenases (P450s) . The focus of A. albopictus mosquito larvae more tolerant to commercial insecticides through P450 induction, and the study found evidence that it did . Aedes  biomass . Overall (P450s) . It posst it did .Aedes mosquito species, A. albopictus and A. triseriatus, and it showed clear interspecific differences. Additional research needs to examine the suite of density dependent and trait-mediated impacts related to vector mosquitoes, including the effects on population performances of a larger number of tire-inhabiting species, on community processes, such as competition, predation, parasitism and mutualism, and on vector competence across a range of disease systems.The effects of tire leachate on mosquitoes are likely to be complex and act at both the immature  and adult life-stages. The main goal of this study was to compare effects of tire leachate on the population performances of two co-occurring 10.7717/peerj.3756/supp-1Data S1Click here for additional data file.10.7717/peerj.3756/supp-2Data S2Click here for additional data file.


The prevalence of thyroid dysfunction is high in Isfahan, an area of iodine sufficient in Iran. The aim of this study is to investigate the incidence of thyroid dysfunctions in adults of metropolitan Isfahan and to determine the role of thyroid autoantibodies.In a population-based cohort study in 2006–2011, we measured TSH, T4, T3, thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TgAb) in 618 out of 2254 people who were euthyroid in 2006. The incidence rates per 1000 person-year (pr) were calculated. The odds ratio (OR), relative risk (RR), and 95% confidence interval (95% CI) were calculated based on logistic regression to quantify the potential predictors of thyroid dysfunction. The receiver-operator characteristic (ROC) analysis along with area under the curve (AUC) was used to determine the optimal cutoff values for baseline TPOAb and TgAb as predictors of thyroid dysfunction.P = 0.01 and 0.817 (0.600–1.035) P = 0.033, respectively. There is a statistically significant association of hypothyroidism and hyperthyroidism with positive TPOAb .Within a 6-year follow-up, the incidence rate of hypothyroidism was 3.3 in women and 2.1 in men while the incidence rate of hyperthyroidism was 3.8 in women and none in men per 1000 (person-year). A cutoff value of TPOAb at 38 IU/mL was obtained to differentiate the patients with hypothyroidism and hyperthyroidism, with specificity of 0.75 and sensitivity of 0.76, and AUC (CI 95%) of 0.882 (0.743–1.02), The incidence rate of thyroid dysfunction is high in Isfahan, and higher TPOAb concentration is its strong predictor. Thyroid diseases are the most common endocrine disorders following the diabetes . ThyroidMany cross-sectional studies have been performed to investigate the prevalence of thyroid disorders in various populations –5. HowevThe prevalence and incidence rate of thyroid disorders are associated with several factors including geographic areas, senility, ethnicity, and the amount of iodine intake , 14. FurThe main aim of the current study is to investigate the incidence of thyroid dysfunction in Isfahani adult population in 2011 who were euthyroid in 2006 in an area, which has been iodine sufficient within last decades , 20. We A population-based cohort study was performed in Isfahan, a metropolitan city located in the center of Iran, from 2006 to 2011. The study is detailed elsewhere , 21.n = 618 out of 2254) who were diagnosed with euthyroid in 2006 were followed up. We have lost contacts with the rest of euthyroid population due to the change of addresses. The people gave informed consent and agreed to participate in the study. The ethics committee of the Isfahan University of Medical Sciences approved the protocol for this study according to Declaration of Helsinki.Roughly one-fourth of people (2).These patients were invited to the Thyroid Clinic of Isfahan Endocrine and Metabolism Research Center (IEMRC) and were examined by a trained general practitioner and an endocrinologist who were collaborator of the 1st phase of the study in 2006. The same laboratory instruments was used to conduct this study by the same personnel who performed the first phase of the research. All data including demographic, age of menopause, number of parity and abortions, past history of thyroid disorders and smoking, use of medications, especially drugs that interfere with thyroid function, and the past medical history were collected. The patients’ thyroids were also examined physically for goiter or nodule. The height and weight were measured and body mass index (BMI) was calculated by dividing weight (kg) to the height square  and thyroid globulin antibody (TgAb), and fasting plasma glucose (FPG) after 8 h of fasting. Serum total T4 and T3 were analyzed by radioimmunoassay . T4 intra- and inter-assay CV were 4.7 and 4.9%, respectively. Normal range of T4 concentration was 4.5–12.0 μg/dL. T3 intra- and inter-assay CV were 5.2 and 3.9%, respectively. Normal range of T3 concentration was 0.92–2.79 nmol/L.Serum TSH concentration was assessed by immunoradiometric assay . Intra-assay and inter-assay coefficient of variation (CV) was 1.5 and 1.9%, respectively. The normal range for TSH was 0.3–3.6 mIU/L. Serum TPOAb and TgAb were measured with Rapid ELISA (Genesis Diagnostic Products Corp.). The intra-assay and inter-assay CV for TPOAb were 7 and 5%, respectively. It was less than 12% for TgAb. TPOAb and TgAb concentrations of more than 75 and 100 IU/mL, respectively, were considered as positive. FPG (mg/dL) was measured by photometric method (Pars Azmon kit Lot number: 94011).Euthyroid was depicted as follows: 0.3 mIU/L ≤ TSH ≤ 3.6 mIU/L, and the definition of hypothyroidism was described as follows: overt  hypothyroidism (TSH > 10 mIU/L), subclinical hypothyroidism (10 ≥ TSH > 3.6 mIU/L). Definition of hyperthyroidism was described as overt  hyperthyroidism  and subclinical hyperthyroidism .131I and were known case of postablative hypothyroidism, according to their medical documents. Therefore, results summarized in this report are obtained from 615 participants with no previously known thyroid dysfunction (undiagnosed thyroid function); however, the data gained from these 3 patients are included in the calculation of incidence rate (n = 618).Within 2006–2011, three of people who were euthyroid in 2006 had got thyroid dysfunction and were on treatment. One of them was a known case of overt hypothyroidism. The other two patients were overt hyperthyroidism who had received Statistical analyses are performed using statistical package for social science . Normality of quantitative data is evaluated using Kolmogrov–Smirnov and Q–Q plot. Logarithmic transformation is conducted for positive skewed variables. Quantitative and categorical data are expressed as mean (SD) or median (interquartile range) [IQR] and frequency (percentage), respectively. Chi-square test is used for comparing categorical data while analysis of variance (ANOVA) is used for continuous quantitative variables. Crude incidence rate of thyroid dysfunctions per 1000 person-years are calculated in total study sample as well as in different age- and gender-specific groups. To determine the association between TPOAb as independent variable and thyroid dysfunction, we have adopted binary logistic regression analysis in different models. In all analyses, thyroid dysfunction is considered as endpoint. In these analyses, after obtaining relative risk (RR) and 95% confidence interval (95% CI) in crude model, adjustments are made for age, gender, smoking, BMI, positive family history to the first model. Additional adjustment is made for number of parity and abortion, age of menopause, having history of goiter or nodule in the second model. The predictive baseline values of TPOAb levels for different thyroid dysfunctions are evaluated using receiver operating characteristic curve (ROC) analysis, and area under the curve (AUC) and its 95% CI are calculated.131I.The total number of participants who were euthyroid in 2006 and enrolled in follow-up study (2006–2011) was 618. Three of the participants were classified as known cases of thyroid dysfunction, one female (46 years old) with overt hypothyroidism on levothyroxine treatment. Two female (57 and 66 years old) patients had known overt hyperthyroidism and were treated with P = 0.002). With increasing age, neither the risk