Kochia scoparia is a troublesome weed across the Great Plains of North America. Glyphosate and dicamba have been used for decades to control K. scoparia. Due to extensive selection, glyphosate- and dicamba-resistant (GDR) K. scoparia have evolved in the USA. Herbicide mixtures are routinely used to improve weed control. Herbicide interactions if result in an antagonistic effect can significantly affect the management of weeds, such as K. scoparia. To uncover the interaction of glyphosate and dicamba when applied in combination in K. scoparia management the efficacies of different doses of glyphosate plus dicamba were evaluated under greenhouse and field conditions using GDR and a known glyphosate- and dicamba-susceptible (GDS) K. scoparia. The results of greenhouse and field studies suggest that the combination of glyphosate and dicamba application controlled GDS, but glyphosate alone provided a better control of GDR K. scoparia compared to glyphosate plus dicamba combinations. Furthermore, investigation of the basis of this response suggested glyphosate and dicamba interact antagonistically and consequently, the translocation of both herbicides was significantly reduced resulting in poor control of K. scoparia. Therefore, a combination of glyphosate plus dicamba may not be a viable option to control GDR K. scoparia. However, because of the extensive and prolonged use of herbicides with the same site of action, a number of weed species evolved resistance to herbicides, which is one of the major threats to sustainable crop production. Additionally, rapid adoption of herbicide-resistant crops, is also contributing to the evolution of herbicide resistance in weeds due to lack of herbicide rotation, thereby increased selection pressure2.Due to wide adoption of no-till agriculture, crop production in North American Great Plains is highly dependent on the use of herbicidesKochia scoparia (L.) Schrad. (K. scoparia), a member of Chenopodiaceae, was introduced to North America for ornamental purpose3. Soon after its introduction, K. scoparia has become highly invasive in crop fields and rangelands3. Because of its ability to tolerate drought, salinity, cold, and prolific seed production as well as tumbling mechanism of seed dispersal6, K. scoparia has turned out to be one of the worst weeds in North American Great Plains. Without timely management of K. scoparia, can cause huge yield loss in crops such as corn, sorghum, wheat, soybean, and sugarbeet7. In the last three decades, the situation has further exacerbated as a result of rapid and wide spread of resistance to acetolactate synthase-, photosystem II-, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)- inhibitors, and synthetic auxins8. The management of K. scoparia, especially those populations that are herbicide-resistant has become an important component in cropping systems in North American Great Plains.9, and to minimize the amount of herbicides applied11. More importantly, tank mixing of different herbicides has been recommended to delay the evolution of herbicide resistance in weed populations14. However, not all herbicides can be used in combinations, due to incompatibility or antagonism between certain herbicide chemical groups15. Combinations of glyphosate plus dicamba have been recommended as burndown application before planting no-till cotton to control horseweed (Conyza canadensis L.)16. In Kansas, glyphosate plus dicamba are usually sprayed together to manage wide spectrum of monocot and dicot weed species including K. scoparia, especially after evolution and spread of glyphosate resistance in weed populations since 20078. However, inconsistent results when glyphosate plus dicamba combination were applied indicate that the interaction between these two herbicides can be species specific. For instance, O’Sullivan and O’Donovan17 reported antagonistic interaction between glyphosate and dicamba, resulting in decreased phytotoxicity of glyphosate on monocot crops such as wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and weeds, like wild oats (Avena fatua L.). Flint and Barrett18 reported that combinations of glyphosate plus dicamba could reduce the efficacy of glyphosate on johnsongrass  due to reduced uptake and translocation. In contrast, Eubank et al.19 suggested addition of dicamba to glyphosate can increase control of horseweed from 70% to over 90%. However, the response of K. scoparia to glyphosate and dicamba combination is not known. When applied in combination, if these two herbicides exhibit an antagonistic interaction, this can result in poor control of K. scoparia, consequently, may accelerate the evolution of glyphosate and/or dicamba resistance because of exposure to less effective doses of these herbicides21, which in turn can weaken the herbicide options for the management of this weed. The glyphosate- and dicamba-resistant (GDR) K. scoparia populations have been reported and wide spread throughout the US Great Plains, including Kansas8. The significance of application of glyphosate and dicamba in combination on K. scoparia control is not known. Hence, it is important to investigate the interaction of glyphosate and dicamba on K. scoparia, to evaluate if use of these herbicides in combination help better control of this weed. Moreover, because growers are expected to rapidly embrace the new glyphosate- and dicamba-resistant crops, it is vital to understand the interaction of glyphosate and dicamba in K. scoparia to maintain the sustainability of the herbicide resistant crops in the K. scoparia infested regions. Therefore, this research was conducted with the following objectives: 1) test the efficacy of glyphosate plus dicamba combinations on GDR K. scoparia in greenhouse and field conditions; and 2) investigate the physiological interaction of glyphosate and dicamba in GDR K. scoparia using radioactive labelled herbicides, by comparing with a known glyphosate- and dicamba-susceptible (GDS) K. scoparia.Combinations of multiple herbicides have been used to control a broader spectrum of weeds−1, respectively. Analyses of herbicide dose response data  and GR50 (effective dose for 50% biomass reduction) of glyphosate for GDR K. scoparia were 978 and 835 g ae·ha−1  in Table −1, Treatment (Trt) 1) and 1400 g ae·ha−1  controlled 0 and 14% of GDR and 82% and 88% of GDS K. scoparia, respectively. When half of the recommended field rate of glyphosate (420 g ae·ha−1) was mixed with 350 g ae·ha−1 of dicamba  had 45 and 95% control of GDR and GDS K. scoparia, respectively. It rendered more control of GDR K. scoparia than glyphosate and dicamba combinations ; and had similar control of Trt 9, which was mixed with 560 g ae·ha−1 of dicamba. Also, the 840 g ae·ha−1 of glyphosate (Trt 5) controlled GDS K. scoparia more effectively than Trt 6 and 7  rendered higher or similar control of the combinations that contain 140 to 1400 g ae·ha−1 of dicamba with the same amount of glyphosate. In the case of combinations with 2100 g ae·ha−1 of glyphosate, the results suggest that 2100 g ae·ha−1 of glyphosate (Trt 15) alone controlled the 95% of GDR K. scoparia, which is higher than Trt 16, 17, and 19 that were mixed with 140, 280 and 1400 g ae·ha−1 of dicamba, respectively. When 700 g ae·ha−1 of dicamba was mixed with 2100 g ae·ha−1 of glyphosate, the control of K. scoparia was similar to the application of 2100 g ae·ha−1 of glyphosate alone. However, all the combinations containing 2100 g ae·ha−1 of glyphosate rendered similar control of GDS K. scoparia except the Trt 19, which was mixed with 1400 g ae·ha−1 of dicamba and rendered only 91% control of GDS K. scoparia.When Trt 10 to 14 were compared, 1260 g ae·haK. scoparia control at 4 weeks after treatment (WAT) with combinations of glyphosate and dicamba are presented in Table K. scoparia, which is better than when treated with dicamba alone . On the other hand, all the treatments with 2100 g ae·ha−1 of glyphosate, including the 2.5 G, 2.5 G + 1.25D (1.25 times of dicamba at label recommended dose), and 2.5 G + 2.5D, rendered 100% control of GDS K. scoparia. But, the treatment 2.5D that contained 1400 g ae·ha−1 of dicamba only provided 84% GDS K. scoparia control at 4 WAT, which is significantly less than the other treatments.The results of K. scoparia, more [14C] glyphosate was absorbed when [14C] glyphosate was mixed with dicamba (hot-GD) than [14C] glyphosate was applied alone (hot-G) at 24 hours after treatment (HAT), e.g., 30.2 and 47.9% of [14C] glyphosate was absorbed at 24 HAT when hot-G and hot-GD was applied on GDR K. scoparia, respectively  when hot-GD was applied than hot-G alone  in both GDR  with hot-GD than hot-G. At all the time points tested, including 24, 72, and 168 HAT, significantly less [14C] glyphosate was translocated to BTL in both GDR , more [14C] dicamba was absorbed at 24 HAT in GDR K. scoparia  applied alone, but this difference was not observed at later time points tested (i.e. 72 and 168 HAT) in GDR , less [14C] dicamba was translocated to ATL at 168 HAT in GDR K. scoparia  to form non-ionized dicamba acid and become more lipophilic. Once dicamba becomes more lipophilic it can be absorbed more quickly via waxy leaf cuticles, which are highly lipophilic29. However, this process could also increase the volatility of dicamba and upsurge the potential of dicamba drift due to presence of acid form of dicamba30, yet not completely absorbed by the plant. On the other hand, glyphosate absorption could have been enhanced by the adjuvants included in Clarity® formulation, but additional study is needed to test this hypothesis.When applied in combination, the absorption of glyphosate and dicamba was enhanced at early hours than treated separately Figs  and 2. EHAT Fig. ; after t14C] glyphosate was translocated and more was retained in treated leaves. This could occur as a result of rapid plant response to dicamba. As an auxinic herbicide, dicamba can cause rapid metabolic and physiological reactions within hours after application, which soon can lead to growth inhibition and reduction of transpiration and carbon assimilation31. Glyphosate is mainly transported via phloem32, which is highly dependent on the source-sink strength33. Therefore, due to weakened source upon dicamba application, the translocation of glyphosate may have been restricted compared to when glyphosate was applied alone. On the other side, reduced dicamba translocation was observed only at later time points when applied in combination. Glyphosate inhibits EPSPS enzyme and shuts down the shikimate pathway, which causes aromatic amino acid synthesis failure and stunts the growth of plants, and ultimately lead to plant death35. Within days, the glyphosate was translocated throughout the plant and shut down the shikimate pathway completely, soon after the carbon assimilation and phloem transport can cease. Therefore, the translocation of dicamba, which is also mainly facilitated by phloem37, would be significantly affected as a result of glyphosate-induced physiological alterations in plants.Translocation of glyphosate was affected by dicamba regardless of time after application. When glyphosate was mixed with dicamba, less  glyphosate and [14C] dicamba in K. scoparia were not affected by spraying plants with formulated herbicides prior to application of [14C] labeled compounds38. In the same research, we also found that less than 5% of dicamba and glyphosate translocated to below ground tissue of K. scoparia38. Therefore, in this study, the K. scoparia plants were not treated with herbicide formulations prior to application of [14C] labeled dicamba or glyphosate, and also the radioactivity in below ground parts of K. scoparia was not tested.In a previous research, we reported absorption or translocation of both [14C] glyphosate working solution (hot-G) with 0.33 kBq·µL−1 of radioactivity was prepared by mixing 93.6 µL of [phosphonomethyl-14C]-glyphosate , 20.5 µL of Roundup WeatherMax® herbicide, 73.5 µL of AMS aqueous solution  and 812.4 µL of water, which was equivalent to 2100 g of glyphosate in a carrier volume of 187 L water with 2.5% (w/v) of AMS. Another mL of [14C] glyphosate plus dicamba combination solution (hot-GD) with 0.33 kBq·µL−1 of radioactivity, which was equivalent to 2100 g of glyphosate and 1400 g of dicamba in a carrier volume of 187 L water with 2.5% (w/v) of AMS, was prepared by mixing 93.6 µL of [phosphonomethyl-14C]-glyphosate, 20.5 µL of Roundup WeatherMax® herbicide, 15.6 µL of Clarity® herbicide, 73.5 µL of AMS aqueous solution , and 796.8 µL of water.One mL of  dicamba (hot-D) and [14C] dicamba plus glyphosate (hot-DG) combination solution were the same as described above for glyphosate vs. glyphosate plus dicamba combination experiments, except that the 1 mL of hot-D working solution  with 0.33 kBq·µL−1 of radioactivity was obtained by mixing 29.3 µL of dicamba-(ring-UL-14C) ethanol solution , 15.4 µL of Clarity® herbicide , 73.5 µL of AMS, and 881.8 µL of water; and the 1 mL of [14C] dicamba and glyphosate (hot-DG) combination solution  with 0.33 kBq·µL−1 of radioactivity was obtained by mixing 29.3 µL of dicamba-(ring-UL-14C) ethanol solution, 15.4 µL of Clarity® herbicide, 20.8 µL of Roundup WeatherMax® herbicide, 73.5 µL of AMS, and 881.8 µL of water.The methods of application and sample collection of both  glyphosate, [14C] dicamba, [14C] glyphosate with formulated dicamba combination, and [14C] dicamba with formulated glyphosate combination containing 3.3 kBq·µL−1 of radioactivity, , were prepared using the same method as described above.Due to the nature of K. scoparia seeds were germinated in trays filled with the commercial potting mixture as described above. Individual seedlings 2 to 3 cm height were transplanted into plastic pots (6.5 × 6.5 × 9 cm) that filled with silica sand  and rinsed in 1% (w/v) of Miricle-Gro water soluble All Purpose Plant Food  and kept in growth chamber. When the K. scoparia seedlings were 6–8 cm height , they were treated with 1 µL droplet of hot-G’, hot-D’, hot-GD’, and hot-DG’ on one newly expanded leaf. At 24, 72, and 168 HAT, K. scoparia plants were gently uprooted, and the roots were washed with water carefully. Then, the whole plant was washed twice with 10 mL of 10% (v/v) ethanol aqueous solution with 0.5% of Tween-20 for 1 minute, and then pressed using a handmade plant press39 and dried at 60 °C for 72 h. The pressed K. scoparia plants were exposed to BAS-IP MS 2040 E Multipurpose Standard Storage Phosphor Screen  for 44 h (the hot-G’ and hot-GD’ treated plants) or 24 h (the hot-D’ and hot-DG’ treated plants), and the screen was read using Bio-Rad molecular imager FX .GDR and GDS K. scoparia. K. scoparia population and herbicide dose were main- and subplot, respectively. Treatments were arranged in a factorial combination with GDR and GDS K. scoparia and different herbicide doses. No interaction between experimental runs was observed; hence, data from the repeated experiments were pooled prior to analysis. Then, visual injury and dry biomass data were subjected to non-linear regression analysis using four parameter log-logistic model40 in R  with the drc package41.Split plot design was used in the experiment of glyphosate and dicamba dose response on GDR and GDS Y refers to the percentage of untreated, C and D are the lower limit and upper limit of the data, respectively, b is the slope, and I50 is the dose required for 50% response of visual injury or biomass reduction, which was used to estimate ED50 (effective dose for 50% control of K. scoparia) and GR50 (effective dose for 50% biomass reduction) values from the visual injury and dry biomass data, respectively.In Eq. K. scoparia population and rate of herbicide combination were the main- and subplot, respectively. Data from the repeated experiments were pooled prior to analysis due to no interaction between experimental runs was found. Two-way analysis of variance was performed in GraphPad Prism 7  using Bonferroni’s multiple comparisons test .Split plot experimental design was also used in greenhouse screening experiments and efficacy study of different glyphosate plus dicamba combinations in field conditions. K. scoparia population and herbicide  were the whole-plot treatment factors, the sampling times as split-plot factor, and the dissected plant parts as the subsampling factor. No interaction between experimental runs was found. Therefore, data of the total amount of absorption, percent of absorption, translocation amount, percent of translocation from repeated experiments were pooled prior to analysis and analyzed separately using SAS software version 9.4  using the MIXED procedure.Randomized completely block design with a split plot and subsampling was used in the absorption and translocation of radioactive herbicides, where, K. scoparia population and sample harvesting time, respectively. Phosphor images were processed in Quantity One software 4.6.9 , and the plant photos were processed in GNU Image Manipulation Program 2.8.20 .Split plot experimental design was used in the phosphor imaging analysis, in which main- and subplot were At least four biological replicates  of each treatment, dose or harvesting time were included for each experiment, and all the experiments were repeated twice in time.


The data of this study was to determine the groundwater quality trend changes in Abhar city (Iran) during one decade (2002–2016). In the first and end year of the study period, the Mean±SD of total hardness , electrical conductivity (as micromhos/cm) and total dissolved solid in the first and end year of the study period were 192.69±56.83, 235.25±84.73 and 606.21±194.69, 744.55±288.52 and 348.79±106.81, 464.71±183.52 respectively. On the basis of Pearson correlation coefficient, the ascending trend of some parameters concentration with time was significant at the level of 95% of confidence limits (α ≤ 0.05). Specifications TableValue of data•2+, Mg2+, +, K+, Cl− and Determination of the physical and chemical parameter including EC, pH, TDS, TH, Ca•Continuing the ascending trend of the parameters concentration and declining the quality of water resources and incompatibility with Iranian drinking water standard can lead to significant health risks.•Tracking the trend changes, investigating the reasons and preventive measures are important1+, Ca2+, Mg2+, K+, EC, TDS, −, and TH as in Abhar County, Zanjan Province, Iran. Data presented here deal with monitoring of physical and chemical including pH, Na22.1X = 49.25–48.35 east longitude and Y = 36.45–35.50 north latitude. The climate of the study area is semiarid, and the precipitation is 300 mm per year. Also the air's highest and lowest temperatures are 38 °C and − 5.15 °C, respectively, with an annual average of 12.7 °C  in 15 years . The imp


Campylobacter is a leading cause of foodborne gasteroenteritis worldwide. Antimicrobial susceptibility testing for Campylobacter spp. is not routinely performed by most clinical laboratories. However, the emergence of resistant isolates strengthens the importance of antimicrobial susceptibility testing and the critical need for epidemiologic surveillance. The aim of this study was to compare the efficacy of Etest and Sensititre kit (a broth microdilution method) as methods for susceptibility tests and the critical need for epidemiologic surveillance. The aim of this study was to compare the efficacy of Etest and Sensititre kit (a broth microdilution method) as methods for susceptibility testing of Campylobacter spp. to tetracycline, erythromycin, and ciprofloxacin.Campylobacter isolates were collected from feces samples and subjected to susceptibility testing by Etest and Sensititre, a broth microdilution kit for tetracycline, erythromycin, and ciprofloxacin. Minimal inhibitory concentration (MIC) results of each method were determined and compared.Sixty-six p < 0.0001); additionally, the correlation between the two methods was highly significant for the three tested antibiotics (p < 0.0001).Similar MIC interpretations for tetracycline, erythromycin, and ciprofloxacin were found in 97%, 98.5%, and 100% of the isolates, respectively, indicating a good level of agreement between Etest and Sensititre (Campylobacter spp. The Sensititre kit has the advantages of high availability and the automation.Both the broth microdilution and the Etest are reliable and convenient methods for testing antimicrobial susceptibility of  Campylobacter is a leading cause of foodborne gastroenteritis worldwide [Campylobacter jejuni and Campylobacter coli [Campylobacter infections result in a self-limited watery diarrhea, accompanied by fever and abdominal cramps [orldwide . In 2015ter coli –5. Usuall cramps . Howeverl cramps . Antibiol cramps , 4. The Campylobacter morbidity, there is a requirement to report Campylobacter infections to the Ministry of Health [Campylobacter infections in the USA was 12.3 per 100,000 population [Campylobacteriosis incidence is globally rising . In Israf Health . While ipulation , in Israpulation .Campylobacter spp. is not routinely performed by most clinical laboratories. However, the emergence of resistant isolates strengthens the importance of AST and the critical need for epidemiologic surveillance [C. jejuni and C. coli, respectively, were resistant to erythromycin. Resistance rates to ciprofloxacin were 25.3 and 39.8%, respectively, for C. jejuni and C. coli isolates [Antimicrobial susceptibility testing (AST) for eillance . For exaCampylobacter spp. are currently available, including disk diffusion, Etest®, and dilution methods (broth and agar dilution) [®  have been developed to facilitate the susceptibility testing. Here we present a comparison of the MIC results, interpreted according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, of 66 Campylobacter isolates from feces cultures for erythromycin, ciprofloxacin, and tetracycline between Etest® and the Sensititre® broth microdilution plate.Several methods for in vitro AST of ilution) . Currentilution) . As thisCampylobacter isolates were collected from feces samples of patients admitted to the hospital and diagnosed as suffering from Campylobacteriosis. Forty-four of the patients were children and 22 were adults. All feces samples were routinely subcultured on Campylobacter selective agar  and incubated at 42 °C for 48 h with CampyGen™  sachet for the generation of microaerophilic conditions.The study was performed at the clinical microbiology laboratory of the Baruch Padeh Medical Center, Poriya, in northern Israel. From May 2015 to February 2017, a total of 66 Campylobacter colonies were identified according to morphologic and biochemical characteristics. Final identification was performed by MALDI-TOF Mass Spectrometry System . All isolates, 50 C. jejuni and 16 C. coli, were sent to a reference laboratory for final confirmation of the species.Following incubation of feces samples, Campylobacter isolates were grown for 24 h at 37 °C in microaerophilic conditions before conducting susceptibility tests. C. jejuni ATCC 33560 was used for quality control.All C. jejuni and 8 mg/L for C. coli.After 48 h, several colonies were suspended in Brain Heart Infusion (BHI) broth to a turbidity of 0.5 McFarland. The suspension was seeded on three Mueller–Hinton agar plates containing lysed horse blood  and then one Etest strip  was put on each agar plate for erythromycin, ciprofloxacin, and tetracycline. Plates were incubated at 37 °C for 48 h in microaerophilic conditions. MIC values were determined after 48 h according to EUCAST guidelines: resistance to tetracycline is defined at MIC values above 2 mg/L; resistance