Clitoria ternatea extract (CTE) has been demonstrated to possess anti-diabetic activity. However, there is no scientific evidence supporting its anti-glycation activity. The objective of this study was to determine the inhibitory effect of CTE on fructose-induced formation of AGEs and protein oxidation. Antioxidant activity of CTE was also assessed by various methods.The accumulation of advanced glycation end products (AGEs) in body tissue has been implicated in the progression of age-related diseases. Inhibition of AGE formation is the imperative approach for alleviating diabetic complications. 3 colorimetric method, and pH differential method, respectively. The various concentrations of CTE were incubated with BSA and fructose at 37°C for 28 days. The formation of fluorescent AGEs, the level of fructosamine, protein carbonyl content, and thiol group were measured. The in vitro antioxidant activity was measured by the 1,1-diphenyl 2-picrylhydrazyl (DPPH) scavenging activity, trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP), hydroxyl radical scavenging activity (HRSA), superoxide radical scavenging activity (SRSA), and ferrous ion chelating power (FICP).The aqueous extract of CTE (0.25-1.00 mg/ml) was measured for the content of total phenolic compounds, flavonoid, and anthocyanin by Folin-Ciocalteu assay, AlCl50 values of 0.47 ± 0.01 mg/ml and 0.58 ± 0.04 mg/ml. Furthermore, the FRAP and TEAC values of CTE were 0.38 ± 0.01 mmol FeSO4 equivalents/mg dried extract and 0.17 ± 0.01 mg trolox equivalents/mg dried extract. However, CTE showed weak scavenging activity on hydroxyl radical and a weak antioxidant iron chelator.The results demonstrated that the content of total phenolics, flavonoids and total anthocyanins in CTE was 53 ± 0.34 mg gallic acid equivalents/g dried extract, 11.2 ± 0.33 mg catechin equivalents/g dried extract, and 1.46 ± 0.04 mg cyanidin-3-glucoside equivalents/g dried extract, respectively. Moreover, CTE (0.25-1.00 mg/ml) significantly inhibited the formation of AGEs in a concentration-dependent manner. CTE also markedly reduced the levels of fructosamine and the oxidation of protein by decreasing protein carbonyl content and preventing free thiol depletion. In the DPPH radical scavenging activity and SRSA, CTE had the ICThe results showed that CTE has strong antiglycation and antioxidant properties and might have therapeutic potentials in the prevention of AGE-mediated diabetic complications. Chronic hyperglycemia is a major cause of complications of diabetes through 5 major mechanisms including polyol pathway, the formation of advanced glycation end products (AGEs), increased expression of AGEs receptor, Protein kinase C isoform activation and hexosamine pathway ,2. In geε-CML) -5. The aε-CML) -8. Fructε-CML) ,10. Intr glucose . Therefo glucose ,13. Ther glucose ,14,15.Clitoria ternatea L. (Family: Fabaceae) commonly known as butterfly pea has been used as a traditional Ayurvedic medicine as a memory enhancer, antistress, anxiolytic, antidepressant, anticonvulsant, tranquilizing, and sedative agent [Clitoria ternatea possesses a wide range of pharmacological activities including anti-oxidant, antimicrobial, anti-inflammatory, antipyretic, anti-helminthic, and analgesic activities [Clitoria ternatea flower exerts anti-hyperglycemic effects in alloxan-induced diabetic rats [Clitoria ternatea extract (CTE) on the inhibition of AGE formation. Therefore, the objective of the present study was to investigate the effect of CTE against bovine serum albumin (BSA) in fructose-induced non-enzymatic glycation. The study also examined the effect of CTE on glycation-induced protein oxidative damages. Antioxidant activity of CTE was also determined in various in vitro models.ve agent . Its flotivities ,17. In atic rats . To the 4), xanthine, xanthine oxidase, 5,5′-dithiobisnitro benzoic acid (DTNB), nitroblue tetrazolium (NBT), 1-deoxy-1-morpholinofructose (DMF), 2,4-dinitrophenylhydrazine (DNPH), thioflavin T reagent -N,N-dimethylaniline chloride), and L-cysteine were purchased from Sigma Chemical Co. . Fructose, Folin-Ciocalteu’s phenol reagent, and gallic acid were purchased from Fluka . All other chemical reagents used in this study were of analytical grade.Bovine serum albumin (BSA), aminoguanidine (AG), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Trolox), 2,4,6- tripyridyl-S-triazine (TPTZ), iron (II) sulfate  and atomizing (90 kPa).The dried flower of d method . Briefly2CO3 and kept for 2 h at room temperature. The absorbance of mixture was measured at the wavelength 760 nm with a spectrophotometer. Total phenolic content was expressed as mg gallic acid equivalents/g dry weight of extract [510 - A700) pH1.0 - (A510 - A700) pH4.5 and the TAC in the testing solution was calculated as cyanidin-3-glucoside equivalents [The CTE was dissolved in distilled water before use. For measurement of total phenolic content, the sample was mixed with Folin-Ciocalteu reagent (previously diluted 10-fold with distilled water), followed by 2% Na extract . Quantif extract . The extivalents .The glycated BSA formation was performed according to a previously published method . In brieAfter incubation for 7, 14, 21, and 28 days, the concentration of fructosamine was determined using a nitroblue tetrazolium (NBT) assay according to a previous study [−1 · cm−1). The results were expressed as nmol carbonyls/mg protein.After incubation for 7, 14, 21, and 28 days, the protein carbonyl content was determined using 2,4-dinitrophenylhydrazine (DNPH) according to a previous study [After incubation for 7, 14, 21, and 28 days, the free thiol concentration of glycated BSA was measured using Ellman’s assay [50 value was calculated from plots of log concentration of inhibitor concentration versus percentage inhibition curves. Ascorbic acid was used as a positive control for this study.DPPH  radical scavenging activity was measured according to the previous method . Briefly+) was induced by adding potassium persulfate (K2S2O4) and ABTS. The mixture was incubated at room temperature for at least 16 hours in the dark. The ABTS°+ solution was diluted in 0.1 M PBS, pH 7.4 to absorbance at 0.700 ± 0.02 nm. The extract was added to ABTS°+ solution for hydrogen atom transfer (HAT). The decrease in the solution absorbance was measured at 734 nm. The TEAC value was calculated from the standard curve prepared by using a Trolox.Assessment of ABTS radical-scavenging activity was done according to a previously published method . The rad3. The extract was added to FRAP solution as oxidizing reagent and incubated for 30 min at 37°C. The increase in the solution absorbance was measured at 595 nm using a spectrophotometer. FRAP value was calculated from a standard curve using FeSO4. FRAP value was expressed as mmol FeSO4/mg dried extract.The reducing power was measured according to a previous method . The fre50 value was calculated from plots of log concentration of inhibitor concentration versus percentage inhibition curves. A trolox was used as a positive control for this study.Hydroxyl radical scavenging activity was measured according a previous method . The abs50 value was calculated from plots of log concentration of inhibitor concentration versus percentage inhibition curves. A trolox was used as a positive control for this study.Superoxide radical scavenging activity was measured according a previous method . The abs50 value was calculated from plots of log concentration of inhibitor concentration versus percentage inhibition curves. EDTA was used as a positive control for this study.The metal chelating power was measured according to a previously published method . The decp < 0.05 was considered to be statistically significant.Data were expressed as the mean ± standard error of mean (SEM) for n = 5. The results were analyzed by one-way analysis of variance (ANOVA) and Duncan’s post hoc analysis. The content of total phenolic compounds in CTE was 53 ± 0.34 mg gallic acid equivalents/g dried extract. The content of flavonoid in CTE was 11.2 ± 0.33 mg catechin equivalents/g dried extract. Moreover, the content of total anthocyanin in CTE was 1.46 ± 0.04 mg cyanidin-3-glucoside equivalents/g dried extract.Figure The level of fructosamine in glycated BSA was gradually increased 2.0-fold to 3.6-fold during 28 days of incubation . The results showed that CTE exhibited less potent antioxidant activity with FICP and HRSA.In DPPH assay, CTE had significant radical scavenging activity with the ICMesona chinensis Benth (Chinese Mesona), most widely consumed as an herbal beverage and a gelatin-type dessert, also showed the percentage inhibition of 39.60-59.42% with concentration of 0.25-1.00 mg/mL at week 4 of incubation [Albumin is a target protein for the glycation reaction due to its abundance in serum . Proteincubation . Our fin3+ and Cu2+) [Abundant evidence exists that an excessive production of reactive oxygen species (ROS) and reactive nitrogen species are generated during glycation and glycoxidation -5 The prnd Cu2+) . When ROnd Cu2+) . Interesnd Cu2+) . TherefoClitoria ternatea flower petal extract revealed the presence of bioactive compounds such as delphinidin-3,5-glucoside, delphinidin-3β-glucoside, malvidin-3β-glucoside, kaemphferol, p-coumaric acid and six major ternatins  [Phytochemical compounds including polyphenolics, flavonoids and anthocyanins, present in plant-based foods, provide beneficial effects on free radical scavenging properties -36. The  and D2) . The pre and D2) ,15,38. I and D2) . FurtherIn the present work, CTE prevents fructose-induced protein glycation and oxidation-dependent damages to protein. It also exhibits an excellent scavenging ability for different forms of free radicals. Antiglycation and antioxidant activity of CTE may be responsible for their usefulness in the management and prevention of AGE-mediated diabetic complication. Further studies are required to investigate the antiglycative effects of CTE on diabetic rats.