of thyroid dysfunction (P = 0.1) nor the level of TSH (P = 0.26) and the increasing level of autoimmune antibodies (P = 0.06) is observed. The risk of thyroid dysfunctions is increased significantly for both women and men, older than 45 years with positive antithyroid antibodies . This risk increased even more when we added menopause as a confounding factor in the model [OR (CI 95%): 3.06 (1.32–7.09 P = 0.009)].The mean (SD) age of 615 out of 618 studied population in 2011 is 47.37 (12.02) [range 25–82 years], 314 were men and 301 were women with the average ages of 48.9 (12.08) and 45.88 (11.78) years, respectively (n = 86), the number of parity (n = 255), and number of abortions (n = 78) in 301 women were 48.3 (5.11) years, 3.2 (1.69), and 1.63 (0.95), respectively. The risk of thyroid dysfunctions was higher in menopausal than non-menopausal females . This risk was increased considerably in subclinical hypothyroidism .The mean (SD) age of menopause , OR (CI 95%): 4.2 (0.48–38.62), P = 0.19] and 92  of total 615 participants. The median (range) of T3 and T4 levels were 1.7 μg/dL (0.40–17 nmol/L), 8.1 μg/dL (1.40–56.6 μg/dL), respectively, in whole population. The median (range) of TSH concentration was 2 (0.01–120) mIU/L in whole population, whereas in subclinical hypothyroidism, overt hypothyroidism, subclinical hyperthyroidism, and over hyperthyroidism these were 4.7 (3.7–9.8) mIU/L, 24 (10–120) mIU/L, 0.08 (0.02–0.2) mIU/L, and 0.03 (0.01–0.05) mIU/L, respectively. The overall prevalence of overt hypothyroidism was 2.3% in total and 1.9% in men and 2.8% in women. However, the overall prevalence of overt hyperthyroidism was 1.9% in total, 3.7% in women and no case in men.Clinical nodule and goiter were identified respectively in 5 .The median of TgAb concentrations in subclinical hypothyroidism, overt hypothyroidism, and overt hyperthyroidism were 62 (23–500) IU/mL, 33 (30–60) IU/mL, and 45 (45–45) IU/mL, respectively. There is no significant difference between various thyroid function groups in terms of TgAb levels (P < 0.05). The multivariate logistic regression analyses adjustments were made for some of important potential confounding factors (Models 1 and 2); however, these associations remained statistically significant (P < 0.05).Table P = 0.01 and 0.817 (0.600–1.035) P = 0.033, respectively.Receiver operating characteristic (ROC) curve analysis was used to determine the cutoff levels of TPOAb and TgAb in 2006 to predict different thyroid dysfunctions in 2011. The areas under the ROC curves for the thyroid dysfunctions occurrence in relation to thyroid autoimmune antibodies are shown in Table This study has estimated the annual incidence of overt hypothyroidism over 6 years to be 2.7 per 1000 and overt hyperthyroidism to be 1.9 per 1000 in Isfahani adults. The incidence of subclinical hypothyroidism and subclinical hyperthyroidism is higher than that of the overt types in both genders after 6 years of follow-up.We found a positive association between almost all types of thyroid dysfunction and positive TPOAb in different models. The median of TPO and Tg autoantibody levels are increased in various thyroid dysfunctions. The areas under the ROC curves for the occurrence of almost all thyroid dysfunction types are significantly based on TPOAb and TgAb levels in 2006. The optimal cutoff point for TPOAb was 38 IU/mL to predict both overt hypo- and hyperthyroidism.Recently, an increased incidence rate of thyroid dysfunction in European population has been reported in a meta-analysis to be 2.59 and 2.26 for hypothyroidism and hyperthyroidism, respectively . MajoritAnother large population study performed in Scotland, during 9-year follow-up (1993–2001), has shown that the annual incidence of primary hypothyroidism varied between 3.90 and 4.89 per 1000 in women and between 0.65 and 1.01 per 1000 in men . For hypFollowing the salt iodization in recent decades in the whole country, Iran has been considered as an iodine-sufficient area , 20, 28;The incidence of thyrotoxicosis is shown to rise in women but not in men in a large population study conducted in Tayside, UK , similarA cohort study conducted in 1999 in Tehran during 6.7-year follow-up showed the incidence rate of hypothyroidism in 1000 subjects py in women and men to be 0.28 and 0.21, respectively, while hyperthyroidism incident rate was 1.4 and 0.21 in 1000 subjects py in women and men, respectively . The incSome variables including positive antithyroid antibodies, positive family history, age, race, and gender have been shown to be correlated to the thyroid dysfunction , 8, 11. Positive thyroid autoantibodies might be a risk factor for developing abnormal thyroid function . Both riMajority of studies have adopted the presence or absence of antithyroid antibodies, the age, and gender of the studied population to predict progression from euthyroidism to overt or subclinical hypo- and hyperthyroidism , 34. HowConsistent with previous researches , 17, 36,Both induced hypothyroidism and hyperthyroidism by autoimmune thyroid diseases have been associated with positive TPO level . IncreasA positive correlation has been shown between the number of parity and development of autoimmune thyroiditis . On the In addition, the family history of goiter predisposes patients to the presence of autoantibodies in the serum , 52. OurA longitudinal study in Australia, with 13-year follow-up, calculated cutoff point of 29 IU/mL for TPOAb in predicting hypothyroidism . A similThe incidence rate of thyroid dysfunction in our study is higher than the reported incidence rate of the world. TPOAb positivity is strong predictor for this incidence rate. The optimal cutoff point for TPOAb in a sample of Isfahani population is slightly higher than previous studies.The prevalence of thyroid dysfunction is studied in Isfahan, an area of iodine sufficient in Iran. We have found that the incidence rate of thyroid dysfunction in our study is higher than previous similar reports, specifically within the hyperthyroidism cases. Positive TPOAb and higher TPOAb concentration are suggested as strong predictors for thyroid dysfunction in Isfahan.


Aspergillus labruscus sp. nov., has been found in Brazil during an investigation of the fungal species present on the surface of grape berries (Vitis labrusca L.) for use in the production of concentrated grape juice. It seems to be associated to V. labrusca, and has never been recovered from Vitis vinifera. This new species belonging to Aspergillus subgenus Circumdati section Nigri is described here using morphological characters, extrolite profiling, partial sequence data from the BenA and CaM genes, and internal transcribed spacer sequences of ribosomal DNA. Phenotypic and molecular data enabled this novel species to be clearly distinguished from other black aspergilli. A. labruscus sp. nov. is uniseriate, has yellow mycelium, poor sporulation on CYA at 25 °C, abundant salmon to pink sclerotia and rough conidia. Neoxaline and secalonic acid D were consistently produced by isolates in this taxon. The type strain of A. labruscus sp. nov. is CCT 7800 (T) = ITAL 22.223 (T) = IBT 33586 (T).A novel fungal species,  Purple grape juice is rich in polyphenol compounds which have been associated with protective effects on the vascular system and with improvement in cognition and neuronal function with agingVitis labrusca L., the most important species for grape juice production2. Grapes have been grown in this region since the 19th century when Italian immigrants arrived bringing with them the culture and tradition of wine production and consumption. These Italian immigrants used their farming knowledge in their new homeland to provide food for consumption and subsistence. However, the climatic conditions of the region were not favorable for growing the European (Vitis vinifera L.) grape cultivars, which led to the introduction of hardier cultivars of American origin (V. labrusca) with better resistance to biotic and abiotic stress. Nowadays “Isabel”, “Isabel Precoce”, “Bordô” and “Concord” are the V. labrusca cultivars extensively grown throughout Rio Grande do Sul. In 2014, 540,000 Kg of V. labrusca grapes were produced in this state3, and used mostly for the production of concentrated grape juice.Serra Gaúcha, in the southern Brazilian state of Rio Grande do Sul is acknowledged as the largest grape producing region in Brazil. The main