to ciprofloxacin is defined at MIC values above 0.5 mg/L; resistance to erythromycin is defined at MIC values above 4 mg/L for ® broth microdilution plate, according to the manufacturer’s instructions. Briefly, each plate was in a dried form and contained a standard panel of serial dilutions of antimicrobial agents. Isolated colonies were suspended in 5 mL cation-adjusted Mueller–Hinton broth with TES buffer (CAMHBT) and turbidity was adjusted to 0.5 McF. The suspension (100 µL) was transferred into 11 mL cation-adjusted Mueller–Hinton broth with TES buffer and lysed horse blood (CAMHBT + LHB) and mixed. The new suspension (100 µL) was transferred to each well, which contained different concentrations of the antibiotic agent. The plate was sealed and incubated at 37 °C for 48 h in microaerophilic conditions. Following incubation, plates were visually read, searching for turbidity as an indicator of microbial growth. MIC values were determined as the lowest antibiotic concentration that inhibited microbial growth. Resistance to the antibiotic agent was determined according to EUCAST guidelines, as mentioned above. Each batch of BMD tests included Campylobacter control strains. The limits of MIC for C. jejuni ATCC 33560 were adopted from the manufacturer’s instructions: for erythromycin, 0.5–2.0 µg/mL; for ciprofloxacin, 0.06–0.25 µg/mL; and for tetracycline, 0.25–2 µg/mL.We performed broth microdilution (BMD) susceptibility test using the SensititreFor calculation of categorical agreement between the two methods, the percentage of agreement was calculated as the percentage of isolates that had the same result interpretation (sensitive or resistant) by the two methods. The level of categorical agreement was analyzed by calculating Kappa coefficients, with the acceptable following values: r < 0 poor agreement; 0 ≤ r ≤ 0.2 slight agreement; 0.21 ≤ r ≤ 0.4 fair agreement; 0.41 ≤ r ≤ 0.6 moderate agreement; 0.61 ≤ r ≤ 0.8 substantial agreement, and 0.81 ≤ r ≤ 1 almost perfect agreement.The very major error rate was defined as the percentage of isolates that had a “susceptible” result by Etest and a “resistant” result by BMD. The major error rate was defined as the percentage of isolates that had a “resistant” result by Etest and a “susceptible” result by BMD.Pearson correlation coefficients were calculated to test the level of agreement. Pearson coefficient range is between − 1 and 1, where zero means no correlation, 0 ≤ r ≤ 1 reflects the strength of a positive correlation, and − 1 ≤ r ≤ 0 reflects the strength of a negative correlation. The higher the coefficient, the stronger the correlation.p < 0.05. The data was analyzed using the SPSS® version 23.Statistical significance was determined with Campylobacter isolates were collected in the current study; of these, 50 Campylobacter jejuni and 16 Campylobacter coli were identified.Sixty-six C. jejuni, were interpreted as sensitive according to Etest and resistant according to Sensititre (one to erythromycin and two to tetracycline). Thus, the very major error rates were 3, 2.5, and 0% for tetracycline, erythromycin, and ciprofloxacin, respectively. No minor or major errors were observed. Kappa coefficients of 0.653, 0.66, and 1 (p < 0.0001) were calculated for tetracycline, erythromycin, and ciprofloxacin, respectively, indicating a substantial level of agreement for tetracycline and erythromycin, and an almost perfect agreement for ciprofloxacin , 98.5% (65/66), and 100% (66/66), respectively (Table p < 0.0001), with Pearson correlation coefficients of 0.645, 0.588, and 0.865 for tetracycline, erythromycin, and ciprofloxacin, respectively.Additionally, the correlation between methods following logarithmic transformation was good and highly significant for all the tested antibiotics (C. jejuni to tetracycline were 100% (50/50) and 96% (48/50), using Sensititre and Etest, respectively. Resistance rates of C. coli to tetracycline were 87.5% (14/16), using both methods.According to EUCAST criteria, 97% (64/66) and 94% 62/66) of our isolates were resistant to tetracycline using Sensititre and Etest, respectively (Table /66 of ouC. jejuni to erythromycin were 4% (2/50) and 2% (1/50), using Sensititre and Etest, respectively. All C. coli isolates were sensitive to erythromycin.Erythromycin resistance rates, according to EUCAST guidelines, were 3.03% (2/66) and 1.5% (1/66), as determined by Sensititre and Etest, respectively. Resistance rates of C. jejuni isolates and 93.75 (15/16) of the C. coli isolates were resistant to ciprofloxacin  of the isolates were resistant to ciprofloxacin; 96% (48/50) of the in Table .Campylobacter resistance to antibiotics is on rise worldwide, emphasizing the importance of AST performance [p < 0.0001). Additionally, no major errors were found and the very major error rates were low (0–3%). The Etest method generally produced higher MIC values, a phenomenon that was previously described [formance , 5, 10. formance , 4. We fC. jejuni and 0% of C. coli strains were resistant to erythromycin . Our results may point to wise/careful use of antimicrobial treatment in campylobacteriosis. In the current study, 95.45% of the isolates (96% of C. jejuni and 93.75% of C. coli) were resistant to ciprofloxacin. This extremely high resistance rate is much higher than the reported resistance levels in the USA and Canada, in which 19–47% of human Campylobacter isolates were resistant to ciprofloxacin [Campylobacter in Thailand and Hong Kong was found to be above 80% [The considerably low resistance rate to erythromycin 1.5–3.1%) in the current study is lower than that found in other countries such as in Bulgaria (31%), Singapore (51%), and Ghana (96%) –3.1% in . In the in Table , the repfloxacin . In compbove 80% , 17, 18,bove 80% .C. jejuni and C. coli strains, respectively (unpublished data). Additionally, ciprofloxacin resistance has increased over the years [C. jejuni and C. coli, respectively (unpublished data).However, our result is similar to the resistance levels found in 2014 by the Israel Ministry of Health that reported 93 and 98% resistance rates among he years ; in IsraC. jejuni was 96–100%, much higher than the reported rate in Israel in 2014, which was 53%. These differences may be explained by demographic characteristics of the study area, northern Galilee, with a rural population having close contact with animals. As Campylobacter infection is a zoonotic disease, resistance to antibiotics is not only influenced by antibiotics use in human but also in animals [Campylobacter strains underscores the need to curb antibiotic use in the veterinary domain and in humans.We found a resistance rate of 94–97% (Etest/Sensititre) to tetracycline among our strains. Interestingly, our resistance rate of  animals . ResistaCampylobacter spp. Obtaining data regarding prevalence and distribution of resistant strains is very important, not only for therapy adjustment but also for epidemiologic surveillance, especially in countries such as Israel, which is characterized by a high prevalence of campylobacteriosis.In summary, Etest and Sensititre kit can be easily adapted for monitoring susceptibility of


Pleurotus species for their total, β and α-glucan content. Pleurotus eryngii was found to have the highest total glucan concentrations and the highest α-glucans proportion. We also found that the stalks (stipe) of the fruit body contained higher glucan content then the caps (pileus). Since mushrooms respond markedly to changes in environmental and growth conditions, we developed cultivation methods aiming to increase the levels of α and β-glucans. Using olive mill solid waste (OMSW) from three-phase olive mills in the cultivation substrate. We were able to enrich the levels mainly of α-glucans. Maximal total glucan concentrations were enhanced up to twice when the growth substrate contained 80% of OMSW compared to no OMSW. Taking together this study demonstrate that Pleurotus eryngii can serve as a potential rich source of glucans for nutritional and medicinal applications and that glucan content in mushroom fruiting bodies can be further enriched by applying OMSW into the cultivation substrate.Mushroom polysaccharides are edible polymers that have numerous reported biological functions; the most common effects are attributed to β-glucans. In recent years, it became apparent that the less abundant α-glucans also possess potent effects in various health conditions. Here we explore several  Mushrooms have a long history of consumption as food, and also have remarkable therapeutic properties which are mainly recognized in oriental countries ,2. Many 6H12O5)n are polysaccharides built of glucose monosaccharides linked by α, β, or mixed α-β glycosidic bonds. β-d-glucans are a naturally-occurring structural component of the cell walls of the fungal mycelium [Pleurotus genus’s (Oyster) pleuran is one of the most important and effective sources of β-glucans [Glucans -β-glucan, there are limited reports on the structure of α-glucans, possibly due to their low concentration in mushrooms and uncertain biological function. However, a few studies report that alkaline-soluble α-(1→3)-ushrooms ,9,10.w/w). However, α-glucans content is much lower. Among four different P. ostreatus strains, the α-glucan content was 3.4–7.9% in the pileus and 3.0–7.6% in stems, compared to 27.4–39.2% β-glucans in the pileus and 35.5–50.0% in the stems [Mushrooms contain consistently high levels of β-glucans, on average 30–40% by weight -d-glucan has been reported only rarely in the literature. Recently, more attention has been given to sulfation, sulfonylation, and carboxymethylation of α-glucan polysaccharides which increase their solubility and antitumor activity [Compared with the intensive investigations of β-activity ,17. Addiactivity ,19,20.Pleurotus ostreatus in a substrate containing 10% of dry olive mill solid waste (OMSW) improved the nutritive value of the treated OMSW for ruminant farm animals. It was shown that while growing on OMSW P. ostreatus utilized the lipid-soluble ingredients of the OMSW. The enhanced utilization of lipids by the mushrooms shifted the degradation of the structural carbohydrates. This effect could imply on glucan concentration of the mushrooms, however glucan extent was not measured in the above mentioned study [P. pulmonarius under different growing conditions. Polysaccharides extracted from P. pulmonarius fruiting bodies grown on straw (FBE) were compared to the glucans extracted from mycelia produced in submerged culture (ME). We found in the abovementioned study [13C and 1H NMR analyses of the FBE preparation showed mixed α-linkages and β-anomeric carbon linkages, whereas the ME polysaccharide demonstrated mainly α-glucan linkages.There has been growing popularity in developing mushroom glucans for medicinal applications or as dietary supplements . Mushrooed study . In the ed study ,24,25,26ed study  previoused study  that thePleurotus cultivars; (2) to measure the effect of relative substrate composition including OMSW on the glucan concentration of Pleurotus in order to delineate a novel methodology to improve the health-promoting effect of Pleurotus.The objectives of this study were: (1) to determine the glucan content, with emphasis on α glucans, in two distinct morphological sections of the fruiting body  of seven different Pleurotus species: P. ostreatus, P. ostreatus var. colombinus, P. pulmonarius, P. pulmonarius var. sajor caju, P. eryngii, P. cornucopiae, and P. salmoneostramineus. The calculated extraction yield of soluble glucans for each mushroom strain is summarized in P. eryngii and P. Pulmonarius. The total dried weight represents 10% of wet weight, i.e., 66 g dried weight comes from 660 g fresh mushrooms.In studies aimed at examining the biological activity of α and β-glucans