Zoogloea sp. N299. This species exhibits a periplasmic nitrate reductase gene (napA). Its specific growth rate was 0.22 h−1. Obvious denitrification and perfect nitrogen removal performances occurred when cultured in nitrate and nitrite mediums, at rates of 75.53% ± 1.69% and 58.65% ± 0.61%, respectively. The ammonia removal rate reached 44.12% ± 1.61% in ammonia medium. Zoogloea sp. N299 was inoculated into sterilized and unsterilized reservoir source waters with a dissolved oxygen level of 5–9 mg/L, pH 8–9, and C/N 1.14:1. The total nitrogen removal rate reached 46.41% ± 3.17% (sterilized) and 44.88% ± 4.31% (unsterilized). The cell optical density suggested the strain could survive in oligotrophic drinking water reservoir water conditions and perform nitrogen removal. Sodium acetate was the most favorable carbon source for nitrogen removal by strain N299 (p < 0.05). High C/N was beneficial for nitrate reduction (p < 0.05). The nitrate removal efficiencies showed no significant differences among the tested inoculums dosage (p > 0.05). Furthermore, strain N299 could efficiently remove nitrate at neutral and slightly alkaline and low temperature conditions. These results, therefore, demonstrate that Zoogloea sp. N299 has high removal characteristics, and can be used as a nitrogen removal microbial inoculum with simultaneous aerobic nitrification and denitrification in a micro-polluted reservoir water ecosystem.Nitrogen is considered to be one of the most widespread pollutants leading to eutrophication of freshwater ecosystems, especially in drinking water reservoirs. In this study, an oligotrophic aerobic denitrifier was isolated from drinking water reservoir sediment. Nitrogen removal performance was explored. The strain was identified by 16S rRNA gene sequence analysis as  During the past few decades, more and more nitrogen has been discarded into the fresh water ecosystem, leading to serious environmental problems ,2,3,4, sThiosphaera pantotropha [Thiosphaera pantotropha [Alcaligenes faecalis [Citrobacter diversus [Pseudomonas stutzeri [Rhodococcus sp. [Robertson and Kuenen’s discovery of aerobic denitrification bacteria totropha  at a dentotropha ,15,16. Mtotropha . Aerobictotropha ,19: nitrtotropha , and dentotropha . There atotropha , ponds [totropha , and soitotropha ; the domtotropha , Alcaligfaecalis ,26, Citrdiversus , Pseudomstutzeri , and Rhoccus sp. .Rhizobium sp. PY8 [Compared with strains isolated in massive amounts from other environmental systems ,22,23, a sp. PY8  had a go sp. PY8 . We carr sp. PY8 , and thenapA gene, to determine nitrogen removal performance in nutrient medium and micro-polluted raw water, and to explore the effect of different factors on nitrate removal. The results can be useful for using aerobic denitrifiers for micro-pollution reservoir bioremediation.To this end, we isolated 196 strains using enrichment and screening processes. We found that the N299 strain has perfect performance on nitrogen removal in aerobic conditions with low pollutant concentration. Therefore, the objectives of the present work were to determine the taxonomic status using the 16S rRNA method, to examine the In the preliminary experiment with enrichment culture isolation, 196 strains were isolated. The isolated strain N299 with high nitrogen removal efficiency was obtained in this study, and was stored on SM (screening medium) slant medium at 4 °C and on SM Glycerin medium at −20 °C.Zoogloea caeni strain EMB43 (Zoogloea caeni strain EMB43 (type culture strain) were in the same group. Therefore, the N299 strain was identified as Zoogloea. A neighbor-joining phylogenetic tree showing the phylogenetic relationships between strain N299 and other previously studied aerobic denitrifiers was also constructed (Zoogloea sp. N299 and Comamonas testosteroni strain GAD4 (aerobic denitrifier) were in the same group. Therefore, the strain was affiliated with Zoogloea sp. N299. The nucleotide sequence of N299 was submitted into GenBank nucleotide sequence databases under the accession number KP717093, and the strain was also deposited to the China General Microbiological Culture Collection Center (CGMCC) under the number 4.7825.The N299 strain was a gram-negative, aerobic, rod-shaped bacterium, and its size was approximately (0.5–1.0) × (1.5–3.0) μm . The 146in EMB43 . A neighin EMB43 . This restructed . ResultsnapA was obtained  of the strain N299 increased from 0.004 to 0.062. a = 0.064, c = 0.004, and μ = 0.22 h−1, with a correlation coefficient of 0.9905. The generation time for N299 was 3.15 h.Zoogloea sp. N299 demonstrated obviously denitrification performance. As shown in 510 of the N299 strain increased from 0 to 0.095, which was lag phase. The N299 strain resulted in simultaneous organic matter and nitrate removal. It was suggested that utilization of organic matter and degradation of nitrate nitrogen took place simultaneously in a true heterotrophic process in logarithmic growth phase. The denitrification of N299 became weak at low C/N (0.97/1) in 72 h, and the denitrification stopped. Because carbon is essential for cell growth and nitrate reduction processes, the optimal quantity of carbon is a key parameter in the denitrification process.Under aerobic conditions , 510 and TOC levels at initial 3.76 mg/L nitrite. The removal of nitrite and TOC correlated strongly with the growth rate of isolate N299, as shown in Few aerobic denitrifiers using nitrite as a sole nitrogen source were identified. Considering the nitrogen level of oligotrophic reservoir and inflow rivers, therefore, the initial nitrite was fixed 3–4 mg/L, and the denitrification activity of N299 strain using nitrite as sole nitrogen was assessed. In order to explore the nitrification potential of N299, strain N299 was cultured in a high ammonia-medium. Changes of various components in the flask culture are shown in To investigate the performance of bioremediation in micro-polluted source water, we conducted a sterilized reservoir source water experiment. The initial conditions are shown in From the sterilized reservoir source water experiment, it was shown that the N299 had a perfect adaptation to low C/N level source water and could easily survive in oligotrophic reservoir source water. This experiment studied denitrification of source water that included indigenous bacteria by N299 bacteria. The initial conditions of experiments were shown in Zoogloea sp. N299 could utilize glucose, sodium succinate, sodium citrate, and sodium acetate to remove nitrate (p < 0.05). Strain N299 exhibited the best nitrate removal ability (78.86% ± 0.29% in 72 h) when sodium acetate was used as the sole carbon source. Accordingly, sodium acetate was employed as the carbon source in the following experiments.Carbon compounds usually serve as energy and electron sources for denitrification bacteria. Strain  nitrate . The nitp < 0.05). The nitrate removal percentage increased from 45.25% ± 0.35% at 10 °C to 78.86% ± 0.29% at 30 °C, in 72 h. Moreover, the excellent adaptability to 10–30 °C presented by strain N299 was beneficial for nitrogen removal in the natural water system.As shown in p < 0.05). Maximal nitrate removal occurred at C/N ratio of 10, and reached 85.18% ± 0.80%, in 72 h. However further decrement in C/N yielded a slight decrease in nitrogen removal efficiency.The influence of different C/N ratios on nitrate removal by strain N299 was shown in v/v) on nitrate removal by strain N299 was shown in p > 0.05). The nitrate removal rates were from 77.23% ± 0.41% to 78.86% ± 0.29% among inoculums dosage (v/v) of 2%–10%, in 72 h.The influence of different inoculums dosage (p < 0.05)  . The nit−1, 10−2, 10−3, 10−4, 10−5, 10−6, 10−7 dilution), 0.2 mL of each bacterial suspension was blended with SM agar plate (liquid SM medium with the addition of 2% agar) and incubated at 30 °C for three days. Purified isolates were obtained via repeated streaking on fresh agar plates. To detect the denitrification performance, isolates were cultivated in SM medium with NaNO3 as the sole nitrogen source. Strain N299 with high nitrogen removal efficiency was obtained in this study.Many studies have focused on the wastewater treatment, however, heterotrophic microorganisms often required high concentrations of organic carbon ,42. In cZoogloea sp. N299. A phylogenetic tree was constructed based on the 16S rRNA gene sequence of strain N299 and other previously studied aerobic denitrifiers  [Pseudomonas denitrificans (0.19–0.23 h−1), and T. pantotropha (0.28–0.45 h−1) under different growth conditions [A. faecalis No.4 (0.2 h−1) [According to its physiological properties and 16S rRNA gene sequences, the strain was identified as in CF-S9 , Providetrain YL , Marinobain GAD3 , Alcaligtrain NR , Comamonain GAD4 , Agrobacain LAD9 , and Baco et al. , and Kon [et al. , NAP1 an primers  that corain N299 , which f.05 h−1) , Pseudomnditions ,50, and 0.2 h−1) , these r510 of the N299 strain  was increased 0.093 during the first 24 h with 1.86 mg/L nitrite and 1.78 mg/L TN consumed, respectively, suggesting that extracellular reduction had occurred, which was in agreement with co-respiration of oxygen and nitrite, as proposed by Robertson and Kuenen [Pseudomonas stutzeri C3 [et al. [et al. [et al. [et al. [et al. [Rhodococcus sp. CPZ24 [Rhizobium sp. PY8 reached 58.17% in 16 days in the same medium, the N299 strain demonstrated some nitrification ability without nitrate and nitrite accumulation.In the pure culture medium  medium), the N299 strain demonstrated its strong denitrification performance and some nitrification ability without nitrate and nitrite accumulation. Using nitrate as the sole nitrogen medium, the removal rate of nitrate reached 75.53% ± 1.69% in 72 h. The removal rate of TN and TDN reached 46.79% ± 0.30% and 72.30% ± 0.52%, in 120 h. Low concentrations of nitrite (less than 6%) relative to nitrate were detected during the entire culturing period, with no nitrite accumulation. However, Xiang-Yang Xu’s study  showed td Kuenen . As the  [et al. , Li et a [et al. , Chen et [et al. , and Yu  [et al. . Considep. CPZ24 , which set al. [et al. [et al.) [et al.) [et al. [In the oligotrophic reservoir source water system. The sterilized raw water experiment showed that TN removal reached 46.99% ± 3.28%. In the non-sterilized reservoir water experiment, the TN of the bacteria system reached 44.88% ± 4.31% in 120 h. because there was a quantity of aerobic denitrifiers in the reservoir ecosystem. Therefore, the TN removal rate of the control also reached 32.77% ± 3.20%, consistent with Carter et