features of this region include low levels of sunlight and acid soils with good drainage. Various grape cultivars are widely used in the Serra Gaúcha region, but some 90% of the growing area is planted with vines of th largest exporter by volume of grape juice and is one of the few countries in the world where V. labrusca is grown on a commercial scale. About 90 million liters of grape juice were sold in 2014.More recently, in addition to Rio Grande do Sul, the Brazilian states of Pernambuco (Petrolina region), Paraná, São Paulo and Minas Gerais have also become significant producers of grape concentrate for juice. This kind of juice is exported to many countries, and used to make grape nectar and grape beverages. Indeed, Brazil is the 10Aspergillus section Nigri, also called “the black aspergilli”, have been shown to occur frequently throughout the world on grapes for wine production, such as V. vinifera8. However, black aspergilli populations on grapes for the production of concentrated grape juice, and especially V. labrusca, have yet to be described. Some black aspergilla are important in biotech processes and also in biodeterioration, but some species in this section can produce carcinogenic mycotoxins ochratoxin A9 and fumonisins, especially fumonisin B210.Fungi belonging to et al.11 recently provided an updated accepted species list for the genus Aspergillus, and 27 species are included in Aspergillus section Nigri. The authors discussed different approaches to species identification in Aspergillus and recommended DNA markers as a reliable means of identification. Information on living ex-type culture collection numbers and GenBank accession numbers for available representative ITS, CaM, BenA and RPB2 sequences were listed, and CaM sequences were proposed as important identification markers for Aspergillus section Nigri.Samson Aspergillus taxon in Aspergillus section Nigri was found on the surface of grape berries. This species was found only in the region of Serra Gaúcha, Rio Grande do Sul, and on one grape variety, “Bordô”. It is described here as Aspergillus labruscus sp. nov. and this species does not produce ochratoxins or fumonisins.During an investigation of the fungal species in grapes for juice production, a new Aspergillus section Nigri due to the subtle morphological differences between species, and therefore DNA sequence information is increasingly being used for species identification and diagnosis.It is very difficult to identify fungi belonging to Nigri have been identified mainly using CaM gene sequences, because the internal transcribed spacer (ITS) of the nuclear ribosomal (nrDNA), accepted as the official DNA barcode for fungi12, is insufficient for correctly identifying all Aspergillus section Nigri species11. The CaM gene sequence contains more variation than the ITS and the nucleotide sequence database is complete for all accepted species11.Fungi belonging to section CaM gene sequence was determined for 275 isolates of Aspergillus section Nigri found on the surface of the V. labrusca grape berries for four different Brazilian regions, to identify the species. All CaM gene sequences were compared using the Basic Local Alignment Search Tool (BLAST) against the NCBI database to recognize fungal species that have similar DNA sequences. The majority of gene sequences were similar (sequence identity ≥99%) to one of the 27 species belonging to Aspergillus section Nigri, but the CaM gene sequences from 23 isolates were found to be significantly different from all other sequences of species described so far . Using the BLAST tool it was found that the CaM gene sequences from these isolates are most similar to those from Aspergillus homomorphus, but with only 85% of sequence identity.Thus, as an initial step, part of the et al.11 provided an updated accepted species list for the genus Aspergillus, now containing 339 species, and to enhance the scientific value of the list, it includes information on living ex-type culture collection numbers and GenBank accession numbers for available representative ITS, CaM, BenA and RPB2 sequences. We generated a CaM-based phylogram of the 23 sequences not assigned to any described species using the BLAST tool, and those retrieved from GenBank for each Aspergillus section Nigri on Samson’s updated list. The CaM-based phylogram placed our isolates on a branch clearly separated from all other species of Aspergillus section Nigri , and lists only verified sequences13. For Aspergillus, all ex-type sequences were included in the RefSeq database. Using the BLAST tool with the “sequences from type material” (RefSeq) option , the ITS sequence from our novel isolates was found to be most similar to those from A. homomorphus CBS 101889, Aspergillus aculeatus CBS 172.66 and Aspergillus japonicus CBS 114.51, all of which have only 85–88% sequence identity. Both, A. aculeatus and A. japonicus belong to the A. aculeatus clade in Aspergillus section Nigri, and A. homomorphus belongs to the A. homomorphus clade in section Nigri11. Interestingly, the ITS amplicon length from all isolates of the putative novel species (540 bp), obtained with the ITS1-ITS4 primer-pair14, was smaller than that of all other Aspergillus section Nigri. They exhibit a 38–39 bp deletion in the ITS1 region when compared to A. aculeatus, A. homomorphus, A. japonicus and A. saccharolyticus , region and partial BenA gene may vary in the number of introns, and the PCR sometimes results in the amplification of paralogous genes15, this locus has been frequently used for molecular identification of Aspergillus. Thus, the DNA sequences from the six isolates of the putative novel species were determined for a portion of BenA. Comparative BLAST sequence analyses of the six isolates revealed that they are more similar to A. homomorphus ex-type strain CBS 101889, with 85% of sequence identity. A BenA-based phylogram placed the six isolates on a branch undoubtedly separated from all other species of Aspergillus section Nigri  = ITAL 22.223 (T) = IBT 33586 (T). The GenBank accession numbers of the ITS barcodes and alternative identification markers BenA and CaM deposited for this paper are present in Table In conclusion, the ITS, A. labruscus were examined for extrolites and they all produced secalonic acid D and neoxaline . The ex type strain produced two as yet not structure elucidated extrolites, that have only been observed in A. labruscus. Metabolite analysis indicated that A. labruscus does not produce ochratoxins and fumonisins.Six strains of ne Table . The phyA. labruscus (CCT 7800) on different media and temperatures. Micromorphology characters are as follows: uniseriate, spherical conidial heads, brown colored stipes smooth with thick wall (3.2–6.1 µm), vesicle 43 × 60 µm, phialides 7.2–7.8 × 3.8 µm, conidia not uniform in size and shape, spherical to ellipsoidal 6.5–8 × 6.1–6.9 µm, black and conspicuously rough. Figure Colony diameters after 7 days at 25 °C were as follows: CYA: 70–77 mm, MEA: 56–60 mm, YESA: 69–79 mm, OAT: 42–45 mm, CREA: 3–4 mm and CYA + 5% NaCl: no growth. On CYA at 10 °C conidia germinate, at 15 °C, 20 °C and 30 °C growth is evident with colony diameters of 9–17 mm, 59–71 mm and 56–86 mm, respectively. The species does not grow on CYA at 37 °C and 42 °C while at 33 °C the colony diameters vary from 0 to 22 mm, indicating that at this temperature the growth response varies among the isolates. At 25 °C in CYA, the species showed yellow mycelium, poor sporulation, abundant salmon to pink sclerotia production, and reverse pale yellow in color. Figure A. labruscus is similar to Aspergillus sclerotioniger because both have yellow mycelium, poor sporulation on CYA at 25 °C, abundant sclerotia and rough conidia. However, they differ because A. sclerotioniger has biseriate conidiophore, yellow sclerotia and produces ochratoxin A11 while A. labruscus is uniseriate, has pink to salmon sclerotia and does not produce this mycotoxin.Morphologically A. labruscus is most similar to A. homomorphus, A. saccharolyticus and A. aculeatus, the yellow mycelium, salmon to pink sclerotia and large spherical to ellipsoidal conidia (6.5–8 µm) differentiate A. labruscus from these species. Furthermore, A. labruscus differed from A. homomorphus and A. saccharolyticus because these two species were able to grow on CYA at 37 °C with diameters 18 mm and 7–10 mm, and on CYA + 5% NaCl with diameters of 68 