in different cell lines, it is essential to use soluble glucans in order to be supplemented into the cells’ medium. To our knowledge, this is the first study to evaluate the content of soluble α, β, and total-glucans, after discarding the insoluble fraction of the mushroom glucans. We extracted therefore, soluble glucans  and stalks (S). The measurements are summarized in Since α, β, and total glucans concentrations measured in P. eryngii that use environmentally problematic agricultural waste and at the same time may enable them to synthesize higher amounts of glucans [P. eryngii was grown in a substrate composed of a sterilized mixture of eucalyptus sawdust containing different concentrations of OMSW (from 20% up-to 80% w/w).We investigated several unique cultivation substrates for the growth of  glucans . P. erynP. eryngii in these environmental conditions on the yield of the extracted glucans fraction. There was a dose dependent effect of relative amounts of eucalyptus sawdust and OMSW on the yield of extracted glucan fraction . The highest α and total glucan content was observed in P. eryngii, with values ranging between 4.5 ± 0.40 % for α-glucans and 48.9 ± 4.82% for total glucan content. β-glucan content obtained by difference between total and α-glucans was 43.47 ± 4.21%. In contrast, P. salmoneostramineus exhibited the lowest glucan concentrations: 0.29 ± 0.04% for α glucans; to 17.09 ± 0.18% for β-glucan content; and 17.34 ± 0.21% for total glucan content .P. ostreatus (0.24–0.38% and 1.6% fresh weight) and P. eryngii. (0.22–0.38% and 3.1% fresh weight) measured according to an enzymatic analysis based on direct hydrolysis by lichenase and β-glucanase. Synytsya et al. [In the present study, we used the enzymatic methods based on the Megazyme kit© ,29 to mea et al. ,33 appeaa et al. .P. eryngii. To this end, we modulated glucans levels by cultivating the mushrooms in a substrate containing different levels of olive mill solid wastes (OMSW). Here we used the solid fraction of a three-phase olive oil mill, this substrate is different in its composition from the two-phase mill or olive pruning residues that are more toxic and were investigated in other studies [Pleurotus on substrate containing OMSW greatly stimulated the β-1,3-glucan synthase activity of P. ostreatus. Reverberi et al. [Pleurotus. In fact, oxidative stress may occur in the presence of OMSW, due to either the presence of oxidizing phenol compounds or the reactive oxygen species formed during laccase activity [Tremella spp. and lentinan in L. edodes [P. ostreatus. Additionally, Elisashvil et al. [We consequently aimed at enhancing the glucan content in  studies . The rea studies , who foui et al.  claimed activity . In fact. edodes . OMSW co. edodes  recentlyl et al. , demonstl et al. ,37.P. eryngii grown on sterilized mixture of eucalyptus sawdust and OMSW at different concentrations responded in the upregulation of the levels of all glucans, being minimal at 20%, median at 60%, and maximal at 80%. The effects were more distinct in stalks than in caps. The effect of α-glucans has been associated with increased insulin sensitivity and improved insulin resistance in peripheral target tissues, therefore they have been demonstrated to induce an anti-diabetic effect [In our study, the c effect . These mPleurotus mushroom species and strains used in this study are summarized in The w/w) up to 80% (w/w). The mixture was wetted to 51% water content and packed into 15 L polypropylene bags, containing a microporous filter, 2 kg wet substrate/bag. The bags were autoclaved at 121 °C for 1 h, closed and cooled to 25 °C for inoculation with spawn. The culture was incubated at 25 °C for 14–21 days. For fruiting, the bags were opened and the temperature was reduced to 16 °C with a relative humidity of 90%, 12 h daily light and CO2 concentration of 600–800 ppm.The mushrooms were grown on a sterilized mixture of eucalyptus sawdust and olive mill solid waste (OMSW). The OMSW is the solid fraction from a three-phase olive mill. The olive mill waste-water was discharged. OMSW was added at concentrations from 20% . Fruiting bodies were collected according to their cap opening and their cap color. After harvest, fruiting bodies were weighed; dried and whole fruiting bodies, stalks, or caps samples were milled to pass a 1.0 mm screen using a Retsch centrifugal mill preparation unit in order to allow for glucan analyses.Pleurotus eryngii mushrooms cultivated in 80% and 20% OMSW are summarized in The proportion of stalks and caps in wet and dried 2O at 121 °C for 30 min (in autoclave). The extract was then centrifuged at 13,000× g at 10 °C for 15 min. Ethanol (EtOH) was added to the supernatant, to a final concentration of 67% (v/v), and the mixture was stored overnight at 20 °C. The float was taken out, and the viscous fraction was removed and lyophilized and glucan content analyzed on the lyophilized material.Dried powder from the different mushroom strains was prepared as mentioned in the previous section. Each 100 g of powdered fruiting bodies originated from each of the strains was extracted with 1000 mL dHPleurotus fruiting bodies  for 1 h at 40 °C. The absorbance of the resulting color complex was measured at 510 nm using a spectrophotometer . Total Glucan (% w/w) and α-Glucan (% w/w) were calculated, and by difference between those two, the β-glucan content (% w/w) was finally calculated. Glucan content was expressed as percentage (w/w) of the fruiting body dry weight. The control provided by the kit consists of glucans extracted from the yeast Saccharomyces cerevisiae [Measurement of total glucan content is performed following a previous solubilization in ice cold 12 M Hrevisiae ,29.p < 0.05.Analyses of the experimental data was performed using the two-way ANOVA test. All data are expressed as mean ± standard deviation (SD). The level of statistical significance was set at Pleurotus eryngii can provide a rich source for glucan extraction  for nutritional and medicinal applications and that glucan content in mushroom fruiting bodies can be further enriched by applying OMSW into the cultivation substrate.The present study demonstrate that the edible mushrooms


Participants who selected “Japanese” and “Healthy” diets showed an obvious reduction in FPG while the FPG of participants from Group A declined slightly. “Japanese” and “Healthy” diets also obtained the highest DDP scores, and thus can be considered suitable for T2DM treatment in China. The results of the newly formed dietary patterns, “Japanese” and “Healthy” diets, confirmed the profound efficacy of nutritional intervention combined with health education for improving dietary behavior and glycemic control although health education played a more important role. The present study is encouraging with regard to further exploration of comprehensive diabetes care.Diabetes mellitus is very common in elderly Chinese individuals. Although nutritional intervention can provide a balanced diet, the sustaining effect on at-home dietary behavior and long-term plasma glucose control is not clear. Consequently, we conducted a long-term survey following one month of experiential nutritional intervention combined with health education. Based on the Dietary Guidelines for a Chinese Resident, we found that the food items met the recommended values, the percentages of energy provided from fat, protein, and carbohydrate were more reasonable after one year. The newly formed dietary patterns were “Healthy”, “Monotonous”, “Vegetarian”, “Japanese”, “Low energy”, and “Traditional” diets. The  Diabetes mellitus (DM), one of the most serious chronic diseases in humans, affected approximately 347 million individuals worldwide in 2011 ; the preDisease self-management, support, and nutrition therapy are important components of diabetes care and necessary to ensure favorable outcomes in all people with DM . NutritiClinicalTrials.gov) over the course of one month following a long follow-up survey. We hope that the results provide valuable information regarding long-term, comprehensive diabetes care.In order to reflect the actual dietary behavior of T2DM patients living at home after centralized intervention, and further develop an effective protocol for comprehensive, sustained T2DM management, we conducted a randomized, single-blind, multi-arm parallel trial for older adults with T2DM at a community health center in Baotou City, Inner Mongolia, China  was provided a standard diet, and “intervention oat groups” (Groups B and C) were provided the same diet coupled with a 50 or 100 g/day oat substitution for the equivalent grain  and plasma glucose (PG). Assessments were conducted at baseline and at the one-year follow-up survey. During the one-month centralized management period, participants were required to refrain from medication and food for at least 12 h prior to blood collection. All examinations were performed by the same certified clinical staff  at the same laboratory. Standardized questionnaires were used to obtain self-reported socioeconomic, demographic, cultural, and biological information.N-DDP scores were utilized to evaluate the characteristic of dietary patterns . The DDPAll data was tested to ensure normal distribution in SPSS 18.0  prior to subsequent analysis .t-test was conducted to compare the changes before and after intervention within groups. Differences in response among groups were analyzed via one-way ANOVA, where results are presented as means and standard deviations. Duncan’s post hoc test was performed for multiple comparisons when changes were significantly different among groups.A paired sample p < 0.001. The associations between interventional effects (including dietary patterns and PG) and sociodemographic factors were assessed according to the odds ratio (OR) with the logistic regression model [p < 0.05). All statistical procedures were performed using SPSS 18.0 software .The dietary results were analyzed via principal component analysis (PCA) with the correlation matrix as input . The Kaion model . The finOf the total participants (299), 54% were female and 46% were male, 4.3% were illiterate, 9.8%, 26.2%, and 31.7% received primary, middle, and high school education, respectively, and 28% received college education or higher. A percentage of 25% were aged from 51 to 60 years, 62.2% from 61 to 70 years, and 12.2% were over 70 years old.p < 0.05). Oil, salt, and fish intake met the suggested values after the one-year intervention, as well, though dairy and fruit intake were still lower than the suggested values and red meat intake continued to exceed the recommended values [The daily intake of energy and nutrients was estimated based on 24-h dietary recall . Intake d values .Nutrient intakes are reported in PCA was utilized to evaluate the refinement solution. The varimax rotation was applied for the final solution as it generated the strongest loadings on each of the final six factors  with the most meaningful interpretation. These six factors explained 62% of the total variance in the data; the each rotated dietary factors explained 13.7%, 10.7%, 10.5%, 9.5%, 9.4%, and 8.1% of the total variance, respectively .For the food items it made good theoretical sense to justify the inclusion of these factors, the final six dietary factors are summarized in Fourteen food items were ultimately valid and foods with scores above 0.20 were lumped into the same category, where higher scores in the dietary category represented higher consumption of these foods. According to the three highest scores, the dietary patterns were defined as “Traditional”, “Vegetarian”, “Japanese”, “Low energy”, “Healthy”, and “Monotonous”, respectively .As shown in p < 0.05) (p < 0.05) from 9.35 ± 3.1, 9.83 ± 2.6 and 9.48 ± 2.8 mmol/L in Group A, B, and C to 7.66 ± 2.5  from 18.14 ± 5.2, 19.36 ± 5.3 and 17.36 ± 5.1 mmol/L in Group A, B, and C to 11.51 ± 4.7, 12.05 ± 4.5 and 11.05 ± 4.3 mmol/L in Group A, B, and C, respectively. Also, HbA1c decreased obviously (p < 0.05) from 8.02% ± 0.93%, 8.30% ± 1.02% and 8.35% ± 1.04% in Group A, B, and C to 7.32% ± 1.24%, 7.34% ± 1.04% and 7.26% ± 1.30% in Group A, B, and C, respectively  than it did in Group A (14.29%) (p < 0.05).One-year later, PG and HbA1c decreased significantly ( < 0.05) . FPG decectively b. It was2h-PG decreased significantly (p < 0.05) after the one-year intervention. Participants who selected “Japanese” diet presented the lowest FPG among the six patterns (p < 0.05). The 2h-PG value at baseline for participants who selected the “Japanese” and “Healthy” diets were relatively high. “Healthy” diet yielded higher 2h-PG than “Traditional ”diet (p < 0.05), while the new 2h-PG values after the one-year intervention were close to normal. p < 0.05). At baseline, the HbA1c value of “Healthy” diet was highest and was significantly lower than “Vegetarian” diet after one year (p < 0.05). Reduction of FPG, 2h-PG, and HbA1c are shown in p < 0.05) and “vegetarian diet” (6.21%) (p < 0.05), and “Vegetarian” diet resulted in the highest 2h-PG among all six diets (p < 0.05). FPG reduction was most obvious in “Japanese” diet.Differences in the participants’ dietary patterns have an impact on their PG levels . FPG and2h-PG reduction. Most notably the said reduction in 2h-PG exceeded 12 mmol/L for female participants. The effects of all six dietary patterns on 2h-PG were significant. The 2h-PG for participants who selected “Japanese” diet decreased even exceeding 12 mmol/L after one-year intervention .The rising epidemic of obesity and increasing prevalence of diabetes worldwide has been partially ascribed to poor eating habits . This st2h-PG, and HbA1c control.The intake of oil, salt, and fat decreased after one year due to the one-month experiential intervention including a structured diet and health education, these results reflect the sustainable efficacy of intensive experiential management. The diets among the three groups showed the same intake in fiber, though oats intake varied from group to group. Importantly, the participants in Group A may have consumed increased levels of other cereals due to their improved health knowledge of nutrition through the health education they received, reflecting the importance of health education. This indeed helps to improve attitudes and encourage healthy dietary habits . By virt2h-PG, and HbA1c decreased considerably, which proved oat-based dietary supplementation helpful in the treatment of T2DM [R2 > 0.85) (data not shown), which suggests that the main contributor to PG and HbA1c was oats (fiber) consumption. These results are in accordance with the previous reports that oat fiber benefits low glycemic response [R2 > 0.90), indicating that β-glucan has a glucose-lowering effect because, oat β-glucan increases the insulin sensitivity index [+ K+-ATPase and Ca2+ Mg2+-ATPase activity [As oats intake increased, FPG, response ,25,26,27response ,29,30. Tty index . Oat β-gactivity , and mayactivity .In this study, “Healthy” diet included staple food (rice) as well as non-staple food , and also contained high quality protein not only from plant (beans) but also from animal . However, it is worth mentioning that the so called “healthy diet” pattern had been composed of different foods in previous studies ,23.Although the diets in this study have varied in other reports, the “Healthy” diet is typically low in fat and rich in vitamins, minerals, and fiber, all of which are considered protective against non-transmissible chronic diseases . The “Ja2h-PG values in women decreased up to 12 mmol/L. We believe these differences are at least partially attributable to gender differences in health-consciousness. It has been well-documented that women pay more careful attention to health, beauty, and nutrition than men. Women also may be more likely than men to focus on changes in living quality, nutrition, and habits which led to the positive results [Previous research has indicated that dietary behavior is dramatically influenced not only by sociodemographic factors, such as gender, age, education level, and marital status, but also by the quantity and quality of health-related information available . It is t results ,44,45. T results ,47. This results .Participants in this study between the ages of 51 and 70 years tended to select the “Traditional” diet. We believe this may be due to the prevalence of conservative thought in this population, as well as a general difficulty in simulating new concepts , togethep = 0.049) in a two-month educational intervention program [Conversely, Group A participants (without oat intervention) showed relatively good FPG results. The main benefit was the effect of health education, which is in accordance with previous studies on the subject . These h program .2h-PG in participants who selected “Japanese” diet and “Healthy” diet. These two dietary patterns contained balanced nutrition and relative energy in the form of carbohydrates from sources such as fruits, vegetables, whole grains, beans, and milk. Also worthy of note is that “Japanese” and “Healthy” diets contained certain high-quality foods as in a “Mediterranean diet”, which has been associated with lower rates of cardiovascular disease, cancer, diabetes mellitus, hypertension, inflammation, Alzheimer’s disease, depression, and all-cause mortality [We observed substantial reduction in FPG and ortality ,57,58,592h-PG decreased significantly after one year, but did not quite reach the target values, this may suggest further limitations, but also may imply that the intervention and education period was too short to be fully effective. Further, our sample was composed of Chinese individuals with T2DM, and therefore the results we derived might not apply to populations in other countries with other dietary patterns. Additionally, we did not determine whether self-reporting a specific behavior  was influential in changing the said behavior [The “Healthy” diet was the most often selected by participants in this study, which implies that the one-year nutritional intervention was quite effective in tandem with health education. The diet with the greatest benefit for diabetes sufferers, the “Japanese” diet, was chosen slightly less often, possibly suggesting certain limitations in the effectiveness of the education regimen. In addition, FPG and behavior .2h-PG, and HbA1c decreased, which indicated the feasibility and sustainable efficacy of intensively experiential management with nutritional intervention, coupled with health education, for the elderly in China with T2DM. Undoubtedly, health education plays a particularly important role. Moreover, oat intake showed the obvious effect for hypoglycemics, the formed “Japanese” and “Healthy” diets could be suitable for T2DM, treatment due to the higher DDP scores and hypoglycemic effects.The results of this study demonstrated that participants did indeed improve their diet after intervention; FPG,


In the United States, ponatinib has received accelerated approval for adults with T315I-positive chronic myeloid leukemia (CML) or T315I (gatekeeper mutation)-positive, Philadelphia chromosome-positive, acute lymphoblastic leukemia (Ph + ALL), and patients with CML or Ph + ALL for whom no other TKI therapy is indicated. The objective of this phase 1, mass balance study was to evaluate the absorption, metabolism, and excretion of [14C]ponatinib dose was administered orally to six healthy male volunteers, and absorption, metabolism, and excretion were assessed.A single 45-mg [N-demethylation), and four methylated products accounting for 20.5, 17.7, 8.3, and 8.4% of the radioactive dose, respectively.86.6 and 5.4% of the dose was recovered in feces and urine, respectively, during days 0–14 postdose. Median time to maximal plasma radioactivity was 5 h and mean terminal elimination half-life of radioactivity was 66.4 h. Ponatinib and its inactive carboxylic acid metabolite M14, the two major circulating radioactive components, accounted for 25.5 and 14.9% of the radioactivity in 0–24 h pooled plasma, with elimination half-lives of 27.4 and 33.7 h, respectively. Major metabolites in urine were M14 and its glucuronides, which, together with other M14-derived metabolites, represented 4.4% of the dose; ponatinib was not detected in urine. In feces, major radioactive components were ponatinib, M31 (hydroxylation), M42  contains supplementary material, which is available to authorized users. The use of tyrosine kinase inhibitors (TKIs) that target BCR–ABL has markedly improved the outcomes in patients with chronic myeloid leukemia (CML) . Second-Cmax) of 145 nM observed at 4–8 h (median time to Cmax was 4.8 h), and a mean terminal elimination half-life (t1/2) of approximately 22 h [In phase 1  and phasely 22 h .14C]ponatinib after administration of a single oral 45-mg dose in healthy male subjects. Herein, we provide detailed quantitative information about the total dose excreted, routes of excretion, and distribution and structural characterization of metabolites in plasma, urine, and feces.The objective of this phase 1, open-label, mass balance study was to evaluate the absorption, metabolism, and excretion of Ponatinib hydrochloride . The protocol and informed consent for this study were reviewed and approved by the Celerion Institutional Review Board. Informed consent was obtained from all individual participants and the study was conducted in accordance with the ethical standards of the Declaration of Helsinki.Recruiting of healthy subjects, housing, administration of [2 (range 20.2–29.9) participated in the study. Each subject received a single oral dose of 45 mg [14C]ponatinib  with 240 mL of water, in the fasted state on the morning of day 1. Whole blood, urine, and feces samples, and toilet tissue wipes, were collected up to the time of discharge . The subject release criterion was defined as follows: the combined urine and fecal radioactivity excretion for two consecutive days should fall below 1% of the dose administered. Most patients were discharged 11–14 days following dose administration.Six healthy male volunteers, 25–45 years of age, with a mean body weight of 83.3 kg (range 64.5–94.4) and a mean body mass index of 26.9 kg/m2EDTA tubes; 2 mL of whole blood was aliquoted for total radioactivity (TRA) analysis, and the rest of the blood was centrifuged to obtain plasma for TRA analysis, metabolite identification, and liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantitation of ponatinib and two metabolites (M14 and M42). Urine and feces collected from each subject were pooled at predose and every 24 h thereafter. Fecal homogenates were prepared by adding water (four times the feces weight) and homogenizing. Following collection, samples were stored at −80 °C and shipped from the clinical site over dry ice to the following laboratories: plasma, to Advion Biosciences, Inc.  for quantitation of ponatinib and N-desmethyl metabolite M42 (AP24567); whole blood, plasma, urine, and feces, to ABC Laboratories for determination of TRA; and plasma, urine, and feces, to ARIAD Pharmaceuticals, Inc.  for metabolite profiling and quantitation of M14.Detailed procedures on sample collection, storage, and shipment are described in Online Resource 1. A brief summary is provided here: 12 mL of whole blood was collected from each subject at various time points into K14CO2 was trapped in Carbosorb  in combination with Permafluor, followed by liquid scintillation counting to determine the radioactivity. Individual samples were counted for 5 min for whole blood and fecal matrices, and 10 min for plasma and urine matrices. Predose