al. . The TN [et al.)  showed t[et al.)  showed r [et al.  suggeste [et al. ,53 in DO [et al. ,55, and  [et al. . Howeveret al. [et al. [p > 0.05), which demonstrated a positive influence on the practical application. The nitrate removal rate reached 78.86% ± 0.29% at pH 7 and 74.53% ± 0.29% at pH 8, not significantly, which was consistent with Pseudomonas stutzeri strain T1 [In the experiments analysing the effects of different factors on nitrate removal, acetate, due to its simple and small molecular structure, can be directly assimilated by heterotrophic microorganisms, as reported by Chen et al. , and the [et al.  showed t [et al. ,42. It w [et al. ,58. Henctrain T1 . Namely,Reservoir sediment samples were collected from the Zhoucun drinking water reservoir . In June 2011, surface sediments were collected at a deep layer of 0 to 10 cm using a sterilized Petersen stainless steel grab sampler . The res3COONa (0.5 g/L); NaNO3 (0.1 g/L); K2HPO4·3H2O (0.1 g/L); CaCl2 (0.05 g/L); MgCl2·6H2O (0.05 g/L) [−1 dilution (1 mL enrichment sludge suspension added to 9 mL sterile distilled water); 10−2 dilution (1 mL 10−1 dilution suspension added to 9 mL sterile distilled water); 10−3 dilution (1 mL 10−2 dilution suspension added to 9 mL sterile distilled water); 10−4 dilution (1 mL 10−3 dilution suspension added to 9 mL sterile distilled water); 10−5 dilution (1 mL 10−4 dilution suspension added to 9 mL sterile distilled water); 10−6 dilution (1 mL 10−5 dilution suspension added to 9 mL sterile distilled water); 10−7 dilution (1 mL 10−6 dilution suspension added to 9 mL sterile distilled water). The resultant bacterial suspension was streaked on a screening medium plates and incubation temperature of 30 °C for 3 days. A screening medium (SM) [3COONa (0.1 g/L); NaNO3 (0.02 g/L); K2HPO4·3H2O (0.02 g/L); CaCl2 (0.01 g/L); MgCl2·6H2O (0.01 g/L). Separate colonies were picked and purified by repeated streaking on fresh agar plates. The isolates were harvested and cultivated in SM medium with NaNO3 as the sole nitrogen source in order to detect the aerobic denitrifying bacteria performance. An isolate N299 with high nitrogen removal efficiency was obtained in this study, and SM slant medium at 4 °C and on SM Glycerin medium at −20 °C.The 100 mL sludge sample was added into 700 mL heterotrophic enrichment denitrification broth medium (HEDM) at pH 7.0–7.5: CH.05 g/L) ,40. Ever.05 g/L) . The temium (SM)  plate atZoogloea sp. N299 was obtained via PCR. The PCR used the primers [2+) (2.5 μL), DNA template (0.5 μL), dNTP (each 2.5 mM), Taq DNA Polymerase (0.2 μL), and sterile nuclease free water to 25 μL. The PCR was carried out as follows: 94 °C for 4 min for one cycle and then 30 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 15 s, and extension at 72 °C for 1 min. After a final extension at 72 °C for 10 min, reactions were stored at 4 °C. The Seqman was used to align the sequences. Homology searching of the sequences in GenBank was performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A neighbor-joining tree was constructed in MEGA5.0 program using neighbor-joining (NJ) method with the maximum composite likelihood model and 1000 bootstrap replicates [The 16S rRNA sequence of  primers : 7F 5'-Cplicates . CulturenapA was amplified with the forward primers NAP1: 5'-TCTGGACCATGGGCTTCAACCA-3' [2 (25 μL), NAP1 (1 μL), NAP2 (1 μL), and sterile nuclease-free water to 50 μL. Conditions used for this reaction were as per [The AACCA-3' , and NAPAACCA-3' . The PCRe as per : initial510 in a shake flask experiment in which 400 mL of the liquid SM medium was put in 1000 mL shake flasks, inoculated with 4 mL of strain pre-culture, and then cultivated at 30 °C. During incubation, 3 mL culture was removed periodically for the determination of cell optical density. The aerobic denitrifying bacteria N299 was pre-cultured for 24 h in 50 mL liquid SM medium (without agar) in a 100 mL Erlenmeyer flask at 30 °C and 120 rpm in order to be activated [t is time (h); y(t) is bacterial cell density at t h (OD); μ is the maximum specific cell growth rate (h−1); and a is the maximum bacterial cell density (OD); c is the bacterial cell density (t = 0). Correlation analysis using OriginPro  yielded.The growth characteristics of the isolated strain N299 were determined through measuring ODctivated . Accordiv/v) into 150 mL liquid SM, short-SM, and HNM of 250 mL Erlenmeyer flask at 30 °C, 120 rpm, respectively. The nitrate, nitrite, TN, TDN, TP, TOC concentrations, and cell optical density (OD) were measured to reflect the denitrification performance of the N299 strain. All parameters were measured in triplicate (n = 3). The SM medium included, at pH 7.0–7.5: CH3COONa (0.1 g/L), NaNO3 (0.02 g/L), K2HPO4·3H2O (0.02 g/L), CaCl2(0.01 g/L), and MgCl2·6H2O (0.01 g/L). A short SM medium [3COONa (0.1 g/L), NaNO2 (0.018 g/L), K2HPO4·3H2O (0.02 g/L), CaCl2 (0.01 g/L), MgCl2·6H2O (0.01 g/L). Heterotrophic nitrification medium (HNM) [3COONa (0.5 g/L), NH4Cl4 (0.1 g/L), K2HPO4·3H2O (0.1 g/L), CaCl2 (0.05 g/L), and MgCl2·6H2O (0.05 g/L).The pre-cultured N299 strain was inoculated in 10%   was alsov/v) into 150 mL sterilized oligotrophic reservoir source water and non-sterilized oligotrophic source water of 250 mL Erlenmeyer flask at 30 °C at 120 rpm. The TN, TDN, TOC, cell optical density, pH, and DO were measured to reflect the denitrification performance of N299. All parameters were measured in triplicate (n = 3).In order to investigate whether adding agents could purify the sterilized oligotrophic reservoir source water and to study whether bacteria in source water affected the N299’s denitrification, sterilized reservoir source water and non-sterilized source water experiments were carried out. The pre-cultured N299 was inoculated in 10% (v/v) and pH. Glucose, sodium succinate, sodium citrate, and sodium acetate were used to explore the effects of carbon source on nitrate removal. To observe the effect of temperature on nitrate removal, the experiment was performed within the range of 10–30 °C. The effect of C/N (sodium acetate as carbon source) on nitrate removal was examined by adjusting the ratio between 1 and 10 with a fixed amount of 3.54 mg/L NO3−-N. The influence of inoculums dosage (v/v) on nitrate removal was conducted by changing the inoculums dosage to 2%, 3%, 5% and 10%. The effect of pH on nitrate removal was examined in 6, 7, 8, 9 and 10. All parameters were measured in triplicate (n = 3).The heterotrophic aerobic denitrification characteristics of isolated strain were determined under different culturing conditions, including carbon source, Temperature, C/N, inoculums dosage (510) using a spectrophotometer  [N--diaminoethane photometry method [The optical density of the culture broth was measured at 510 nm  . Nitritey method . TN and y method . TP was y method . TOC dety method ,40. Phyly method .p < 0.05) using SPSS software .Data are presented as means ± SD (standard deviation of means), and analyzed by one-way ANOVA with Tukey’s HSD test  and high DO (7–8 mg/L) conditions, the N299 showed an efficient denitrification performance and strong adaptability to tolerate low C/N and high DO. The strong adaptability of strain N299 to neutral or slightly alkaline pH and low temperature conditions make it a promising candidate for treating micro-polluted source water in natural regions. It could utilize the organics of water to remove nitrogen to survive. This study thus provides significant information for bioremediation of micro-polluted reservoir source water.The newly isolated indigenous aerobic denitrifier, N299 strain was named as


The risk of prostate cancer-specific mortality (PCSM) following a diagnosis of prostate cancer may improve after patients have survived a number of years after diagnosis. We sought to determine long-term conditional PCSM for patients with stage T4, N1, or M1 prostate cancer.We identified 66,817 patients diagnosed with stage IV  prostate cancer between 1973 and 2011 using the Surveillance, Epidemiology, and End Results (SEER) database. Conditional five-year PCSM was evaluated for each group of patients at 5, 10, and 15 years of survival according to the Fine & Gray model for competing risks after adjusting for tumor grade, age, income level, and marital status. Race-stratified analyses were also performed.p < 0.001 in all cases). In patients with N1 disease, 5-year PCSM increased within the first five years and decreased thereafter, from 18.9 % at diagnosis to 21.4 % (p < 0.001), 17.6 % (p = 0.055), and 13.8 % (p < 0.001), respectively. In patients with metastatic disease, 5-year PCSM improved from 57.2 % at diagnosis to 41.1, 28.8, and 20.8 %, respectively (p < 0.001). White race was associated with a greater increase in conditional survival compared to non-white race among those with T4 or N1 disease. There were 13,345 patients with T4 disease, 12,450 patients with N1 disease, and 41,022 patients with M1 disease. Median follow-up among survivors in the three groups was 123 months (range: 0–382 months), 61 months (range: 0–410 months), and 30 months (range: 0–370 months), respectively. Conditional PCSM improved in all three groups over time. Among patients with T4 disease, 5-year PCSM improved from 13.9 % at diagnosis to 11.2, 8.1, and 6.5 % conditioned on 5, 10, or 15 years of survival, respectively (While patients with T4, N1, or M1 prostate cancer are never “cured,” their odds of cancer-specific survival increase substantially after they have survived for 5 or more years. Physicians who take care of patients with prostate cancer can use this data to guide follow-up decisions and to counsel newly diagnosed patients and survivors regarding their long-term prognosis. Stage IV prostate cancer, consisting of stage T4 (invasion of adjacent organs), N1 , or M1 (metastatic spread) disease . When a Some data suggests that patients with T4, N1, or M1 disease are unique in the degree to which prognosis improves as patients survive their disease –8. In adThe Surveillance, Epidemiology, and End Results (SEER) database collects cancer diagnostic, treatment, and survival data from 18 SEER registries, accounting for approximately 28.0 % of the US population . We usedPatients were included if they were diagnosed with stage T4N0M0, N1M0, or M1 prostate cancer between 1973 and 2011. In total, this approach identified 66,817 men. We collected information from SEER on tumor characteristics, including clinical staging information and grade. We also extracted patient characteristics, including survival information, age at diagnosis, marital status at