mm and 17 mm, respectively.Although molecular data revealed that A. labruscus. In total, 23 isolates of this new species were found on the surface of grape berries, V. labrusca, cv. Bordô grown in Rio Grande do Sul (Serra Gaúcha). The origins of representative A. labruscus isolates are shown in Table A. labruscus on the samples from different vineyards of Rio Grande do Sul and the range of contamination. Serra Gaúcha has a distinct climatic condition for vineyards, with an annual average temperature of 17 °C, rainfall of 1,700 mm and relative humidity of 76%. While most of the world’s vineyards grow the European cultivars (V. vinifera) which produces fine wine, in Brazil due to the high humidity conditions, this species was affected by fungal disease and did not adapt to Brazilian conditions. Thus, the rustic North American cultivars of V. labrusca were introduced, which showed high disease resistance, and today 80% of Brazilian vineyards grow the cultivar V. labrusca, especially in the State of Rio Grande do Sul.Among a total of 89 grape samples collected from different regions and grape cultivars analyzed for fungal contamination, 5 samples (5.6%) revealed the presence of A. labruscus has never been found on V. vinifera grapes. A similar situation is known for peanuts where the species Aspergillus arachidicola, has only been found on the wild type species Arachis glabrata, but not on the domesticated peanut, Arachis hypogea18.It is interesting to note that A. labruscus was able to grow at temperatures of 15 °C to 33 °C, with no growth at 37 °C. Most members of Aspergillus section Nigri can grow at 37 °C and even higher20, including the closely species A. homomorphus and A. saccharolyticus. A. labruscus has never been found before in European vineyards and grape products21 indicating that A. labruscus have an association with V. labrusca rather than V.vinifera. Data on the growth temperature of A. labruscus show that this species is adapted to colder temperatures, which may be due to its North American origin and therefore well-adapted to the Serra Gaúcha region which is colder than the other regions in Brazil. In addition, data to be published later will show that this species has not been found in V. labrusca cv. Bordô vineyards in the North Brazilian region.Data on growth temperatures under laboratory condition as presented above showed that 20 and the three closely related species A. labruscus, A. homomorphus and A. saccharolyticus were examined for the production of two extracellular enzymes: tannase and caseinase , is designated as the holotype of A. labruscus. It was isolated from the surface of grape berries (Vitis labrusca) grown in Rio Grande do Sul State, Brazil, in February, 2013, by Fungaro MHP and Sartori, D. Cultures derived from this type include ITAL 22.223  and IBT 33586 .Diagnosis: This species differs from all species in Aspergillus section Nigri24 by its yellow mycelium, poor sporulation on CYA at 25 °C, abundant salmon to pink sclerotia and no growth at 37 °C on CYA. A profile of secondary metabolites differing from that of the closely related species A. homomorphus and A. saccharolyticus and a distinctive DNA sequence of BenA and CaM genes and ITS regions.Description: Colony on CYA, after 7 days at 25 °C: 73–84 mm diameter, yellow mycelium, poor sporulation, abundant salmon to pink sclerotia, and pale yellow reverse. On MEA, after 7 days at 25 °C: colony of 58–78 mm diameter, abundant sporulation and pale yellow reverse. The species does not grow on CYA at 37 °C and CYA at 42 °C after 7 days, but can grow on CYA at 15 °C (diameter of 9–17 mm) and germinate at 10 °C. Conidiophores uniseriate with spherical vesicles 40 × 60 µm, stipe smooth-walled and brown colored 3.2–6.1 µm, phialides 7.2–7.8 × 3.8 µm, conidia not uniform in size and shape, spherical to ellipsoidal with 6.5–8.0 µm, black and coarsely roughened.ITS Barcode: KU708544 .Extrolites: All isolates produced secalonic acid D and neoxaline.Other isolates examined: ITAL 21.217, ITAL 22.224, ITAL 22.225, ITAL 22.227, ITAL 22.228, ITAL 28.255, ITAL 28.256, ITAL 28.257, ITAL 28.264, ITAL 28.267, ITAL 28.270, ITAL 28.272, ITAL 28.273, ITAL 28.275, ITAL 28.283, ITAL 28.286, ITAL 28.289, ITAL 28.294, ITAL 31.311, ITAL 31.312, ITAL 31.316, ITAL 39.381 all from the surface of grape berries (V. labrusca) grown in the Rio Grande do Sul State, Brazil.Etymologyn = 22), São Paulo (n = 21), Paraná (n = 16) and Rio Grande do Sul (n = 30). In 2014, twenty bunches were collected from each field, close to harvest time between late January and early February in Paraná and Rio Grande do Sul, in April in Pernambuco, and in December in São Paulo. The V. labrusca grape cultivars studied were “Bordô”, “Cora”, “Concord”, “Isabel”, “Isabel Precoce”, “Violeta”, “Coder”, “Rudder”, “Niagara” and “Muscadine”. These are the grape cultivars most widely used for juice production in Brazil. The samples were collected as described in Serra et al.25, across two diagonal transects. A total of 100 berries of each sample was plated according to Pitt and Hocking26 but without surface disinfection. All Aspergillus section Nigri fungi were transferred to CYA  and incubated at 25 °C for seven days.The 89 grape samples were collected from four Brazilian states: Pernambuco , according to the manufacturer’s instructions. As an initial step to identify the isolates, partial amplification and sequencing of the CaM gene were performed as described in detail by Taniwaki et al.27. The CaM sequences obtained were aligned with type or neotype strain sequences from all Aspergillus section Nigri species obtained from the database maintained by NCBI (http://www.ncbi.nlm.nih.gov/). Alignment was performed using ClustalW28. MEGA6.0 software29 was used to construct a Maximum Likelihood (ML) tree, based the Tamura-Nei model30. Of the twenty three isolates none were phylogenetically closely related to any of the species described so far, six were further investigated using the ITS1–5.8S–ITS2 region of nrDNA (ITS) and partial BenA gene sequences. Partial amplification and sequencing of the BenA gene and ITS region were performed as described in Taniwaki et al.27. The ITS and BenA sequences obtained were aligned with the type or neotype strain sequences from all Aspergillus section Nigri species using the above-mentioned approaches.A total of 275 A. labruscus (Table A. homomorphus (IBT 21893 and IBT 21894) and two isolates of A. saccharolyticus (IBT 28231 and IBT 30881 = CBS 127449) were analysed for small molecule extrolites by extracting 3 agar plugs from 7 day incubated cultures at an incubation temperature of 25 °C in darkness31. The extracts were analyzed by ultra high performance liquid chromatography using diode array detection (UHPLC-DAD)32 and compared to authentic small molecule extrolite standards33.Six isolates of us Table , two isoet al.11. Briefly, a spore suspension was prepared in 0.5% agar dissolved in water. Petri dishes were inoculated in a three-point pattern. The following culture media (formulated) and incubation temperature were used: Czapek yeast autolysate (CYA) agar at 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 33 °C, 37 °C, and 42 °C; malt extract agar (MEA), oatmeal agar (OAT), creatine sucrose agar (CREA) and CYAS (Czapek yeast autolysate agar with 5% NaCl), at 25 °C. For comparison, two isolates of A. homomorphus (IBT 21893 and IBT 21894) and two isolates of A. saccharolyticus (IBT 28231 and IBT 30881 = CBS 127449) were also grown on CYA at 37 °C and CYAS at 25 °C.Morphological characterization was performed based on Samson A. labruscus were examined under light optical microscopy and scanning electron microscopy. The slide for light optical microscopic observations was prepared using lactic acid and lactophenol cotton blue. For each structure 3 replicate measurements were performed. For scanning electron microscopy, a 0.5 × 0.5 cm plug was fixed for 24 h at 4 °C in 2% glutaraldehyde in 0.1 M of NaPO4 buffer. The plug was washed three times for 15 min (each) in 0.1 M phosphate buffer (NaPO4) and post-fixed in osmium tetroxide 1% buffer for 2 h at 25 °C in the dark. The plug was then washed again three times in 0.1 M phosphate (NaPO4) buffer for 15 min and dehydrated in ethanol series  for 10 min. It was then placed in a critical point dryer  and subsequently sputter-coated with gold . The observation was performed using a FEI Quanta 200® microscope.After seven days, macro and micromorphological characters of A. labruscus (Table A. homomorphus (IBT 21893 and IBT 21894) and two isolates of A. saccharolyticus (IBT 28231 and IBT 30881 = CBS 127449) were tested for production of two extracellular enzymes: (i) tannase, growing the isolates on tannin sucrose agar (TAN) as described by Thrane34 and (ii) caseinase growing on PRO agar , 1 mL; yeast extract, 3.0 g; agar, 20.0 g, water, 1 L). These isolates were one point inoculated on each medium and incubated at 25 °C for 7 days and examined for production of enzyme haloes.Six isolates of us Table , two isoSupplementary Figure S1