blood, plasma, urine, and feces samples served as respective control samples to determine and subtract the background radioactivity in the analysis.TRA analysis was carried out at ABC Laboratories. The radioactivity in plasma (duplicate per time point) and urine samples (triplicate per time point) was quantitated by direct counting in a Beckman LS6000SC or LS6500 liquid scintillation counter . Whole blood (duplicate per time point) and fecal homogenate (triplicate per time point) samples were oxidized, and the resultant ® v6.4 .Ponatinib and M42 (AP24567) were determined using a validated LC/MS/MS method previously described . M14 wasStructural characterization of the metabolites and quantitative determination (using radioactivity) of ponatinib and metabolites in pooled plasma, urine, and feces were carried out using LC/MS/MS methods coupled with offline radioactivity detection.For each subject, a 0–24 h pooled plasma sample was prepared according to the scheme outlined in the literature . In addig at 4 °C for 6 min. The supernatants were transferred into new polypropylene tubes and kept on ice. The resulting plasma protein pellets were extracted twice to ensure good recovery and the extracts were combined. The combined extracts were loaded onto Ostro sample preparation plates , dried, eluted with 0.2 mL of cold acetonitrile, and all eluates were collected and evaporated under nitrogen . The dry residues were reconstituted in 0.1 mL of methanol, and a 0.005-mL aliquot was removed for determination of radioactivity. The remainder of the supernatants were transferred to high-performance liquid chromatography (HPLC) vials and stored at −80 °C until analysis.All pooled 0–24, 5, 10, 12, and 24 h plasma samples were processed as follows. Since the radioactivity in the plasma samples was low, unlabeled ponatinib and M14 were added to enhance the recovery. Plasma samples (2.4 mL) were spiked with 1.2 μL of a methanol solution containing ponatinib and M14 (each at 1 mg/mL) followed by 5 mL of ice-cold 1% formic acid in acetonitrile. The samples were vortexed for 30 min and centrifuged at 4500Pooled post-24-h plasma samples were extracted using a slightly modified procedure. These samples were spiked with ice-cold 0.1% methanol solution containing deuterated analogs  of ponatinib, M42, and M14. Appropriate volumes of formic acid were added to achieve a final concentration of 1%. Then, the samples were warmed to 37 °C and loaded to preconditioned Oasis HLB cartridges . These columns were then washed with water, dried under vacuum, and eluted with methanol. The eluates were collected and evaporated under nitrogen. The dry residues were reconstituted in 0.1 mL of methanol, and a 0.005-mL aliquot was removed for determination of radioactivity. The remainder of the supernatants were transferred to HPLC vials and stored at −80 °C until analysis.Predose and 0–72-h urine samples  were prepared. Urine samples (5 mL) were centrifuged, the supernatant was transferred to a 15-mL polypropylene tube and placed on ice; the residues were extracted with 1 mL of methanol and the methanol extracts were added to the supernatants. The combined extracts were evaporated under nitrogen and reconstituted in 0.2 mL of methanol. A 0.010-mL aliquot of the reconstituted solution was removed for determination of radioactivity, and the remainder was transferred to HPLC vials and stored at −80 °C.g). The resulting residues were extracted three more times (once with 2 mL of methanol and twice with 1 mL of methanol containing 1% formic acid) and centrifuged. All supernatants were combined and evaporated under nitrogen, and the residue was reconstituted with 0.5 mL of methanol, vortexed, sonicated, and centrifuged. A 0.010-mL aliquot of the supernatant was removed for determination of radioactivity, and the remainder of the supernatant was transferred to an HPLC vial and stored at −80 °C.The predose and 0–144-h pooled fecal homogenate samples  were prepared. One-gram samples of the fecal homogenates were transferred to 50-mL polypropylene tubes, extracted with 4 mL of methanol, and centrifuged  was used along with a C-12 guard column (4 × 2 mm). LC conditions were as follows: solvent A was 10 mM ammonium bicarbonate in water (pH 10.0) and solvent B was 100% methanol and the flow rate was 0.8 mL/min. The following gradient was employed (B%): 0–5 min (20%), 32 min (42%), 33 min (60%), 55 min (70%), 56–60 min (95%), and 62–70 min (20%).A Finnigan Surveyor HPLC system , consisting of an LC pump plus an autosampler and a UV–Vis plus detector, was used mostly for profiling . A PhenomenexReconstituted solvent extracts (0.050–0.10 mL) of plasma, urine, or feces were injected onto the HPLC column. The post-column eluate was split after UV detection into two streams (0.64 and 0.16 mL/min). The 0.64-mL stream was diverted to a fraction collector  to be collected in 96-well plates at 0.16 min/well , and the 0.16-mL stream was directed to the mass spectrometer. The solvent in the 96-well plates was removed at 40 °C using a Genevac evaporator .The radioactivity counts per minute (CPM) in each well was determined using a MicroBeta scintillation counter . The resulting CPM vs. time data were processed using Excel . The CPM in each well was background-subtracted . Radiochromatographic profiles were obtained by plotting the resulting net CPM values against time after injection. Radioactive peaks in the chromatographic profiles were identified, and the radioactivity in each peak was summed and reported as a percentage of TRA in the HPLC run.1) or Q-Exactive (MS1 and MS2) mass spectrometers (Thermo Fisher Scientific Inc.) Exact conditions are described in the Online Resource 3.The remaining eluate stream of 0.16 mL/min from the HPLC runs was diverted to the inlet of the mass spectrometer. The samples were analyzed using a heated electrospray ionization probe. Full-scan positive ion MS data were collected using Finnigan Exactive , and 0.2 nM, respectively. The elimination curves of ponatinib, M14, M23, and M42 are nearly parallel, indicating that their terminal elimination half-lives are very similar. The TRA appeared to decrease in plasma with a longer half-life than that of the parent drug. This was likely due to the contribution of non-identifiable radioactive degradation products slowly released from deep body compartments .The concentration–time profiles of TRA, ponatinib, and metabolites M14, M23, and M42 are illustrated in Fig. The cumulative recovery of radioactive dose in urine, feces, and total dose excreted is presented in Fig. 2 fragmentation pattern of ponatinib (AP24534) is shown in Fig. + was observed at m/z 533.2278, and the main fragment ions were observed at m/z 433.1269, 260.0814 (100% abundance), 232.0855, 205.0749, and 101.1075. The fragment ion at m/z 433.1269 was formed by the neutral loss of N-methylpiperazine moiety (100 amu). The amide bond cleavage produced a major fragment ion at m/z 260.0814, and further fragmentation of m/z 260.0814 resulted in fragments of m/z 232.0855 and 205.0749 by successive losses of CO (28 amu) and HCN (27 amu). The ion at m/z 101.1075 is from the protonated N-methylpiperazine group. The fragmentation pattern of ponatinib was compared with those of its metabolites for identification of metabolites (Online Resource 5). The proposed metabolite structures were supported by exact mass measurements of their [MH]+ ions. The metabolites of ponatinib predominantly underwent fragmentation analogous to that of the parent drug, which allowed localization of structural changes in the metabolites. Metabolite profiling of plasma, urine, and feces is shown in Fig. The metabolite structures were characterized by mass spectral fragmentation patterns (Online Resource 5) and, where possible, were confirmed by comparison of chromatographic retention times and mass spectra of the reference standards . The MS14C]ponatinib to healthy human volunteers is presented in Fig. A representative radiochromatographic profile of metabolites in pooled 0-24 h plasma after administration of [N-desmethyl ponatinib (M42), and ponatinib N-oxide (M36) accounted for 7.0, 6.0, 2.1, 0.5, and 0.5% of the plasma radioactivity, respectively. In addition to M14, the amide hydrolysis would yield a putative aromatic amine (M19). However, M19 was not observed in any human samples, but only trace levels of acetylated M19  were observed in MS traces in human plasma, urine, and feces. Radiochromatograms of the 5-, 10-, and 12-h plasma extracts are shown in Online Resource 4A, 4B, and 4C, respectively, and the corresponding data are presented in Table Metabolite profiles of pooled plasma samples taken at several time points during 0–144 h were qualitatively similar among the six subjects Table . Ponatinatinib M4, and ponN-despiperazinyl acid.As a result of low levels of radioactivity in post-24-h plasma samples, quantitation by radioactivity was limited to ponatinib and two of the metabolites  were the two largest radioactive metabolites in human feces, and accounted for 20.4 and 9.6% of the radioactivity, respectively  of the dose was recovered in feces and only a small percentage (5.4%) was recovered in urine. Low levels of radioactivity were observed in plasma even at 264 h postdose plasma, whereas ponatinib, M14, and M42 levels in plasma were at or below detectable limits 144 h postdose.Following oral administration of a single dose of 45 mg [N-dealkylation of the despiperazinyl group, was a minor metabolite (7% of plasma TRA). Dealkylation of the piperazinyl group has been observed in an another TKI, imatinib [N-desmethyl ponatinib were the most abundant; several minor metabolites that resulted from two or more metabolic modifications were also identified. Four methylated ponatinib metabolites  totaling 9.7%  were only observed in feces, and these metabolites were likely generated as a result of metabolism of ponatinib by methyl transferases [In 0–24 h human plasma, ponatinib and M14 were the major radioactive components, with M14 present at 14.9% of the plasma TRA. M23, the product of imatinib . M42, a imatinib . Among tsferases , 16. NeaCmax and area under the concentration–time curve from zero to infinity (AUC0–∞) by 78 and 47%, respectively [N-demethylation (8.3%), methylation (8.4%), and amide bond hydrolysis (4.4%), among which hydroxylation and N-demethylation were mostly CYP3A4-mediated. These data indicate that ponatinib was readily absorbed and eliminated through multiple pathways following administration of a single oral dose in healthy male subjects.Overall, the proposed in vivo metabolism of ponatinib in humans