diagnosis, race, median family income in the county of diagnosis, cause of death, and length of survival.T was determined by identifying patients who survived for S > T months and generating a new survival variable S’ = S - T. Survival analysis was then performed on patients who survived past time S using S’ as the survival variable and compared to the general cohort using S as the survival variable. This procedure was repeated for T = 5, 10, or 15 years. We also analyzed subgroups of patients with M1 disease based on whether they had M1a disease (distant lymph node involvement), M1b disease (bone involvement), or M1c disease (other site involvement with or without bone involvement) [race*SurvivalT, where SurvivalT represents whether or not a patient survived past time T. P-values were reported as statistically significant if less than α = 0.05 after correction for multiple comparisons, when applicable [Stata/MP 13.1 was used for all statistical analyses. Patient demographic data were summarized for patients with T4, N1, or M1 disease. Prostate cancer-specific mortality was estimated using the Fine & Gray model  for compplicable . SpecifiThere were 13,345 patients with T4 disease, 12,450 patients with N1 disease, and 41,022 patients with M1 disease. Median follow-up among survivors in the three groups was 123 months (range: 0–382 months), 61 months (range: 0–410 months), and 30 months (range: 0–370 months), respectively. Other baseline characteristics of the three cohorts and of those who survived for 5, 10, or 15 years are represented in Table p < 0.001 in all cases). In patients with N1 disease, 5-year PCSM slightly worsened within the first five years, from 18.9 % at diagnosis to 21.4 % conditioned on 5 years of survival (p < 0.001), but then declined to 17.6 % (p = 0.055) and 13.8 % (p < 0.001) conditioned on 10 and 15 years of survival, respectively.Conditional adjusted prostate cancer-specific mortality improved among patients with T4, N1, or M1 disease over time . In subgroup analyses  had improved 5-year PCSM from 55.0 % at diagnosis to 42.4, 34.4, and 17.0 % at 5, 10, and 15 years of survival, respectively . For the subgroup of patients with M1b disease , 5-year PCSM improved from 59.7 % at diagnosis to 49.1, 36.0, and 29.7 % at 5, 10, and 15 years of survival . For the subgroup of patients with M1c disease , 5-year PCSM improved from 62.6 % at diagnosis to 46.5 % and 39.6 % at 5 and 10 years of survival, respectively (p < 0.005 in both cases), with no significant difference at 15 years of survival .In patients with metastatic disease, 5-year PCSM improved from 57.2 % at diagnosis to 41.1, 28.8, and 20.8 % conditioned on 5, 10, and 15 years of survival, respectively , while white patients had similar mortality as the overall cohort, with adjusted hazard ratios of 1.10 (p < 0.001), 0.898 (p = 0.022), and 0.712 (p < 0.001), respectively. While non-white patients with T4 disease had improved 5-year PCSM after having survived 5, 10, or 15 years, their improvements were 20-39 % smaller than those of white patients .In order to determine whether race interacted with survival, we repeated our initial analyses after stratifying by race and by modeling an interaction term. Among patients with M1 disease, conditional mortality was similar between white and non-white patients (data not shown). However, among patients with N1 disease, non-white patients did not have a significant reduction in conditional mortality at 5, 10, or 15 years of survival compared to diagnosis  generally improved among all three groups conditioned on 5, 10, or 15 years of survival , even after adjusting for patient-specific factors like tumor grade, age, marital status, county-wide median income, and race. Interestingly, we found that this relationship was stronger in white patients than among non-white patients with T4 or N1 disease.These data may be of great interest to physicians and patients. Our survival analyses imply that cancer-specific mortality is not static after diagnosis with advanced prostate cancer. While it may not be appropriate to label patients with advanced-stage prostate cancer as “cured” after surviving for several years, many of these patients are at significantly reduced likelihood of cancer-specific death the longer they survive. Physicians should use results to better counsel patients and provide appropriate follow-up and surveillance. For example, we found that cancer-specific mortality drops sharply among patients with T4 disease after 5, 10, and 15 years of survival. Therefore, it would be appropriate for clinicians to increase the time between follow-up appointments, imaging, and laboratory tests once patients survive to 5 years. On the other hand, we found that patients with N1 disease did not have a reduction in PCSM until 10 years. Therefore, physicians should monitor the N1 patient who has survived for 5 years just as closely as they would monitor the newly diagnosed one.Patients can also use this data to better understand how their long-term prognosis changes over time. Most cancer-specific mortality data is reported from diagnosis. Therefore, it can be difficult to re-assess prognosis for the fortunate patient who is able to survive 5, 10, or even 15 years after diagnosis. Our data can help patients understand their changing risk of PCSM as time passes. For example, a patient newly diagnosed with metastatic prostate cancer would likely be counseled that their risk of dying within five years is 50 % or more , 17.In addition to our findings that conditional survival improved among all three groups of patients, our secondary analyses may point to racial disparities in long-term prostate cancer care. We found that among patients with T4 disease, non-white patients had smaller improvements in survival than white patients; among those with N1 disease, non-white patients had no improvement at all. While others have shown that minorities are at higher risk of cancer-specific mortality , our resTo our knowledge, other studies examining conditional survival from prostate cancer have not specifically examined patients with T4 or N1 disease, two important subsets of patients with whom some clinicians may have limited experience due to their relative rarity , 9. SomeDespite our sample size, our study has some limitations. First, the SEER database does not contain data about disease recurrence or receipt of salvage therapy. Therefore, we were not able to analyze these important secondary outcomes. Second, the SEER database has previously been reported to have coding errors in cancer stage , so we mIn order to better understand how the cancer-specific mortality risk of advanced prostate cancer changes over time, we studied the conditional mortality of patients with stage T4, N1, or M1 prostate cancer. We found that in all three groups , cancer-specific mortality generally improved after survival to 5, 10, or 15 years. Among those with T4 or N1 disease, non-white patients had smaller improvements compared to white patients or no improvement at all. Our results should be used to counsel prostate cancer survivors on their changing risk profile and to tailor follow-up and cancer surveillance over time. In addition, physicians should pay special attention to minority patients, as they may be at risk for relatively poor conditional mortality, possibly due to worse long-term follow-up.


Bauhinia racemosa aerial parts was tested for antimicrobial activity against different bacterial and fungal strains and for antioxidant activity and also total content of polyphenols with phytochemical analysis of the extract was determined. The results have showed that the extract has a significant antimicrobial activity, it inhibited the growth of Bacillus subtilis and also it was highly active against Candida albicans suggesting that it can be used in the treatment of fungal infections. The extract has a good antioxidant activity, it has shown high values of the trolox equivalent antioxidant capacity and polyphenol content while it has shown a lower value of oxygen radical absorbance capacity. Phytochemical analysis has shown that it has interesting phytochemical bioconstituents, include flavonoids, tannins and others, and the deep phytochemical study results in the isolation of methyl gallate, gallic, kaempferol, quercetin, querection 3–O–α–rhamnoside, kaempferol 3–O–β–glucoside, myricetin–3–O–β– glucoside, querection–3–O–rutinoside (Rutin). All compounds were identified by different spectroscopic analyses  and Mass Spectrometry (MS). Methanol 70% extract of  Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae are most common. With the continuous use of antibiotics, microorganisms have become resistant, in addition to these problems; antibiotics are sometimes associated with adverse effects on host which include hypersensitivity, depletion of beneficial gut, mucosal microorganism, immunosuppression and allergic reactions  damage, gastritis, cancer and AIDS. Antioxidants or molecules with radical scavenging capacity are intent to execute a potential effect against free radical damage. It has been mentioned the antioxidant activity of plants might be due to their phenolic or/and flavonoid biocompounds ; 13C-NMR spectra: Varian Unity 400 (100 MHz) . MS . (Silica gel (0.063-0.200 mm for column chro matography) and Sephadex LH-20 . Solvent mixtures, BAW . Paper Chromatography (PC) Whatman No.1  sheets for qualitative detection of flavonoids and sugars.UV/VIS: Shimadzu UV-visible recording spectrophotometer model-UV 240 . Plant materialBauhinia racemosa were collected from Orman garden, Giza, Egypt in April 2011. The plant was identified by Dr. Mohammed El–Gebaly, Department of Botany, National Research Centre (NRC) and by Mrs. Tereez Labib Consultant of Plant Taxonomy at the Ministry of Agriculture and director of Orman botanical garden, Giza, Egypt. A voucher specimen No. 2345 is deposited in the herbarium of Orman garden, Giza, Egypt. Aerial parts of Prepartion of the extractB. racemosa was extracted with methanol 70% at room temperature several times until exhaustion. The extract was concentrated under reduced pressure to give 54 g of crude extract.700 g of air dried powder from the aerial parts of Antimicrobial assaysStaphylococcus aureus ATCC 29213, Bacillus subtilis ATCC), Gram–negative  and fungal strain . The methanol extract of B. racemosa was tested for its antimicrobial activity using a qualitative screening assay of the antimicrobial properties by the adapted disk diffusion method, Kirby–Bauer method , Gram–negative  and fungal strains . The microbial