In single metal systems, all adsorbents exhibited good to excellent adsorption capacities toward lanthanides and actinides. In a simulated multicomponent mixed solution study, higher selectivity and efficiency were observed for Th4+ over other metal cations, with ginkgo leaves providing the highest adsorptivity (81.2%) among the seven biosorbents. Through optimization studies, the selectivity of Th4+ biosorption on ginkgo leaf was found to be highly pH–dependent, with optimum Th4+ removal observed at pH 4. Th4+ adsorption was found to proceed rapidly with an equilibrium time of 120 min and conform to pseudo–second–order kinetics. The Langmuir isotherm model best described Th4+ biosorption, with a maximum monolayer adsorption capacity of 103.8 mg g–1. Thermodynamic calculations indicated that Th4+ biosorption was spontaneous and endothermic. Furthermore, the physical and chemical properties of the adsorbent were determined by scanning electron microscopy, Brunauer–Emmett–Teller, X-ray powder diffraction, and Fourier transform infrared analysis. The biosorption of Th from a real sample  was studied and an efficiency of 90.4% was achieved from nitric acid at pH 4 using ginkgo leaves.Low–cost biosorbents  were evaluated in the simultaneous removal of La Monazite samples were obtained from Lingshou County jade product processing plant and sieved to a mesh size of 1250. Monazite powder (600 mg) was digested with dilute nitric acid solution (pH 1) in a beaker with magnetic stirring. After stirring for 12 h and filtering, the filtrate was adjusted to pH 4 and diluted to a final volume of 250 mL for use in experiments. The filtrate  was contacted with biosorbent (0.04 g). After shaking for 120 min at 120 r/min, the sorbent was filtered. Metal ion concentrations in the filtrate were determined directly by ICP-OES. Metal ions sorbed on the biomass, R (%), were calculated using the equation shown in the experimental procedures. Experiments were performed in triplicate for each biosorbent.3+, Ce3+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Yb3+, and Lu3+), two actinides (UO22+ and Th4+), four transition metal ions  and an alkali earth metal (Sr2+), were measured. Biosorption percentages are shown in 4+, and UO22+. For example, the adsorption efficiencies for Th4+ were 92.0% for GLP, 75.3% for OLP, 86.0% for BLP, 80.1% for MLP, 59.4% for HLP, 88.7% for WSP, and 70.9% for GFP, while the adsorptivity of transition metal ions and Sr2+ were markedly lower, at 14.0–42.0% for GLP, 28.2–34.9% for OLP, 23.7–38.9% for BLP, 13.8–18.6% for MLP, 9.1–14.2% for HLP, 10.6–21.5% for WSP, and 18.7–24.5% for GFP. Despite the successful biosorption of individual metal ions under controlled conditions, these efficiencies may differ in solutions containing multiple metal ions. Therefore, to test the potential selectivity for Th4+ adsorption among competing ions, a multicomponent mixed solution containing Th4+ and 15 other types of metal ion was prepared. The results are shown in The adsorption efficiencies of GLP, OLP, BLP, MLP, HLP, WSP, and GFP for sixteen metal nitrates, including nine lanthanides  value of 81.2%. This indicated that GLP was more selective for Th4+ adsorption than for that of other metal cations. For OLP, BLP, MLP, HLP, WSP, and GFP, similar lower adsorption efficiencies were observed for almost all metal cations examined. The R (%) values for Sm3+, Eu3+, Yb3+, Pr3+, Nd3+, Ce3+, Gd3+, Lu3+, La3+, Co2+, Y3+, Zn2+, Sr2+, Ni2+, and UO22+ were the range of 1.3–23.0% for OLP, 1.0–25.5% for BLP, 0.8–22.2% for MLP, 0.4–20.7% for HLP, 0.5–10.8% for WSP, and 0.1–4.0% for GFP. The exception was Th4+ adsorption, which presented much higher adsorptivities than other cations, of 49.1% on WSP and 62.7% on GFP. Therefore, for all seven biosorbents, the selectivity for was highest for Th among all metal cations tested. This might be due to Th4+ being tetravalent, which results in a stronger electrostatic force toward the carbonyl groups in the biosorbents than for trivalent cations [4+ from mixtures of actinides, lanthanides, and other metal ions.The results showed that the adsorptivities of all cations were lower in the mixed solution than in the single–metal solutions. However, the adsorption efficiency for Th ions remained higher than that of other metal cations. Notably, GLP showed an adsorptivity of 0.3–5.2% for most metal ions, except for Th cations . TherefoTo assess the adsorption capacity of GLP toward metal ions, various nitrate metal salts were screened in a mixed aqueous medium using the same adsorption technique. The results are shown in –1, the removal rate reached more than 90%. This demonstrated that GLP had high Th4+ selectivity in the presence of competing ions and, therefore, some potential for practical application. However, the origin of this selectivity is not yet fully understood. The preferential biosorption of Th4+ over other metal ions by GLP might be due to the rich flavonoid content of GLP, which endows GLP with a multitude of carbonyl groups that provide a coordination site that binds highly–charged Th4+ better than other metal ions [Further intriguing results were obtained using different GLP concentrations for the biosorption of mixed aqueous ions, as shown in tal ions .Having selected GLP for further experiments, the effects of various experimental parameters  on adsorption were investigated using a “one variable at a time” method .4+ biosorption from aqueous solution by GLP and determine changes in uptake levels in the pH range 1–6. 4+ biosorption.Among all other parameters, the aqueous solution pH is the most important, as it effects both metal ion speciation and the charge at adsorption sites in the biological adsorbents , 54. Acc–1 with an increase in pH from 1 to 4, and remained almost constant above pH 4. This trend in biosorption was due to competition at binding sites between hydrogen ions and Th ions [3O+), which restricted metal cation approach through electrostatic repulsion [+ ions and the increase in negatively charged ligand sites resulted in improved metal ion biosorption [The results showed that, in general, the adsorption efficiency increased steadily with increasing pH . At low  Th ions . At low epulsion , 56. At sorption . Based oContact time is another important parameter for the successful application of biosorbents in practical and rapid adsorption . The tim–1) added to the analyte solution was investigated. As shown in –1 were used to determine the effect of GLP concentration on the biosorption rate.Next, the amount of GLP , with increasing initial Th4+ concentration leading to a clear decrease in adsorption efficiency from 92.8% to 62.2%, while the amount of Th4+ adsorbed per unit mass of GLP increased from 21.9 to 80 mg g–1  is the qe of a complete monolayer, which is a constant related to adsorption capacity, and KL (L mg–1) is a constant related to binding site affinity and adsorption energy.The Langmuir adsorption isotherm assumes an adsorbate monolayer covering a homogeneous adsorbent surface, with an equal adsorption activation energy for each molecule adsorbed on the surface. This model has been successfully applied to many adsorption processes, including solute adsorption from liquid solutions, for which it is the most widely used adsorption isotherm . The LanF and 1/n are Freundlich constants related to the adsorption capacity and adsorption intensity of the adsorbent, respectively.The Freundlich isotherm describes multilayer adsorption on a heterogeneous surface where the heat of adsorption has a nonuniform distribution , and is 2 values are listed in 4+ on GLP followed the Langmuir isotherm equation more closely. This suggested that the metal ions were adsorbed as a monolayer on the biosorbent surface. The maximum Th4+ uptake capacity (qm) of GLP was found to be 103.8 mg g–1.All isotherm equation parameters and R4+ biosorption on GLP.Rate–determining steps of biosorption processes are usually described using kinetics models . Four wie and qt are the amount of solute adsorbed (mg g–1) at equilibrium and time t (min), respectively, and k1 is the pseudo–first–order rate constant for adsorption (min–1).The pseudo–first–order equation is expressed as :qt=qe(12 is the pseudo–second–order rate constant for adsorption (g mg–1 min–1).The pseudo–second–order equation is expressed as :qt=k2qeThe Elovich equation is expressed as :qt=A+Bli is the intra–particle diffusion rate constant (g mg–1 min–1/2), C (mg g–1) is a constant dependent on boundary layer thickness, with larger C values obtained for increased boundary layer effects.The intra–particle diffusion model is expressed as :qt=Kit12) obtained using these models are shown in These rate constants and correlation coefficients , enthalpy (∆H°), and entropy (∆S°) [D (mL g–1) is the distribution coefficient. Changes in enthalpy (∆H°) and entropy (∆S°) were estimated using the following equation [The thermodynamic parameters corresponding to biosorption, including changes in Gibbs free energy (py (∆S°) , were capy (∆S°) :lnKD=ΔSequation , 71:ΔG°∆H° and ∆S° were calculated from the slope and intercept, respectively, of a plot of ln KD vs. 1/T, as shown in ∆H°, ∆S°, and ∆G° values are shown in 4+ adsorption by GLP had ∆G° values of less than zero. ∆G° is used to determine the preference and driving force of a process, with the negative ∆G° values for Th4+ biosorption on GLP indicating a thermodynamically feasible and spontaneous process [∆H° values suggested that Th4+ complexation with the biosorbent was dominant over other opposing enthalpic factors  [∆S° values indicated that randomness at the solid/solution interface increased irregularly during Th4+ biosorption [As shown in  process , 72. Thedration) , 74 and dration) . The possorption .4+ adsorption, as presented in 4+ adsorption. 4+ in the pores. 4+–loaded GLP, in which pores are partially blocked with the smooth large particles due to Th4+ deposition. Notably, significant adsorption had occurred on the GLP surface.Scanning electron microscopy (SEM) has been used to characterize adsorbents and elucidate possible adsorption mechanisms in many studies , 78. In 2 g–1) was larger than that after biosorption (1.4786 m2 g–1). The GLP specific surface area had changed slightly after biosorption due to metal ion adsorption, as confirmed by SEM analysis, which showed that the adsorbents had no well–defined holes in their morphology (only a few surface pores) and a small surface area. This confirmed that this process was essentially surface adsorption, not pore adsorption [The BET surface area of GLP before biosorption  and ester (–COOR) groups [–1 was attributed to asymmetric C–O stretching vibrations in carboxylate groups (–COO–). The peak at 1068 cm–1 was attributed to C–OH stretching vibration in alcohols and carboxylic acids.The pattern of metal ion adsorption on biomaterials is governed by their active groups and bonds . FT–IR sd lignin . The peaellulose . The pea) groups . The pea4+ biosorption on GLP was likely due to electrostatic attraction between these groups and Th4+ cations.The FTIR spectrum clearly indicated that carboxyl and hydroxyl groups were abundant in GLP. Therefore, Th–1 associated with C = O stretching vibrations in GLP was shifted to 1718 cm–1 in the complex, suggesting that carbonyl oxygens were involved in coordination with Th4+. Changes in peaks at 3412, 2926, 1653, and 1068 cm–1 indicated that hydroxyl, carbonyl, and carboxylic groups were also involved in adsorption. Furthermore, sharp peaks were observed at 1383 cm–1 and 1035 cm–1 that corresponded to the υTh–O band of Th ions, providing direct evidence for Th4+ adsorption on GLP under these conditions [After Th adsorption, the sharp band at 1733 cmnditions . These rMonazite, is among the main ore minerals containing light rare earth elements (REEs) and Th . AlthougA real sample containing monazite mineral was studied in a batch experiment. The results obtained  showed t3+, Ce3+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Yb3+, and Lu3+), actinides (UO22+ and Th4+), transition metal ions  and an alkali earth metal (Sr2+). Compared with other metal cations, excellent lanthanide and actinide adsorption efficiencies were obtained for GLP, WSP, and GFP in single metal systems. Furthermore, these biosorbents, especially GLP, exhibited a noticeable selectivity for Th4+, a representative actinide, over lanthanides, transition metal ions, and alkali earth metals under acidic conditions in a mixed–ion aqueous solution. In biosorption studies, effective Th4+ biosorption by GLP was achieved under the following conditions: pH 4; contact time, 120 min; biosorbent concentration, 2 g L–1; particle size, 150–200 mesh; initial Th concentration, 100 mg L–1; temperature, 298 K. Biosorption followed the Langmuir adsorption isotherm model, with a maximum capacity of 103.8 mg g–1. Th4+ biosorption from aqueous solution by GLP obeyed pseudo–second–order kinetics. Thermodynamic calculations indicated the feasible, exothermic, and spontaneous nature of the biosorption process. More importantly, the good sorption efficiency Th4+ of GLP allowed its use in water samples containing monazite mineral, which is of great significance for the recovery and utilization of radioactive contaminants containing Th ions to protect the environment from their radioactivity and toxicity.In summary, we have demonstrated the preparation of three low–cost biosorbents, namely, ginkgo leaf powder, walnut shell powder, and grapefruit peel powder, and their biosorption of lanthanides (La


Trametes versicolor capable of degrading pesticides has been revealed. The conditions for degrading of five selected pesticides including chlorpyrifos, chlorothalonil, pyrimethanil, atrazine and isoproturon with the purified laccases from Trametes versicolor were optimized. The results showed that the optimum conditions for the highest activity were pH at 5.0 and temperature at 25 °C. The best mediators were violuric acid for pyrimethanil and isoproturon, vanillin for chlorpyrifos, and acetosyringone and HBT for chlorothalonil and atrazine, respectively. The laccase was found to be stable at a pH range from 5.0 to 7.0 and temperature from 25 to 30 °C. It was observed that each pesticide required a different laccase mediator concentration typically between 4.0–6.0 mmol/L. In the experiment, the degradation rates of pyrimethanil and isoproturon were significantly faster than those of chlorpyrifos, chlorothalonil and atrazine. For example, it was observed that pyrimethanil and isoproturon degraded up to nearly 100% after 24 hours while the other three pesticides just reached up 90% of degradation after 8 days of incubation.A high capacity of laccase from  Pesticides are important chemicals and widely used in modern agriculture to control pests and improve the commodity yield and quality. Up to 80% of crop yields could have had been lost without the proper uses of pesticides1Bioremediation is an innovative technology that has the great potential to alleviate such problems by decreasing the pesticide residues in food and reducing the pollutant levels in the environment. Therefore, this work focuses on the microorganisms of naturally occurring microbial consortia for pesticide degradationRhus vernicifera by Yoshida913141618Laccase belongs to the family of blue copper-containing polyphenol oxidase. It was first described in latex obtained from Japanese tree Trametes versicolor for degrading five selected pesticide, including chlorpyrifos, chlorothalonil, pyrimethanil, atrazine, and isoproturon, with the presence of different mediators at different pH, temperature and mediator concentrations.In order to better understand the pesticide degradation by laccases, this research group optimized the conditions of laccase from white-rot fungus The results of relative activity are given in Trametes versicotor with an appropriate mediator promoted the degradations of five selected pesticides including atrazine, chlorothalonil, isoproturon, pyrimethanil and chlorpyrifos. Comparing to the treatments of CK and without a mediator, the optimizing mediators were HBT for pesticide atrazine. Its degradation rate was up to 75.0% with