Gelsemium elegans (GE) is a kind of well-known toxic plant. It can be detoxified by Mussaenda pubescens (MP), but the detoxification mechanism is still unclear. Thus, a detoxification herbal formula (GM) comprising GE and MP was derived. The Caco-2 cells monolayer model was used to evaluate GM effects on transporting six kinds of indole alkaloids of GE. The bidirectional transport studies demonstrated that absorbance percentage of indole alkaloids in GE increased linearly over time. But in GM, Papp (AP→BL) values of the most toxic members, gelsenicine, humantenidine, and gelsevirine, were lower than that of Papp (BL→AP) (P < 0.05). The prominent analgesic effect members, gelsemine and koumine, were approximately 1.00 in γ values. Nowhere was this increasing efflux more pronounced than in the case of indole alkaloids with N-O structure. In the presence of verapamil, the γ values of humantenidine, gelsenicine, gelsevirine, and humantenine were decreased by 43.69, 41.42, 36.00, and 8.90 percent, respectively. The γ values in presence of ciclosporin were homologous with a decrease of 42.32, 40.59, 34.00, and 15.07 percent. It suggested that the efflux transport was affected by transporters. Taken together, due to the efflux transporters participation, the increasing efflux of indole alkaloids from GM was found in Caco-2 cells. Gelsemium elegans Benth. (GE) belongs to the family Loganiaceae and is widely distributed in Northern America, East Asia, and Southeast Asia [ast Asia . It is gast Asia . Howeverast Asia –4. The past Asia –8. Chiefast Asia , 10. Desast Asia , there hMussaenda pubescens Ait. f. (MP) belongs to the Rubiaceae family and is widely distributed in valleys, shady hillsides, and shrub jungle of east, south, and southwest China. It has been used as a Chinese folk medicine for thousands of years. It is not only used to treat common cold, heatstroke, diarrhoea, and some inflammatory diseases, such as laryngopharyngitis and acute gastroenteritis [nteritis , but alsnteritis . For insnteritis . Taking Thus, the constituents of GE in the absence or presence of MP were comparatively analyzed by HPLC method. There were no significant differences in categories and contents of the constituents . In lighCiclosporin and verapamil were purchased from Sigma Chemical Company . HPLC-grade water was obtained from a Milli-Q water purification system . HPLC-grade acetonitrile and methanol were obtained from Merck . HPLC-grade formic acid was obtained from Tedia . HPLC-grade ammonium acetate was obtained from Aladdin Co., Lid . Cell culture grade dimethyl sulfoxide (DMSO) was purchased from Sigma Chemical Company . Hank's balanced salt solution (HBSS), trypsin-EDTA, heat-inactivated fetal bovine serum (FBS), nonessential amino acid solution, and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco Laboratories . All other chemicals and solvents used were at least of analytical grade.−1. The entire plant collection and dried powders extraction of MP were similar to those described previously [−1. Concentrations of GE dichloromethane part and MP aqueous part were all converted according to the material dosage. Two parts were filtered and evaporated under reduced pressure to obtain GE dichloromethane and MP aqueous extracts with typical yields of 0.67 ± 0.071% and 5.78 ± 0.20%, respectively. The compatibility proportion in GM was 1 : 40 (GE dichloromethane extract: MP aqueous extract).Fresh roots collection and dried powders extraction of GE were similar to those described previously . The coneviously . Then thμm)  was applied for all analyses. The mobile phase was composed of A  and solvent B (acetonitrile) with a linear gradient elution: 0–0.5 min, 98-98% A; 0.5–1.5 min, 98–80% A; 1.5–3.0 min, 80–70% A; 3.0–4.0 min, 70–55% A; 4.0–4.1 min, 55–98%; 4.1–6.0 min, 98-98% A. The mobile phase flow rate was 0.3 ml·min−1, the column temperature was controlled at 40°C and sample injection volume was 2.0 μl. ESI/MS/MS was operated using electrospray ionization in positive mode with the following parameters: capillary voltage, 3.5 kV; cone voltage, 50 V; desolvation temperature, 500°C; source temperature, 150°C; cone gas (nitrogen), 150 L·h−1; desolvation gas (nitrogen), 900 L·h−1. Under the mass spectrometric conditions, the maximum stable response of the precursor ions and the major product ions of the analytes were achieved. The maximal peak area in every product ion was chosen as quantitative ion pair. The rest were all qualitatively ion pairs. The chemical profiles of extracts were illustrated in Identification of gelsemine, humantenidine, koumine, gelsenicine, gelsevirine, and humantenine in GE dichloromethane extract or GM extract was achieved by LC-ESI-MS/MS method. The analyses were performed using a triple-quadrupole Quattro micro mass spectrometer  equipped with an electrospray ionization (ESI) interface to obtain structural information. The raw data were detected by Waters 2998 photodiode array detector and processed with Empower TM software. A Waters ACQUITY UPLC C18 column , which has a surface area of 4.2 cm2. Culture medium was changed every two days for 14 days and daily thereafter for the next 7 days. In our culture condition, cells monolayer, after seeding approximately between 19 and 21 days, usually was available for transport experiments.Human colon adenocarcinoma cell lines (Caco-2) were obtained from the cell bank of Chinese Academy of Sciences . Cells were routinely grown in DMEM supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) glutamine, 1% (v/v) nonessential amino acid solution, and 100 U/mL penicillin-streptomycin solution under a humidified atmosphere of 5% CO2 [The integrity of the cells monolayer was monitored by measuring transepithelial electrical resistance (TEER) across the cells monolayer. Briefly, the cells monolayer was washed three times with blank HBSS after culture medium was aspirated. Then TEER values of cells monolayer were measured at 37°C by using a Millicell-ERS meter . Cells monolayer was considered intact and suitable for transport assays when the TEER values were more than 550 Ω cm2 .monolayer is the resistance of the cells monolayer along with the filter membrane, TEERblank is the resistance of the filter membrane, and A is the surface area of the membrane.The TEER value was calculated with the following equation :(1)TEER=5 cells/ml) and were incubated in the culture medium for 24 hours at 37°C under a humidified atmosphere of 5% CO2. Then the culture medium was removed and cells were bathed in culture medium supplemented with 0, 0.0156, 0.031, 0.062, 0.125, 0.25, or 0.50 mg·ml−1 extracts for 24 hours. Doses of the extracts, including GE and GM, were converted according to the GE material dosage. After incubation with 20 μL of 5.0 g·L−1 MTT for 4 hours at 37°C, the MTT solution was aspirated. The formazan crystals were dissolved in 150 μL DMSO by agitating the dishes on a micro oscillator. The absorbance (A) at 490 nm in each well was measured by using a microplate reader . The cytotoxicity was calculated according to  in all subsequent experiments. All transport experiments were conducted at 37°C unless specified otherwise. Transport of indole alkaloids across the Caco-2 cells monolayer was studied using monolayer of 19–21 days after seeding. Before the experiment, the monolayer was washed twice and preincubated with the blank HBSS for 1 h. Thereafter the HBSS solution on both sides of the cells monolayer was aspirated.μL of samples was taken for LC-MS/MS analysis at 0, 1, 2, 3, and 4 h after incubation, followed by an immediate replacement of 400 μL of the same solutions. At the end of the transport experiment, integrity of the monolayer was monitored by TEER value.Transport studies were conducted in the absorptive direction  basolateral (BL)) and the efflux direction (BL-AP), separately. For determination of AP to BL transport, 2.5 mL of sample solutions was added to the AP side, respectively, and 2.5 mL of blank HBSS was added to the BL side. For determination of BL to AP transport, 2.5 mL of sample solutions was added to the BL side, respectively, and 2.5 mL of blank HBSS was added to the AP side. The cells monolayer was then put into a shaker at 50 rpm during the transport process. 400 μmol), was added to GM. 10 μmol ciclosporin (a P-gp and MRP2 inhibitor) was also added to GM. The control group was set as well. After the preincubation, the different mixes were performed in Caco-2 cells monolayer. Transport studies were undertaken as mentioned above in the absorptive direction (AP-BL) and the efflux direction (BL-AP), separately. The samples were taken at 0, 1, 2, 3, and 4 h for LC-MS/MS analysis. The absorption and efflux transport apparent permeability (Papp) of indole alkaloids were determined.As efflux transporters (P-gp and MRP2) are expressed on the apical side of Caco-2 cells, the absorption or efflux transport of indole alkaloids across this model was investigated in the presence of efflux transporters inhibitors. A selective P-gp inhibitor, verapamil , Y = 66331X + 13558 (r = 0.9995), Y = 6268X − 1503 (r = 0.9996), Y = 91160X − 10147 (r = 0.9998), Y = 32116X − 3951 (r = 0.9990), and Y = 36977X + 13830 (r = 0.9994), respectively.400 eviously . RegressPapp) for both the absorptive direction (AP→BL) and the efflux direction (BL→AP) studies were calculated according to the following [dQ/dt (μg·min−1) is the drug permeation rate, A (cm2) is the filter surface area, and C0 (μg·mL−1) is the initial concentration in the donor chamber at t = 0 min.All experiments were run in triplicate. Results were expressed as mean ± SD. The apparent permeability coefficients (ollowing :(3)Papp=γ) was calculated with the following equation:The efflux ratio , cytotoxicity was all below 5%. In other words, cell survival rates were over 95% in this concentration. Considering an extremely low rate of cell injury and need of detectable concentration (see reference [As shown in eference ), we choDue to detoxification of compatibility of GE and MP in traditional Chinese medicine, the absorptive transport of indole alkaloids was examined. Papp (AP→BL) values of six indole alkaloids were lower than that of Papp (BL→AP). There was significant difference in the values of humantenine, gelsenicine, and gelsevirine (P < 0.05). It implied that the herbal formula could increase the efflux of indole alkaloids in the small intestine. But the cause of increasing efflux of indole alkaloids was still unclear. Therefore the detoxification herbal formula effects on efflux transporters were studied in the Caco-2 cells monolayer.As indicated in Papp coefficients of indole alkaloids across Caco-2 cells monolayer in the AP→BL and BL→AP directions were presented in γ) was calculated in Papp values of 6 kinds of indole alkaloids were all increased whether in AP-BL direction or in BL-AP direction.  were carried out. The Papp coefficients of indole alkaloids across Caco-2 cells monolayer in the AP→BL and BL→AP directions were presented in γ) was calculated in Papp value of 6 kinds of indole alkaloids was all increased whether in AP-BL direction or in BL-AP direction  were carried out. The Papp of humantenidine, gelsenicine, and gelsevirine was approximately 2.0 times higher than that of the absorptive Papp  was carried out in the process of transport assays to confirm the effect of efflux transporters. It can be seen from Tables