strains were identified using a VITEK I automatic system. VITEK cards for the identification and the susceptibility testing (GNS–522) were inoculated and incubated according to the manufacturer’s recommendations. In our experiments there were used bacterial suspensions of 1.5x108 UFC/ mL or 0.5 McFarland density obtained from 15–18 h bacterial cultures developed on solid media. The antimicrobial activity was tested on Mueller–Hinton medium recommended for the bacterial strains and Yeast Peptone Glucose (YPG) medium for Candida albicans. Solutions of the extract in DMSO (dimethyl sulfoxide) having 2048 μg/ mL concentration were used.The quantitative assay of the antimicrobial activity was performed by broth microdilution method , 25 in 9r method . The quar method , in ordeQualitative screening of the antimicrobial properties of the extractnd variant, 10 μL of the tested extract solutions were placed in the agar wells cut in the solid culture medium seeded with the microbial inoculum. In the 3rd variant of the qualitative antimicrobial activity assay, 10 μL of the extract solutions were spotted on Petri dishes seeded with bacterial/yeast inoculum. In all the three variants, the Petri dishes were left at room temperature to ensure the equal diffusion of the compound in the medium or to allow the drop of solution to be adsorbed in the medium and afterwards the dishes were incubated at 37 °C for 24 hours. The solvent used was also tested in order to evaluate a potential antimicrobial activity.The antimicrobial activity of the extract was investigated by qualitative screening of the susceptibility spectrum of different microbial strains to the tested extract solubilised in DMSO (1 mg/mL) using adapted variants of the diffusion method. In the 1st variant, 10 μL of the extract solution were equally distributed on the paper filter disks placed on Petri dishes previously seeded “in layer” with the tested bacterial strain inoculums. In the 2Quantitative assay of the antimicrobial activityFor the quantitative assay of the antimicrobial activity of the extract by the microdilution method , 27 in lAntioxidant assays Extraction: 0.2 g of extract with 10 mL Millipore water boiled was sonic, centrifuged and filtered. Evaluation of antioxidant activity of the extract, using methods Oxygen Radical Absorbance Capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC) and determination of total polyphenols content:Oxygen Radical Absorbance Capacity (ORAC)This method determines peroxil radical inhibition capacity, inducing oxidation highlighting the classical radical release; H atom transfer ORAC values were reported as Trolox equivalents, is expressed as micromol TE/DW. The intensity was monitored at 485 nm and 525 nm for 35 min.Trolox equivalent antioxidant capacity method (TEAC)+  by antioxidants. ABTS is oxidized by radicals peroxil or other oxidants to its radical cation ABTS+, intensely colored (λmax = 734 nm). Antioxidant capacity is expressed compounds tested as potential, to discoloration by direct reaction with it radical ABTS+.This method is based the neutralizing capacity the radical anion ABTSThe total content of polyphenolsThe blue compounds formed between phenols and Folin–Ciocalteu reagent phenolic compounds are independent of structure, thus developing complex between metal center and phenolic compounds. Absorption was recorded at a wavelength of 765 nm. Total phenol content was expressed as gallic acid equivalents .Qualitative phytochemical analysis3 and Mayer᾽s reagents for detecting of tannins and alkaloids, respectively  in Butanol–Acetic acid–Water 4:1:5 (BAW) and 15% acetic acid were combined to give 4 fractions . Fraction I (950 mg) was subjected to sub–column of silica gel eluted with dichloromethane: ethyl acetate (60:40) gave compound 1  and elution with dichloromethane: ethyl acetate (90:10) gave compound 2 . Fraction II (830 mg) was subjected to sub–column of silica gel eluted with dichloromethane: ethyl acetate (95:5) to give compound 3  and elution with ethyl acetate solvent gave compound 4 . Compound 5  and 6  were obtained from elution with ethyl acetate: Methanol (90:10) from fraction III (640 mg) and their purification were carried out on sephadex LH–20 column which eluted with methanol (50%). Compound 7  and 8  were isolated from fraction IV (1.2 g) by sub–column of silica gel eluted with ethyl acetate: methanol (60:40) and their purification were carried out on sephadex LH–20 column which eluted with methanol (50%).The extract 54 g was defatted with Acid hydrolysis of flavonoidsSolutions of 5 mg of compounds 5, 6, 7 and 8 in 5 mL 10% HCl were heated for 5 h. The reaction mixture was extracted with Ethyl acetate. The Ethyl acetate fraction (aglycone) and the aqueous fraction (sugars) were concentrated for identification. The sugars were identified by TLC (acetonitrile–water 85:15) by comparsion with authentic samples., estimation of the main bioactive constituents of B. racemosa aerial parts. The major bioactive components of B. racemosa are methyl gallate, gallic acid, kaempferol, quercetin, quercetin 3–O–α–rhamnoside, kaempferol 3–O–β–glucoside, myricetin 3–O–β–glucoside and quercetin 3–O–rutinoside. The structure of bioactive components was elucidated by different spectroscopic analyses.The present investigation was focused to evaluate the antimicrobial activity (expressed in μg/mL) and antioxidant capacity  as can be seen in Structure Elucidation of the isolated compoundsMethyl gallate (1): white amorphous powder: UV λmax (MeOH): 275. 1H–NMR : δ 6.94 , 3.73 . 13C–NMR : δ 166.8 (–COO), 146 , 138.9 (C–4), 119.8 (C–1), 109 , 52 (–OCH3).Gallic acid (2): white amorphous powder. UV λmax (MeOH): 273. 1H–NMR : δ 7.15 .13C–NMR : δ 167.2 (–COOH), 145 , 137.7 (C–4), 121 (C–1), 109.1 .Kaempferol (3): Yellow powder. UV λmax (MeOH): 265, 320, 366; (NaOMe): 276, 317, 406; (AlCl3): 262sh, 269, 310sh, 367; (AlCl3/HCl): 263sh, 268, 320sh, 344, 425; (NaOAc): 274, 306, 382; (NaOAc/H3BO3): 267, 368. 1H–NMR : δ 8.11 , 6.96 , 6.47 , 6.19 . EI–MS: m/z 286.Quercetin (4): Yellow powder. UV λmax (MeOH): 255, 267, 371; (NaOMe): 270, 320, Citrus Aurantium & Labor Pain 1077 3): 270, 455; (AlCl3/HCl): 264, 303sh, 315sh, 428; (NaOAc): 257, 274, 318, 383; (NaOAc/H3BO3): 259, 387. 1H–NMR : δ 7.74 , 7.55 , 6.92 , 6.42 , 6.15 . EI–MS: m/z 302.420; (AlClQuercetin 3–O–α–rhamnoside (5) Yellow crystals: 1H–NMR  δ ppm 7.26 , 6.83 , 6.49 ,6.14, 5.25  0.78 . 13C–NMR : δ ppm 177.42 (C–4), 167.45 (C–7), 161.40 (C–5), 157.01 (C–2), 157 (C–9), 149.19 (C–4'), 145.57 (C–3'), 134.12 (C–3), 131.97 (C–6'), 121.40 (C–1'), 115.71 (C–2'), 115.40 (C–5'), 103.10 (C–10), 101.97 (C–1''), 99.98 (C–6), 94.47 (C–8), 71.47 (C–4''), 70.94, 70.85, 70.62 , 17.78 (C6'').Kaempferol 3–O– β–glucoside (6): Yellow crystals. UV λmax (MeOH): 266, 364; (NaOMe): 274, 327sh, 401; (AlCl3): 274, 304, 349, 396; (AlCl3/HCl): 274, 345, 394; (NaOAc): 274, 305, 393; (NaOAc/H3BO3): 267, 352. 1H–NMR  δ 8.0 , δ 6.9 , δ 6.5 , 6.2 δ , 5.4 , 3.80–3.10 .Myricetin 3–O–β–glucoside (7): 1H–NMR of  δ 7.16 , δ 6.13, 6.35 δ , 5.45 , 3.90–3.20 . 13C–NMR : δ ppm 177.85 (C–4), 164.83 (C–7), 161.71 (C–5), 156.81 (C–2), 156.71 (C–9), 146.49 (C–3'), 145.87 (C–5'), 137.97 (C–4'), 133.95 (C–3), 120.49 (C–1'), 109 , 104.37 (C–10), 101.4 (C–1''), 99.22 (C–6), 93.00 (C–8), 78.04 (C–5''), 77.04 (C–3''), 74.44 (C–2''), 70.36(C–4'') 61.52 (C–6'').Quercetin 3–O–rutinoside (Rutin) (8): 1H–NMR : δ ppm 7.54 , 6.85 , 6.38 , 6.19 , 5.35 , 5.33 , 5.02,, 4.39 , 3.90–3.20 , 0.99 . 13C NMR: δ ppm 177.85 (C–4), 164.70 (C–7), 161.68 (C–5), 157.14 (C–2), 156.95 (C–9), 148.92 (C–4'), 145.25 (C–3'), 133.76 (C–3), 122.12 (C–6'), 121.66 (C–1'), 116.73 (C–2'),115.72 (C–5'),104.41 (C–10), 101.66 (C–1'''), 101.23 (C–1''), 99.24 (C–6), 94.16 (C–8), 74.58(C–3''),72.33(C–5''), 72.2 (C–4'''), 71.05(C–2''), 70.8(C–2'''), 70.87(C–3''') 70.49(C–4'') 68.74 (C–6'') 18.19(C–6''').Antimicrobial activity of B. racemosa extracti.e. paper filter disks impregnated with the tested extract solution and disposal of the respective solutions in agar wells, the reading of the results was performed by measuring the microbial growth inhibition zones around the filter disks impregnated with the testing extract and around the wells, respectively. The most efficient qualitative method proved to be the direct spotting of the tested solutions on the seeded medium, the results being very well correlated with the results of the  MIC quantitative assay. For the quantitative methods of the antimicrobial activity of the tested extract by the microdilution method in liquid medium, the MIC was read by wells observation: in the first wells containing high concentrations of extract, the culture growth was not visible, the microbial cells being killed or inhibited by the tested extract. At lower concentrations of the tested extract, the microbial culture becomes visible. The lowest concentration which inhibited the visible microbial growth was considered the MIC (μg/mL) value for the extract. In the next wells, including the standard culture growth control wells, the medium become muddy as a result of the microbial growth. In the sterility control wells series, the medium had to remain clear. From the last well without any visible microbial growth and from the first one that presented microbial growth, Gram stained smears were performed for the results confirmation. In B. racemosa extract. Our results have shown that the extract was highly active against C. albicans, suggesting its possible use in the treatment of fungal infections, also it exhibited antimicrobial activity on B. subtilis and it has shown a moderate antimicrobial activity against K. pneumoniae but it was not active on other bacterial strains. For the antimicrobial qualitative methods, Antioxidant activity B. racemosa and Total polyphenol content B. racemosa extract showed high TEAC value 201 ± 3.6 mM TE/g  , alkaloids and carbohydrates (Table 3). Phenolic compounds form one of the main classes of secondary metabolites. They display a large range of chemical structures and are responsible for the major bioactivity of plants. Flavonoids have shown a significant antimicrobial activity  and Trolox equivalent antioxidant capacity method (TEAC) assays. In (TEAC) assay,  mM TE/g , as well mM TE/g  which wamM TE/g)  these reactivity , 33, tanactivity , as wellactivity . B. racemosa methanol extract yielded eight known phenolic compounds which were isolated and purified by standard methods. Compounds 1, 2 were white amorphous powder, showed chromatographic properties and colour reactions (positive FeCl3 and KIO3 tests) indicative of galloyl esters  and tannins .Chemical and Chromatographic separations of l esters . Compounure data -39. On t