HBT as a mediator. But with other mediators, the rates were found to be ranging from 21.7% to 38.9%. For isoproturon and pyrimethanil, the best mediators were found to be acetosyringone, ABTS, HBT and violuric acid. Syringaldehyde and vanillin were the best mediators for chlorpyrifos only. As can be seen in White-rot fungi with an appropriate mediator are able to degrade the pesticides. The results are presented in In order to obtain the optimum conditions for pesticide residues in the biodegradation, pH of culture solution, incubation temperature and mediator concentration were studies. The pH values for laccase were determined by measuring the pesticide degradation rates in a citric acid-dibasic sodium phosphate buffer at varying pHs (pH 3–7). As can be seen in Albatrella dispansusCantharellus cibariusCoriolus hirsutusLentinula edodesTricholoma giganteumHericium erinaceumPanus tigrinusTrametes versicolor in this study. The laccases from C. cibarius and H. erinaceum which had an optimum temperature at 50 °C, while C. hirsutus and R. lignosus were at 45 °C and 40 °C, respectively. Laccase requires a temperature of 40 °C to exhibit its maximal activity. The high activity observed at 35–45 °C for Rigidoporus lignosus (38). As shown in Trametes versicolor to reach its maximal activity. When the temperature was at 20 and 30 °C, it also showed a good stability of laccase activity in this study.Temperature, pH and mediator play important roles in laccase activity and its catalyzed pesticide degradation. In the literature, most of the fungal laccase optimum pH is 5.0 (form 4.0 to 6.0). For example, the pH optimum was at 5.0 for the laccase from the edible wild mushrooms, including Trametes versicolor with an appropriate mediator could accelerate the degradations of five selected pesticides compared to the treatments of without a mediator. A number of studies reported the capacity of removal of pesticides in contaminated agricultural environment by LMS. Purified phenol oxidase (laccase) from the white rot fungus Pleurotus ostreatus (Po) together with the mediator of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) indicated complete and rapid oxidative degradations of the nerve agents O-ethyl S-methylphosphonothiolate (VX) and O-isobutyl S-methylphosphonothiolate (RVX) and the organophosphorus insecticide analog diisopropyl-Amiton. A molar ratio of 1:20 for OP/ABTS and 0.05 M phosphate at pH 7.4 provided the highest degradation rate of VX and RVXTrametes versicolor showed incomplete but effective degradation of VX, O-ethyl-S-[2-(diisopropylamino)ethyl] phenylphosphonothioate (PhX) and RVX in the presence of ABTS2+, Tween 80 with laccase, respectivelyTrametes villosa. But it was transformed to a significant extent, when a mediator was present31et al. reported that the optimal pH for degradation of VX and RVX was 7.4 as compared to DiPr-Amiton that is degraded more rapidly at pH 8.0et al. showed that the pH optimum of catechol oxidation by Pleurotus oastreatus 0432 laccase was 6.0The results demonstrated that the laccase from Mediator concentration is also a factor in laccase-catalyzed pesticide degradation. The optimal molar ratio of ABTS/OP for VX and RVX degradation was 1:20, whereas, the rate of diPr-amiton degradation rate reached its peak at a molar ratio of 1:10Gas chromatography was an Agilent GC 7890  equipped with a μ-ECD detector and an analytical column HP-5ms J&W Ultra Inert capillary column . High performance liquid chromatography  contained a 1200 SL HPLC system with an Agilent ZORBAX SB-C18  analytical column.Chlorpyrifos (purity: 99.9%), chlorothalonil (purity: 97.5%), pyrimethanil (purity: 97.5%), atrazine (purity: 99%), isoproturon (purity: 99%) were purchased from Dr. Ehrenstorfer Company (Germany).Trametes versicolor were purchased from Sigma (USA). Organic solvents, n-hexane, acetone, acetonitrile, methanol, sodium chloride and petroleum ether, were of analytical grades and purchased from Kermel Chemical Reagent Co., Ltd . Pesticide standard stock solutions (1000 mg/L) were prepared in acetone and stored in a refrigerator at 4 °C. HBT, ABTS, violuric acid, and acetosyringone were purchased from Oddo’s Biological Co., Ltd . Syringaldehyde and vanillin were purchased from Aladdin . Guaiacol was purchased from Chinese Medicine Group Chemical Reagent Co., Ltd. .Laccase purified from + radicals from the enzymatic oxidation of ABTS at 25 °C using a spectrophotometer. The assay mixture contained 200 μL of 0.5 mmol/L ABTS, 2700 μL of sodium acetate buffer (pH 4.5) and 100 μL of the enzyme-containing sample. One unit of laccase activity (U) was defined as the amount of enzyme that formed 1 μmol ABTS per min, using the extinction coefficient ε 420 nm of 36,000 /M/cmLaccase activity was measured at 420 nm by generation of ABTS2Laccase was added in citric acid-dibasic sodium phosphate buffers with the pH at 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0. The incubation system were in tubes put in an incubator, and the temperature was controlled at 25 °C. Samples were collected at 1, 2, 4, 6 and 8 days intervals after laccase application for the detection of laccase activity. Laccase activity was controlled in 50 U/L of each treat at the beginning. Laccase was separately added to citric acid-dibasic sodium phosphate buffers with the pH at 5.0. The incubation system were in tubes put in the incubator, and the temperature was controlled at 20, 25, 30, 35, and 40 °C, separately. Samples were collected at 1, 2, 4, 6 and 8 days after Laccase application for the detection of laccase activity.Degradation experiment were carried out in 10 mL tubes in citric acid-dibasic sodium phosphate buffers containing 20.0 mg/L pesticides in the dark at 30 °C in the incubator. The enzyme activity was in the control of 0.05 U/mL by an ultraviolet spectrophotometer. Samples of atrazine, chlorothalonil, chlorpyrifos, and pyrimethanil, isoproturon were collected at 1, 2, 4, 6, 8 days and 0, 2, 6, 10, 16, 24 h after incubation, respectively. Then the residues were measured by HPLC or GC. All of the treatments including controls were in triplicates.For chlorothalonil, pyrimethanil, chlorpyrifos and isoproturon, 2.0 mL of the buffer solution was added into a 10 mL glass tube, aloowed by adding 2.0 mL of petroleum ether to the extract. The mixture was vortexed for 2 min and wait for the layer separation. The supernatant fluid  was transferred into a clean glass tube. Repeat the above steps 2 additional times. Then the supernatants were combined. Three (3.00) mL of the supernatant was transferred into another tube and dried under a stream of nitrogen (40 °C). The pesticide residues were dissolved in 1 mL of appropriate organic solvent* and filtered through a 0.22 μm nylon filter into an autosampler vial. .2 at 1 mL/min, injection volume 1.0 μL, and splitless mode. A linear calibration curve was used and the calibration range was 0.01–5 mg/L. The retention time of chlorpyrifos was approximately 5.8 min.The conditions for the analysis were: detector temperature 300 °C, injector temperature 250 °C, oven temperature program starting at 120 °C, 13.5 min at 120–270 °C (ramp 20 °C/min), carrier gas, NThe mobile phase containing acetonitrile/water  was delivered at a flow rate of 0.8 mL/min. The injection volume was 20 μL and detection wavelength was 222 nm. The calibration curve was obtained using peak area versus concentrations and the calibration range was 0.01–5 mg/L. The retention time of atrazine was approximately 4.2 min.The mobile phase containing acetonitrile/water  was delivered at a flow rate of 0.3 mL/min. The injection volume was 10 μL and detection wavelength was 245 nm. The calibration range was 0.01–5 mg/L. The retention time of isoproturon was approximately 10.4 min.The mobile phase contains methanol/water  flow rate 0.7 mL/min, injection volume 20 μL and detection wavelength 240 nm. The calibration range was 0.01–5 mg/L. The retention time of chlorothalonil was approximately 9.7 min.The mobile phase contained methanol/water , flow rate 1.0 mL/min, injection volume 20 μL and detection wavelength 270 nm. The calibration range was 0.01–5 mg/L. The retention time of pyrimethanil was approximately 4.6 min.How to cite this article: Jin, X. et al. Conditions Optimizing and Application of Laccase-mediator System (LMS) for the Laccase-catalyzed Pesticide Degradation. Sci. Rep.6, 35787; doi: 10.1038/srep35787 (2016).