Background: α‐Synuclein is a major component of pathologic inclusions that characterize Parkinson's disease. PRX002 is an antibody that targets α‐synuclein, and its murine parent antibody 9E4 has been shown in preclinical studies to reduce α‐synuclein pathology and to protect against cognitive and motor deteriorations and progressive neurodegeneration in human α‐synuclein transgenic mice. Methods: This first‐in‐human, randomized, double‐blind, placebo‐controlled, phase 1 study assessed the impact of PRX002 administered to 40 healthy participants in 5 ascending‐dose cohorts (n = 8/cohort) in which participants were randomly assigned to receive a single intravenous infusion of study drug  or placebo (n = 2/cohort). Results: PRX002 demonstrated favorable safety, tolerability, and pharmacokinetic profiles at all doses tested, with no immunogenicity. No serious adverse events, discontinuations as a result of adverse events, or dose‐limiting toxicities were reported. Serum PRX002 exposure was dose proportional; the average terminal half‐life across all doses was 18.2 days. A significant dose‐dependent reduction in free serum α‐synuclein (unbound to PRX002) was apparent within 1 hour after PRX002 administration, whereas total α‐synuclein (free plus bound) increased dose‐dependently, presumably because of the expected change in kinetics following antibody binding. Conclusions: This study demonstrates that serum α‐synuclein can be safely modulated in a dose‐dependent manner after single intravenous infusions of an anti–α‐synuclein antibody. These findings support continued development of PRX002, including further characterization of its safety, tolerability, pharmacokinetics, and pharmacodynamic effects in the central nervous system in patients with Parkinson's disease. © 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. Parkinson's disease is a neurodegenerative disorder that manifests a spectrum of motor, psychiatric, cognitive, sleep, and autonomic signs and symptoms. The key underlying motor manifestation is caused by a slow and progressive degeneration of dopamine‐producing neurons in the substantia nigra.Currently available treatments for Parkinson's disease target the dopaminergic features of the disease but do little to address its nondopaminergic symptoms and fail to treat the underlying neurodegeneration and progressive decline in neurologic function. In addition, nonmotor symptoms of the disease , which can be direct or indirect outcomes of dopaminergic and other neuronal loss, are often resistant to dopamine replacement strategies and may be exacerbated by treatment under some conditions.The aggregation‐prone α‐synuclein protein is the major component of Lewy bodies and Lewy neurites, which are neuropathologic hallmarks of Parkinson's disease and other neurodegenerative diseases.Although the etiology of Parkinson's disease is yet to be determined, substantial clinical and nonclinical data suggest that soluble aggregated forms of α‐synuclein  self‐propagate and may spread between interconnected nervous system regions and contribute to disease progression. For instance, the pattern of Lewy pathology in patients with Parkinson's disease is generally consistent with disease propagation over interconnected neuronal networksWe have developed a monoclonal immunoglobulin G1 antibody, PRX002, designed to preferentially target soluble and insoluble aggregated forms of α‐synuclein. PRX002 is derived from the murine monoclonal antibody 9E4, which was developed based on results of immunization experiments that showed that antibodies directed against carboxyl terminus epitopes of α‐synuclein were the most effective at reducing both the neuronal accumulation of α‐synuclein and the behavioral deterioration in animal models.ClinicalTrials.gov, NCT02095171). The study was performed at Worldwide Clinical Trials Early Phase Services, LLC, San Antonio, Texas. Participants were enrolled into 1 of 5 dose‐level cohorts in which they received PRX002  or placebo administered as a 60‐minute intravenous infusion. The study duration was 16 weeks, comprising a 4‐week screening period and a 12‐week follow‐up period after administration of a single dose of the study drug. The study protocol was approved by the investigational review board for the study site. All participants provided written informed consent. The study was conducted according to International Committee on Harmonisation Good Clinical Practice guidelines and the principles of the Declaration of Helsinki.This was a single‐center, randomized, double‐blind, placebo‐controlled, single ascending‐dose, phase 1 study in healthy volunteers  unless approved by the sponsor and the investigator; and no vaccination within 30 days before baseline.Key enrollment criteria included the following: 21 to 65 years of age; body weight > 45 to ≤ 110 kg and body mass index 18 to 32 kg/mWithin each cohort, participants were randomly assigned in 2 blocks. In the first block, 2 participants received PRX002 or placebo 1:1 and served as sentinel participants to assess safety and tolerability before the second block was randomly assigned. In the second block, 6 participants were randomly assigned to receive PRX002 or placebo 5:1 at least 7 days after the sentinel participants were dosed and their safety data were reviewed.The study participants, investigators, sponsor, and all staff members involved in the conduct of the study were blinded to treatment assignment. The only personnel who had access to the randomization code before database lock were the pharmacist and a bioanalyst/pharmacokineticist not involved in the clinical assessment of the participants. Randomization codes were maintained in a secure location to which only the nonblinded staff had access.All participants were screened within 4 weeks before administration of the study drug. Eligible participants were confined in the study unit from the day before dosing until 24 hours after dosing. Participants returned at intervals for outpatient visits until the final assessment at week 12. Dose escalation for the second cohort was based on the evaluation of 4 weeks of safety data from the first cohort; for each subsequent cohort, dose escalation was based on 2 weeks of safety data from the preceding cohort.PRX002 is a humanized IgG1 monoclonal antibody. The manufacturing process for PRX002 drug substance used a recombinant Chinese hamster ovary cell line grown in a suspension culture. The manufacturing campaign consisted of cell culture and harvest operations followed by a multiple‐step purification and formulation process. Manufactured PRX002 had the expected structure and biological activity demonstrated by the results of an extensive and comprehensive evaluation using an array of accepted analytical techniques. All drug substances and drug products were produced under good manufacturing practice conditions. PRX002 was supplied by the sponsor in sterile vials containing 200 mg lyophilized PRX002, which was reconstituted with water for injection. Reconstituted PRX002 was mixed with normal saline to obtain a total volume of 250 mL. The study drug was prepared by a nonblinded study pharmacist for intravenous infusion over 60 ± 10 minutes. Normal saline (250 mL) was used as the placebo.All participants were asked to return 3 days, 7 days (week 1), 14 days (week 2), 28 days (week 4), 42 days (week 6), and 84 days (week 12) after dosing for clinical, safety, and pharmacokinetic assessments. Immunogenicity was assessed before dosing and at 2, 4, and 12 weeks after dosing. Free and total serum α‐synuclein concentrations were assessed before dosing and at 1 to 3 days and at 1, 2, 4, 6, and 12 weeks after dosing. Serum PRX002 concentrations were assessed at the following times: before dosing; 30 minutes after the start of the infusion; at the end of the 60‐minute infusion; 0.25, 0.5, 1, 2, 4, and 8 hours after the end of the infusion; 1 and 3 days after dosing; and 1, 2, 4, 6, and 12 weeks after dosing. Cerebrospinal fluid was not collected in this healthy volunteer study.inf), observed terminal rate constant, terminal elimination half‐life, total body clearance, and volume of distribution.The primary study objectives were to evaluate the safety, tolerability, and pharmacokinetics of a single intravenous infusion of PRX002, and the secondary objective was to assess immunogenicity. Safety was assessed in all participants who received the study drug (PRX002 or placebo); assessments included adverse events (AEs), serious AEs, severity of AEs, laboratory tests, vital signs, electrocardiography, and physical and neurologic examinations and were reviewed before dose escalation. Pharmacokinetic and pharmacodynamic sampling was performed on all participants. Serum PRX002 was measured using a validated, quantitative sandwich electrochemiluminescence (ECL) assay (lower limit of quantification 39.0 ng/mL) developed by Prothena. Serum PRX002 pharmacokinetic parameters were estimated using noncompartmental techniques , including maximum concentration, time to reach maximum concentration, area under the serum concentration‐time curve from time 0 hours to last quantifiable concentration calculated using linear trapezoidal summation, area under the serum concentration‐time curve from time 0 hours to infinity . Samples with high (>600 μg/mL) hemoglobin levels were excluded from α‐synuclein measurements.Baseline and demographic characteristics, safety, and pharmacokinetic data were summarized using descriptive statistics. Total α‐synuclein and free serum α‐synuclein concentrations and change from baseline concentrations at each sampling time were summarized at each dose level using descriptive statistics and were assessed using a repeated‐measures analysis performed separately for free or total serum α‐synuclein. Two linear fixed‐effects models, adjusted for baseline as a covariate, were fit to results from 10 postbaseline time points. Treatment, time, and their interaction, estimated using a restricted maximum likelihood method, were included in the model. Given that the treatment‐by‐time interaction was significant for both parameters, the sequential trend test was performed at each time point for both the free and total α‐synuclein levels. All tests, including main and interaction effects, were conducted at the 2‐sided significance level of .05.When relevant, participants who received placebo were pooled across cohorts. Participant disposition, discontinuation, and treatment‐emergent AEs were summarized using the frequencies and percentages of participants (by treatment and dose where appropriate).A total of 40 healthy volunteers were randomly assigned to receive placebo or PRX002 from October 2014 to February 2015. Participants ranged in age from 21 to 58 years; the median age was 37.0 years  to 85,920 h·μg/mL (30 mg/kg PRX002). Average terminal half‐life across all doses was 18.2 days. Total body clearance was similar across doses, ranging from 0.2553 mL/h/kg (1 mg/kg PRX002) to 0.3492 mL/h/kg (30 mg/kg PRX002). Volume of distribution values ranged from 124.1 mL/kg (0.3 mg/kg) to 240.6 mL/kg (10 mg/kg PRX002).Serum PRX002 exposure generally increased in parallel with PRX002 doses over the range of 0.3 to 30 mg/kg Fig. ; Table 3P < .0001; Fig. A dose‐ and time‐dependent, statistically significant reduction in free PRX002 unbound) serum α‐synuclein of up to 96.5% was apparent within 1 hour after the end of the infusion, which was the first time point sampled after infusion  and a higher proportion of them were not white (42.5%) compared with a more typical patient population with Parkinson's disease, safety and tolerability will continue to be closely assessed in future studies. Moreover, although no anti‐PRX002 antibodies were detected in this study, it remains important to continue to assess this end point in ongoing and future studies in which patients will receive multiple doses of the study drug.ClinicalTrials.gov, NCT02157714).This phase 1 clinical trial in healthy volunteers demonstrates the safety of a single intravenous dose of PRX002 and its ability to significantly reduce the levels of free α‐synuclein in serum. These results support the design of the ongoing, multiple‐ascending dose, double‐blind, phase 1 study evaluating PRX002 in patients with Parkinson's disease (1. Research Project: A. Conception, B. Organization, C. Execution; 2. Statistical Analysis: A. Design, B. Execution, C. Review and Critique; 3. Manuscript Preparation: A. Writing the First Draft, B. Review and Critique.D.B.S.: 1A, 2C, 3A, 3BM.K.: 1A, 2C, 3A, 3BD.K.N.: 1A, 2C, 3A, 3BS.G.G.: 1A, 2C, 3A, 3BM.G.: 1A, 2C, 3A, 3BW.Z.: 1A, 2C, 3A, 3BJ.S.: 2C, 2C, 3A, 3BG.A.: 2B, 3A, 3BS.O.: 1A, 2C, 3A, 3BG.G.K.: 1A, 2C, 3A, 3BD.B.S. reported employment, leadership, stock ownership, intellectual property, and travel for Prothena. M.K. reports employment, leadership, stock ownership, and travel for Prothena. D.K.N. reports employment, stock ownership, and travel for Prothena. S.G.G. is a consultant for Prothena. M.G. is a consultant and reports stock ownership in Prothena. W.Z. reports employment, stock ownership, and travel for Prothena. J.S. reports employment, stock ownership, and travel for Prothena. G.A. has nothing to disclose. S.O. reports employee status and stock ownership in F. Hoffmann‐La Roche Ltd. G.G.K. reports employment, leadership, stock ownership, travel, and patents/intellectual property with Prothena.