Zymoseptoria tritici. We fused the gene for enhanced green-fluorescent protein (egfp) to the promoter region of Z. tritici nitrate reductase , 1,4-β-endoxylanase A , l-arabinofuranosidase B , galactose-1-phosphate uridylyltransferase 7  and isocitrate lyase . This was followed by quantitative analysis of cytoplasmic reporter fluorescence under induced and repressed conditions. We show that Pnar1, PlaraB and Pex1A drive very little or no egfp expression when repressed, but induce moderate protein production when induced. In contrast, Pgal7 and Picl1 show considerable egfp expression when repressed, and were strongly induced in the presence of their inducers. Normalising the expression levels of all promoters to that of the α-tubulin promoter Ptub2 revealed that PlaraB was the weakest promoter (∼20% of Ptub2), whereas Picl1 strongly expressed the reporter (∼250% of Ptub2). The use of these tools promises a better understanding of essential genes, which will help developing novel control strategies that protect wheat from Z. tritici.Development of new fungicides, needed for sustainable control of fungal plant pathogens, requires identification of novel anti-fungal targets. Essential fungal-specific proteins are good candidates, but due to their importance, gene deletion mutants are not viable. Consequently, their cellular role often remains elusive. This hindrance can be overcome by the use of conditional mutants, where expression is controlled by an inducible/repressible promoter. Here, we introduce 5 inducible/repressible promoter systems to study essential genes in the wheat pathogen  When α-tubulin was expressed under the Pex1A promoter, cells grew normally, but growth was significantly impaired when Pex1A was repressed. This demonstrates the use of conditional promoters in functional analysis of essential genes in Z. tritici.Here, we establish 5 homologous inducible/repressible promoters in the wheat pathogen . maydis . Secondlus niger . We esta awamori . Moreoveoformans . Finally. grisea . Using e22.1Escherichia coli strain DH5α was used for the maintenance of plasmids. Agrobacterium tumefaciens strain EHA105 (A. tumefaciens-mediated transformation of Z. tritici. E. coli and A. tumefaciens were grown in DYT broth or DYT agar at 37 °C and 28 °C respectively. The fully sequenced Z. tritici wild-type isolate IPO323 (n EHA105  was usede IPO323  was used2.2U. maydis Nar1 sequence (XP_759994.1), Aspergillus fumigatus LaraB sequence (KEY83958.1), A. awamori Ex1A sequence (CAA55005.1), C. neoformans Gal7 sequence (XP_568349.1) and M. oryzae Icl1 sequence (XP_003712381.1) from the NCBI server (http://www.ncbi.nlm.nih.gov/pubmed). We used these to screen the published sequence of Z. tritici (http://genome.jgi.doe.gov/Mycgr3/Mycgr3.home.html) using the provided BLASTP function and compared the best hits in EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). The domain structure was analyzed in PFAM (http://pfam.xfam.org/search/sequence). The promoter sequences upstream of the open reading frame were obtained from the JIG server (http://genome.jgi.doe.gov/Mycgr3/Mycgr3.home.html).To identify conditional promoters, we obtained the sequences of 2.3S. cerevisiae DS94  and 1000 bp Z. tritici ex1A promoter  and 1,000 bp laraB promoter  and 1000 bp Z. tritici gal7 promoter  and 1000 bp Z. tritici icl1 promoter  fused to tub2 gene for replacement of endogenous tub2 promoter in Z. tritici with ex1A promoter using hygromycin as a selectable marker. A 9533 bp fragment of pCeGFPTub2 (digested with BamHI and HindIII), 1149 bp tub2 promoter  was added to Agrobacterium induction medium agar plates. For selection of transformants, 200 μg/ml hygromycin  was added to the Czapekdox agar containing 5% xylose, along with100 μg/ml cefotaxime and 100 μg/ml timentin. The individual transformants were transferred to complete medium CM  for 3 days at 18 °C with 200 rpm. 3 ml of yeast-like cells were used for fungal genomic DNA extraction as described . 1014 bp sdi1 probe (3′ end of the Rsdi1 gene and sdi1 terminator) was generated by using DIG labeling PCR mix  with primers SK-Sep-10 and SK-Sep-13; To confirm the integration of vectors pCPocedures . Z. tritescribed . Approxiex1ATub2 into the tub2 locus of Z. tritici was confirmed by Southern blot. Z. tritici transformants obtained with plasmid pHPex1ATub2 were grown in complete medium CM with 5% xylose for 3 days at 18 °C with 200 rpm. Approximately 3 μg of genomic DNA of IPO323 and transformants obtained with vector pHPex1ATub2 were digested with SacII, and separated on a 1.0% agarose gel and capillary transferred to a Hybond-N membrane . 1149 bp tub2 probe (α-tubulin promoter) was generated by using DIG labeling PCR mix  with primers SK-Sep-217 and SK-Sep-218; Integration of vector pHP2.8nar1eGFP cells were grown in Nitrate minimal medium NM ; the IPO323_CPlaraBeGFP cells were grown in Aspergillus minimal medium with 2% arabinose (ON) and Aspergillus minimal medium with 1% glucose (OFF); the IPO323_CPgal7eGFP cells were grown in Aspergillus minimal medium with 0.5% galactose (ON) and Aspergillus minimal medium with 2% glucose (OFF); the IPO323_CPicl1eGFP cells were grown in Aspergillus minimal medium with 20 mM sodium acetate (ON) and Aspergillus minimal medium with 2% glucose (OFF) at 18 °C with 200 rpm for 24 h. In order to compare the expression levels of eGFP in strains IPO323_CPnar1eGFP, IPO323_CPex1AeGFP, IPO323_CPlaraBeGFP, IPO323_CPgal7eGFP and IPO323_CPicl1eGFP with eGFP expression under the tub2 promoter in strain IPO323_CeGFP . The fluorescent tags of all strains, with the exception of IPO323_Picl1eGFP, were excited using a VS-LMS4 Laser Merge System with a 488 nm solid-state laser  at 20%. IPO323_Picl1eGFP was excited with 10% of the 488 nm solid-state laser to avoid camera saturation.Fluorescence microscopy was performed as previously described . In brieAverage intensity was analyzed in those images by creating a region of interest (ROI) within a cell covering only a part of the cytoplasm but excluding the nucleus or vacuoles. A copy of the same ROI was placed next to the cell to get the average intensity of the neighbouring background. Both values were transferred to Excel  and the value of the background was subtracted from the value of the cell. Those corrected values were transferred to Prism 4.03  to perform all statistical analysis. All parts of the system were under the control of the software package MetaMorph .2.9ex1ATub2. For better visualisation of the colonies, 1% activated charcoal was added to the media. The IPO323 and IPO323_HPex1ATub2 cells were grown in CM with 5% xylose at 18 °C with 200 rpm for 2 days. The cell density was adjusted to an optical density of 0.6 at 660 nm. 1,000 μl of cell culture was transferred to 1.5 ml Eppendorf tube and centrifuged at 3000 rpm for 3 min. The supernatant was discarded and the cell pellet was resuspended either with CM containing 5% xylose (ON condition) or 5% maltodextrin (OFF condition). The cell cultures were serially diluted (10 times each) with the corresponding media. The serial diluted cultures were then applied as 5 μl droplets on either CM agar with 5% xylose and 1% charcoal or CM agar with 5% maltodextrin and 1% charcoal and grown at 18 °C for 6 days. Photographs of the relative colony densities were taken using a canon digital IXUS 80 IS camera .The complete medium CM agar either with 5% xylose  or 5% maltodextrin  was used to examine the growth habit of IPO323 and IPO323_HP33.1Z. tritici homologues of the U. maydis nitrate reductase (Nar1), A. fumigatusl-arabinofuranosidase B (LaraB) and A. awamori 1,4-β-endoxylanase A (Ex1A), C. neoformans galactose-1-phosphate uridylyltransferase  and M. oryzae isocitrate lyase (Icl1) were identified by BLASTP search (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with the U. maydis Nar1 sequence (XP_759994.1), A. fumigatus LaraB sequence (KEY83958.1), A. awamori Ex1A sequence (CAA55005.1), C. neoformans Gal7 sequence (XP_568349.1) and M. oryzae Icl1 sequence (XP_003712381.1) against the Z. tritici published genome sequence of IPO323 (http://genome.jgi.doe.gov/Mycgr3/Mycgr3.home.html). Most similar to U. maydis Nar1 in Z. tritici was protein 111003 (NCBI accession number: XP_003849160.1), to A. fumigatus LaraB in Z. tritici was protein 70396 (NCBI accession number: XP_003854224.1), to A. awamori Ex1A in Z. tritici was protein 60105 (NCBI accession number: XP XP_003851797.1), to C. neoformans Gal7 in Z. tritici was protein 72281 (NCBI accession number: XP_003852576.1) and to M. oryzae Icl1 in Z. tritici was protein 102083 (XP_003855965.1), respectively. The predicted proteins ZtNar1, ZtLaraB, ZtEx1A, ZtGal7 and ZtIcl1 shared 39.6%, 45.3%, 40.4%, 42.0% and 71.7% aa identity with their homologues and have a similar domain structure. ZtNar1 shares with Nar1 from U. maydis an oxidoreductase molybdopterin binding domain (P = 8.6e−49), a molybdopterin cofactor oxidoreductase dimerisation domain (P = 8.6e−4), a cytochrome b5-like heme/steroid binding domain (P = 3.8e-23), an oxidoreductase FAD-binding domain (P = 3.5e−31) and an oxidoreductase NAD-binding domain (P = 6.4e−05). ZtLaraB shares with LaraB from A. fumigatus an alpha-l-arabinofuranosidase B domain (P = 2.1e−132), and ZtEx1A shares with Ex1A from A. awamori a glycosyl hydrolases family 11 domain (P = 4.6e−83). ZtGal7 shares with Gal7 from C. neoformans two Galactose-1-phosphate uridyl transferase domains  and ZtIcl1 shares an isocitrate lyase family domain with Icl1 from M. oryzae (P = 1.7e−278).The f IPO323  at the J3.2nar1, Pex1A, PlaraB, Pgal7 and Picl1). We choose 1000 bp, which includes the majority of putative promoter sequences in the yeast S. cerevisiae  that drive expression of eGFPs in Z. tritici. All vectors were built on the Agrobacterium binary vector pCAMBIA0380  to enable A. tumefaciens-based transformation into Z. tritici, which is based on the 25 bp imperfect directional repeat sequences of the T-DNA borders . Incorporation by homologous recombination mutates the sdi1 gene and integrates the eGFP constructs into the sdi1 locus . Under these conditions, almost no cytoplasmic fluorescence was observed for Pnar1, PlaraB and Pex1A . Notably, Pex1A-egfp containing cells showed a signal intensity that was not significantly higher than that of untransformed IPO323 , suggesting that the promoter is “tight” under repressed conditions. In contrast, Pgal7 and Picl1 were not repressed entirely and weak GFP