Flow rate was 1.0 mL/min with a column temperature of 30°C. Validation of the method was made in terms of specificity, linearity, accuracy, and intra- and interday precision, as well as quantification and detection limits. The developed method can be used in the laboratory to routinely quantify dexamethasone, ondansetron, granisetron, tropisetron, and azasetron simultaneously and to evaluate the physicochemical stability of referred drugs in mixtures for endovenous use.A simple and rapid high-performance liquid chromatography with diode array detector (HPLC-DAD) method has been developed and validated for simultaneous quantification of five antiemetic agents in infusion samples: dexamethasone, ondansetron, granisetron, tropisetron, and azasetron. The chromatographic separation was achieved on a Phenomenex C HencFrom the literature survey it is evident that various analytical methods were available for the determination of DEX, OND, GRA, TRO, and AZA with other combinations by using high-performance liquid chromatography (HPLC) –31. No m2PO4, triethylamine, and phosphoric acid of AR Grade were obtained from Xilong Chemical Ltd. . HPLC grade acetonitrile was purchased from Fisher Scientific International . Ultrapure water was purified using a Milli-Q system .The working standards of DEX sodium phosphate, OND hydrochloride, GRA hydrochloride, TRO hydrochloride, and AZA hydrochloride were purchased from the National Institutes for Food and Drug Control  and stored at 4°C. The pharmaceutical formulations used in this study were DEX sodium phosphate injection 5 mg/1 mL , OND hydrochloride injection 4 mg/2 mL , GRA hydrochloride injection 3 mg/3 mL , TRO hydrochloride injection 5 mg/1 mL , and AZA hydrochloride injection 10 mg/2 mL . The solution of 0.9% NaCl used to prepare the sample mixtures was from Kelun Pharmaceutical Co., Ltd. . Potassium dihydrogen phosphate KH18 column  and using the mobile phase of acetonitrile-50 mM KH2PO4 buffer-triethylamine . The mobile phase was prepared daily and filtered through a 0.22 μm membrane filter . The flow rate of the mobile phase was kept at 1.0 mL/min. The selected detection wavelengths for DEX, OND, GRA, TRO, and AZA were 241 nm, 310 nm, 302 nm, 285 nm, and 302 nm, respectively. The assay was performed at 30°C and injection volume was 20 μL.An UltiMate® 3000 standard high pressure liquid chromatographic instrument  composed of an UltiMate 3000 quaternary gradient pump, an ASI-100 autosampler, a TCC-100 thermostat column oven, and an ultraviolet detector (DAD) was employed in the study. Data acquisition was carried out using Chromeleon® software. Chromatographic analyses were performed on a Phenomenex CWe accurately weighed and transferred 20 mg of DEX sodium phosphate, 16 mg of OND hydrochloride, 12 mg of GRA hydrochloride, 10 mg of TRO hydrochloride, and 20 mg of AZA hydrochloride working standard into a 100 mL volumetric flask. We added about 70 mL of deionised water and used sonication for dissolving completely and made volume up to the mark with the same solvent to obtain the final concentration of 0.2 mg/mL of DEX sodium phosphate, 0.16 mg/mL of OND hydrochloride, 0.12 mg/mL of GRA hydrochloride, 0.1 mg/mL of TRO hydrochloride, and 0.2 mg/mL of AZA hydrochloride, respectively. The solutions were kept at –20°C until use. Fresh working standard solutions were prepared by diluting the stock solution with deionised water to the required concentrations before use.In order to mimic a concentration range relevant to clinical practice and the conditions commonly occurring in hospitals, four sample infusion solutions were prepared under aseptic conditions in laminar flow hoods.2 mL (10 mg) DEX sodium phosphate injectable solution and 4 mL (8 mg) OND hydrochloride injectable solution were transferred to a 100 mL polyolefin bags and filled with 0.9% sodium chloride injection.2 mL (10 mg) DEX sodium phosphate injectable solution and 3 mL (3 mg) GRA hydrochloride injectable solution were transferred to a 100 mL polyolefin bags and filled with 0.9% sodium chloride injection.2 mL (10 mg) DEX sodium phosphate injectable solution and 1 mL (5 mg) TRO hydrochloride injectable solution were transferred to a 100 mL polyolefin bags and filled with 0.9% sodium chloride injection.2 mL (10 mg) AZA hydrochloride injectable solution was transferred to a 100 mL polyolefin bags and filled with 0.9% sodium chloride injection.The developed analytical method was subjected to validation with respect to various parameters such as linearity, intra- and interday precision, accuracy, limit of quantification (LOQ), limit of detection (LOD), and reproducibility for each analyte.The compatibility and stability studies of the infusion samples were performed at 25 ± 0.5°C; all the solutions were protected from light exposure and checked at predetermined times: 0, 2, 4, 8, 24, and 48 hours. At the specified times, the infusion samples were examined for the changes in appearance and the pH value of the mixture was also determined in a digital Crison phs-3c pH meter. Moreover, the concentrations of the drugs were determined at each analysis by the above described HPLC-DAD method. In the concentrations' analysis, 2 mL samples were collected from each polyolefin bag and were diluted 1 : 5 in deionised water before injection into HPLC system so as to be within the range covered by the calibration curves.18 column gives symmetric and sharp peaks. The best resolution and analysis time was obtained through isocratic elution using a mobile phase consisting of acetonitrile-50 mM KH2PO4 buffer-triethylamine  at a flow rate of 1.0 mL/min. Buffer pH was evaluated in the range from 2.0 to 6.0, and good resolution and peak shapes were achieved at pH 4.0. The present study is aimed at developing a chromatographic system capable of eluting the individual drugs, permitting their separation and simultaneous determination in infusion solutions. On the basis of the literature consulted –31, an aμg/mL, 2.0–80.0 μg/mL, 1.2–36.0 μg/mL, 1.0–50.0 μg/mL, and 2.0–100.0 μg/mL, respectively. The peak area ratio of each sample against respective concentration of five antiemetic agents was found to be linear. The correlation coefficient for all five antiemetic agents was greater than 0.998. Linearity results were presented in S/N) ratio. Dilutions and injections of DEX sodium phosphate, OND hydrochloride, GRA hydrochloride, TRO hydrochloride, and AZA hydrochloride standards were then made until an HPLC chromatograph showed that the three-peak height reached an S/N of approximately 10 : 1 and 3 : 1 for LOQ and LOD solutions, respectively. The LOD and LOQ for both drugs were determined and shown in Linearity was performed by preparing mixed standard solutions of DEX sodium phosphate, OND hydrochloride, GRA hydrochloride, TRO hydrochloride, and AZA hydrochloride at six concentration levels. The linearity of detector response for DEX sodium phosphate, OND hydrochloride, GRA hydrochloride, TRO hydrochloride, and AZA hydrochloride was demonstrated by prepared solutions of over the concentration range of 1.0–100.0 μg/mL for DEX sodium phosphate; 2.0, 16.0, and 32.0 μg/mL for OND hydrochloride; 1.2, 6.0, and 12.0 μg/mL for GRA hydrochloride; 1.0, 10.0, and 20.0 μg/mL for TRO hydrochloride; 2.0, 20.0, and 40.0 μg/mL for AZA hydrochloride) with six determinations per concentration at the same day. Interday precision (6 days) was also estimated as the RSD calculated from three quality control samples in the same way. The calculated RSD values from repeated measurements were summarized in The precision (RSD) of the method was determined as intraday precision and intermediate precision. Intraday precision was estimated by assaying quality control samples at three concentration levels  by spiking a known quantity of DEX sodium phosphate, OND hydrochloride, GRA hydrochloride, TRO hydrochloride, and AZA hydrochloride standard drugs into infusion samples in triplicate. The results for accuracy of the method are given in No precipitation or color change was observed in all of the infusion mixtures during the 48 hours storage period. The results of the HPLC analysis for each of the test drugs are presented in The developed HPLC-DAD method is simple, sensitive, specific, and adequate to the simultaneous quantification of DEX sodium phosphate, OND hydrochloride, GRA hydrochloride, TRO hydrochloride, and AZA hydrochloride in infusion samples. The method was successfully applied to a study of the chemical stability of these infusion mixtures under 25°C for 48 hours. The method might also be suitable for application to other analytical problems, for example, quality control of pharmaceutical formulations or evaluating the chemical stability of referred drugs in mixtures for clinical use. The results of the stability study showed that mixtures of DEX sodium phosphate-OND hydrochloride; DEX sodium phosphate-GRA hydrochloride; DEX sodium phosphate-TRO hydrochloride and AZA hydrochloride in normal saline in 0.9% sodium chloride injection when stored in polyolefin bags protected from daylight were chemically stable for 48 hours at 25°C.