fluorescence was still visible under OFF conditions , all strains showed cytoplasmic fluorescence, indicating eGFP expression from the induced promoters  in laraB was shows the weakest relative expression level when induced . Similar to the plasmids described above, pHPex1ATub2 was built into the Agrobacterium binary vector pCAMBIA0380  for A. tumefaciens-based transformation and is suited for yeast recombination-based cloning  on agar medium containing 5% xylose. Under these conditions, tub2 expression was induced and cells grew normally. However, when colonies were cultivated on 5% maltodextrin-containing agar medium, expression of tub2 was repressed in both mutants and cells did not grow, whereas IPO323 was not affected (ex1A repression appears to be relatively “tight” and repressing α-tubulin under this promoter impairs growth, other genes of interest that may have low native expression levels may not be sufficiently repressed under these conditions. However, these results show that conditional promoters can be useful tools to study the importance of essential genes in Z. tritici.Pto Pex1A A and was spindle . We theraffected D. It is 4Z. tritici. This adds a repertoire of homologous promoters to the established heterologous isocitrate lyase promoter derived from N. crassa (Z. tritici? Pnar1, PlaraB, and Pex1A are relatively “tight” when their repressors are present (gal7 may be a better choice, as its expression level under ON conditions is significantly higher than that of PlaraB, Pnar1 and Pex1A, while the promoter is still clearly repressed in the presence of glucose. Furthermore, experimental conditions could dictate the use of a specific promoter. For example, analysis of sugar metabolic pathways precludes the usage of PlaraB, but suggests the usage of Pnar1. On the other hand, Pex1A is the tightest promoter, as it shows no eGFP fluorescence, suggesting that eGFP expression is strongly repressed under OFF-conditions. Therefore, this promoter may be useful in investing signaling cascade components, which could act in very low concentrations in the cell and, therefore, require a near-complete shutdown of their transcription. PlaraB, on the other hand, shows relatively weak expression under induced conditions. This could be useful to avoid over-expression of genes that have naturally low cellular expression. Finally, Picl1 showed exceptionally strong expression under induced conditions, but was not tightly “shut-down” in the presence of its repressor. Thus, Picl1 may be a useful tool for over-expression studies, which, again, will depend on the native expression level of the genes of interest. Taken together, the set of conditional promoters are a powerful tool to perform in-depth analysis of essential genes in Z. tritici.In this study we established 5 conditional promoters for use in . crassa . Using e present , suggest


The road traffic impact on the concentrations of 13 fractions of particulate matter (PM) and their components was assessed. PM was sampled at two points in Katowice (southern Poland), a background point beyond the effects of road traffic, and a near-highway traffic point. The samples were analyzed for organic and elemental carbon, 8 water-soluble ions, 24 elements, and 16 polycyclic aromatic hydrocarbons (PAHs). The traffic emissions (mainly particles from car exhaust) enriched the ultrafine, submicron, and fine PM particles with elemental carbon. The traffic-caused re-suspension of the road and soil dust affecting the concentrations and chemical composition of the coarse PM fraction. However, for each PM fraction, the carcinogenic equivalent ratios, assumed as a measure of the hazard from 16 PAHs in this paper, were similar at the two sampling points. The traffic emissions from the highway appeared to have a weaker influence on the concentrations and chemical composition of PM in a typical urban area of southern Poland than elsewhere in Europe. The growth of road traffic causes the growth of health hazards from atmospheric aerosols Schwartz . However0.03-0.06, PM0.06-0.108, PM0.108-0.17, PM0.17-0.26, PM0.26-0.4, PM0.4-0.65, PM0.65-1, PM1-1.6, PM1.6-2.5, PM2.5-4.4, PM4.4-6.8, PM6.8-10, PM10-40  at these two sites are discussed in the paper.The goal of the work was to determine the differences in the chemical composition between various fractions of PM and to evaluate the road traffic influence on these differences in a typical urban area in southern Poland. PM was sampled in Katowice, at two sites differing in the traffic contributions to the air pollution. The concentrations, chemical composition, and exactness of the mass reconstruction  of each of thirteen following PM fractions: PMParticulate matter was sampled simultaneously at two points in Katowice, a city in southern Poland with approximately 2,000,000 population. One of the points, UB Fig. , was an In Katowice, especially in winter, traffic PM emissions can be obscured by emissions of PM from combustion of fossil fuels or biomass for residential heating  was used (1 µg resolution). Before each weighing, the substrates were conditioned for 48 h in the weighing room . The substrates and impactors were prepared for exposure in a laminar chamber. After weighing, the exposed substrates were stored in a freezer until analysis.Two kinds of Whatman  substrate filters were used. Alternating between the sample-takings, quartz fiber filters  and nylon membrane filters  were used; the former four and the latter five times. Always the same type substrates were used on all impactor stages, with the same type simultaneously used at both sampling points. Altogether, nine samples (four on quartz and five on nylon filters) of each of the 13 PM fractions were taken at each point over the entire measuring period.2) fragments were cut out from each quartz filter just before the analysis. The PM on one of them was analyzed for organic carbon (OC) and elemental carbon (EC). The remaining fragments, four for each of the 13 fractions and each point, were used to make fraction samples. A fraction sample for a point was made by putting together the four filter fragments containing this PM fraction sample from this point. Each of these 26 fraction samples was analyzed for 16 PAHs.At each point, four samples of each fraction were taken on quartz filters. Two equal 1.5 cm.5 cm2 fr−, NO3−, SO42−, Na+, NH4+, K+, Ca2+, Mg2+) were determined in the extracts.Five samples of each fraction were taken on nylon filters at each sampling point. The PM on each nylon filter was analyzed for elemental composition first , then it was extracted in water and concentrations of the main ions . The analytical procedures, equipment, and quality control for OC, EC, and PAH in PM are described in Rogula-Kozłowska and Klejnowski . Sixteen PAHsejnowski  and Roguejnowski .2 (Se) to 19.6 ng/cm2 (Si).The elemental composition of PM was determined by means of energy dispersive X-ray fluorescence (EDXRF). An Epsilon 5 instrument , calibrated with thin-layer single-element standards  was used to measure total concentrations of the elements. To control the performance of the analytical procedure, samples of reference material  were measured weekly. The recoveries were between 85 % and 120 % of the certified values (except 52 % and 39 % recoveries of V and Co) and the X-ray tube and detector drifts were monitored monthly. The detection limits were from 0.18 ng/cm3 of de-ionized water for 60 min at 15°C, then shaking for about 12 h . The ion content of extracts was determined using an ion chromatograph . The method was validated against the CRM Fluka products nos. 89316 and 89886; the standard recoveries were 92 %–109 % of the certified values, and the detection limits were: 10 ng/cm3 for NH4+, 18 ng/cm3 for Cl−, SO42− and K+, 27 ng/cm3 for NO3− and Na+, and 36 ng/cm3 for Ca2+ and Mg2+.The water extracts of PM were made by ultrasonizing the substrates containing the samples in 25 cmp between 0.17 and 1.6 µm (PM0.17-1.6). They contributed 72 % to the total PM mass at UB and 64 % at HW  traffic-related Fe, Co, Ni, and Mo  of EC bound to the nuclei and accumulation modes at HW and UB . The concentrations of PM0.03-0.17-related Fe, Cu, and Zn were also elevated at HW. The density functions for some PM-related metals , substances synthesized from residues of fuel combustion, and lubricant or fuel additives  . The significantly higher PM0.03-1.6-bound BaP concentration at HW than at UB allows for consideration of BaP as a marker for exhaust emissions in urban areas of Upper Silesia in the periods of lower municipal emissions.Surprisingly, despite the significant PAH content of exhaust OC, the ambient concentrations of fine particle-bound ∑PAH did not differ significantly between the points except for the PMp > 4.4 µm) and of some its components  also were higher at HW. For example, the average concentrations of PM4.4-6.8-, PM6.8-10-, and PM10-40-related Na+ and K+ were almost four times greater at HW than at UB, while that for Ca2+ was approximately two-fold greater.The concentrations of coarser PM  was assumed to be 1.4 of the analytically determined mass [OC]A of organic carbon (OC): [OM] = 1.4[OC]A. Secondary inorganic aerosol, SIA, consisted of SO42−, NO3−, NH4+; [SIA] = [SO42−]A + [NO3−]A + [NH4+]A. [NaCl] = [Cl−]A + [Na+]A.The chemical mass closure of PM was checked at both points. There were six groups of identified chemical PM components, and the seventh one, unidentified matter (UM); their masses together made the total PM mass. The mass [EC] of elemental carbon (EC) was assumed to be the analytically determined mass [EC]+, Ca2+, and Mg2+), were used to define crustal matter (CM) and trace elements (TE). The contents of CM and TE depended on both the PM fraction and the measuring point. These PM-bound elements were divided into two groups based on their enrichment factors EF , FeO and Fe2O3 (except PM0.4-1.6-bound FeO and Fe2O3 at both points), Rb (except PM0.17-1.6-bound Rb at both points), Sr , PM2.5-6.8-bound Ba, and Ba from particles with Dp greater than 10 µm. Except for CO32−, the masses of the compounds (oxides) in CM  were computed stoichiometrically from the masses of their component elements determined analytically; Fe was assumed to be distributed equally between FeO and Fe2O3. [CO32−] was computed from [Ca2+]A and [Mg2+]A . The chemical compounds containing an element with EF ≤ 20 that were known to contribute significantly to the PM mass were included in CM; if there were no such compounds (known) for an element, or the element was probable to occur in numerous compounds, only the element was included in CM. CM included CO+ (only for PM0.4-0.65 and PM0.06-0.108 at UB), Sc, Ti, V, Cr, Mn, Fe (Fe at both points for PM0.4-1.6 only), Co, Ni, Cu, Zn, As, Se, Br, Rb (Rb at both points for PM0.17-1.6 only), Sr (only for PM0.4-1.6 and for PM0.03-0.26 at HW and for PM0.03-1 at UB), Mo, Ag, Cd, Sb, Te, Ba (Ba only for PM0.03-2.5 and PM6.8-10 at both points), and Pb. The mass [TE] of TE was assumed to be the sum of all the analytically determined masses of the elements from TE.The chemical elements with EF > 20 were assumed to be anthropogenic; these many-source origin elements occurred in many PM-bound compounds that were not determined because of their multiplicity. These elements alone were put in TE. TE consisted of KThe mass of unidentified matter [UM] was the deficient mass; it was the difference between the gravimetrically determined mass of PM and [SIA] + [EC] + [OM] + [NaCl] + [CM] + [TE].4.4-6.8, the mass of identified compounds was higher at UB than at HW.In Fig. 0.06-0.65 and in particles with Dp > 10 µm were greatest; whereas at UB, they were greatest in PM0.108-0.65 and PM1.6-4.4. UM consists of the organic compounds and nitrates that evaporate during transportation and storage of PM samples, unidentified compounds, and water  at HW.The EC concentrations were higher at HW, and the EC mass distribution differed from that at UB Table ; Fig. 2.42−, NO3−, NH4+; Table 0.03-0.108 and coarser particles (Dp > 4.4 µm) were on average greater by 4 and 5 % at UB, respectively.Although the concentrations of the components of SIA . The mass contribution of TE to PM0.65-1.6 was greater by about 2 % at HW than at UB. The NaCl, CM, and TE contributions to fine, especially to ultrafine, PM were higher at UB than at HW because ultrafine PM had noticeably lower concentrations at UB  between the two points  differed between the points only in their EC content; PM10—in the EC and, greater by about 1 % at HW, CM contents , while the concentrations of PM2.5 and PM10 at HW were greater by no more than 5 % (for PM10 they were 28.6 µg/m3 at HW and 27.3 µg/m3 at UB).The differences in the chemical composition of PM between the points were mainly in ultrafine and coarse PM. The distributions of the mass among the component groups differed extremely for PM0.17-1.6 contributes most to the PM mass, and traffic does not affect this PM fraction much. The concentrations and the chemical composition of this PM fraction within the Silesian Agglomeration are formed by municipal emissions, big power plants, and industry. The presented results show that human health is not more at risk near a big highway than some distance away. At both sampling points the carcinogenic equivalent ratios (CEQ) for particular PM fractions were close, both CEQ for total PM were equal to 4.2 µg/m3 (Table So, although the number of cars per person grows closer to the European average, traffic emissions are not as significant a PM source in the Silesian Agglomeration as they are in other European regions (e.g. Viana et al. m3 Table .4.4-10 between two sites in such a specific area as the Silesian Agglomeration can be found only by investigating particular fractions of PM4.4-10. However, the experiment should rather be considered as a pilot research and should be repeated on a wider scale at other traffic-affected sites, such as crossroads in a city center, street canyons, etc., to generalize the conclusions.The present pioneering research shows that some differences in the chemical characteristics of PM


Transthyretin-mediated amyloidosis is an inherited, progressively debilitating disease caused by mutations in the transthyretin gene. This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple doses of patisiran (ALN-TTR02), a small interfering RNA encapsulated within lipid nanoparticles, in patients with transthyretin-mediated familial amyloid polyneuropathy (FAP).n = 4), 0.05 (n = 3), 0.15 (n = 3), or 0.3 (n = 7) mg/kg every 4 weeks (Q4W), or 0.3 mg/kg (n = 12) every 3 weeks (Q3W).In this phase II study, patients with FAP were administered 2 intravenous infusions of patisiran at one of the following doses: 0.01  was mild-to-moderate infusion-related reactions in 10.3 % of patients. Four serious AEs (SAEs) were reported in 1 patient administered 0.3 mg/kg Q3W , and 1 patient administered 0.3 mg/kg Q4W had 1 SAE (extravasation-related cellulitis).Patisiran was generally well tolerated and resulted in significant dose-dependent knockdown of transthyretin protein in patients with FAP. Patisiran 0.3 mg/kg Q3W is currently in phase III development.NCT01617967.The online version of this article (doi:10.1186/s13023-015-0326-6) contains supplementary material, which is available to authorized users. Transthyretin (TTR) is a tetrameric protein produced primarily in the liver. Mutations in the TTR gene destabilize the tetramer, leading to misfolding of monomers and aggregation into TTR amyloid fibrils (ATTR). Tissue deposition results in systemic ATTR amyloidosis –3. Over Treatment options for FAP focus on stabilizing or decreasing the amount of circulating amyloidogenic protein. Orthotopic liver transplantation reduces mutant TTR levels , with imRNA interference is a cellular process that controls gene expression, in which small interfering RNAs (siRNAs) mediate the cleavage of specific messenger RNAs (mRNAs) , 17. Lip2; adequate liver and renal function ; and seronegativity for hepatitis B virus and hepatitis C virus. Patients were excluded if they had a liver transplant; had surgery planned during the study; were HIV-positive; had received an investigational drug other than tafamidis or diflunisal within 30 days; had a New York Heart Association heart failure classification > 2; were pregnant or nursing; had known or suspected systemic bacterial, viral, parasitic, or fungal infections; had unstable angina; had uncontrolled clinically significant cardiac arrhythmia; or had a prior severe reaction to a liposomal product or known hypersensitivity to oligonucleotides.Eligible patients were adults (≥18 years) with biopsy-proven ATTR amyloidosis and mild-to-moderate neuropathy; Karnofsky performance status ≥ 60 %; body mass index 17–33 kg/mThis was a multicenter, international, open-label, multiple-dose escalation phase II study of patisiran in patients with FAP. Cohorts of 3 patients received 2 doses of patisiran, with each dose administered as an IV infusion. Cohorts 1–3 received 2 doses of patisiran 0.01, 0.05, and 0.15 mg/kg every 4 weeks (Q4W), respectively; cohorts 4 and 5 both received 2 doses of patisiran 0.3 mg/kg Q4W. All patients in cohorts 6–9 received 2 doses of patisiran 0.3 mg/kg administered every 3 weeks (Q3W). As lipid-based delivery systems have previously been associated with adverse immune events , 25, allThe study procedures  were approved by the ethics committee on human experimentation at each site. All patients provided written informed consent.The primary study objective was to evaluate the safety and tolerability of multiple ascending doses of patisiran. Secondary objectives were to characterize the plasma and urine PK of patisiran, and to assess preliminary evidence of the PD effect of patisiran on serum total TTR protein levels.Serum levels of total TTR protein were assessed for all patients using an enzyme-linked immunosorbent assay (ELISA). Additionally, wild-type and mutant TTR protein were separately and specifically measured in serum for patients with the Val30Met mutation using a proprietary mass spectrometry method . Serum samples were collected at screening, and on Days 0, 1, 2, 7, 10, 14, 21, 22, 23 (Q3W only); 28, 29 (Q4W only); 30 (Q4W only); 31 (Q3W only); 35, 38 (Q4W only); and 42, 49, 56, 112, and 208 of follow-up.Plasma concentration–time profiles were created for TTR siRNA, based on blood samples collected on Day 0 and at the following time points: pre-dose (within 1 h of planned dosing start), at end of infusion (EOI), at 5, 10, and 30 min, and at 1, 2, 4, 6, 24, 48, 168, 336, 504 , and 672 h  post-infusion. Additional samples were collected on Days 84 and 180 for the Q4W regimens, and on Days 35, 91, and 187 for the Q3W regimen. For cohorts 3–9, blood samples on Day 0 at EOI and 2 h post-infusion were also analyzed for both free and encapsulated TTR siRNA. Serum TTR siRNA was analyzed using a validated ATTO-Probe high-performance liquid chromatography (HPLC) assay . PK analyses were conducted using non-compartmental and/or compartmental evaluation of TTR siRNA plasma concentration–time data to determine PK parameter estimates using the validated software program WinNonlin®. Urine samples were analyzed for levels of excreted TTR siRNA, and renal clearance was measured after dosing.Serum levels of vitamin A and retinol binding protein (RBP) were measured by HPLC and nephelometry, respectively, at the same time points specified for total TTR .Safety evaluations included assessment of adverse events (AEs), electrocardiograms (ECGs), arterial oxygen saturation using pulse oximetry, vital signs, clinical laboratory safety tests, and physical examinations. AEs were defined as mild , moderate (sufficient discomfort to interfere with daily activity), or severe . The numbers and percentages of patients with any treatment-emergent AE (TEAE), with any serious TEAE, with any TEAE leading to discontinuation of study medication, or with any TEAE considered a dose-limiting toxicity were summarized by dose cohort and overall. Dose-limiting toxicities included any of the following: any life-threatening toxicity; ALT and AST ≥ 5 × ULN or total bilirubin > 2.0 mg/dL; an infusion reaction that required hospitalization; and any other toxicity that in the opinion of the SRC precluded administration of a second dose.Based on the planned dose escalation scheme, we expected to enroll 27 patients. Patient populations included intent-to-treat  and per-protocol . Safety measures were assessed in the ITT population. No substitutions were made to allow for missing data points.Means and variances for TTR knockdown from baseline were calculated for the PP population, with baseline defined as the average of all pre-dose values. We used analysis of variance (ANOVA) and analysis of covariance (ANCOVA) to analyze the PD data , with Tukey’s post hoc tests of individual pairwise comparisons (between dose levels). Nadir TTR levels were defined as the minimum level per patient during the 28-day period (21-day period for Q3W group) after each dose administration . Relationships between TTR and RBP or vitamin A, relative to baseline, and the relationship between wild-type and V30M TTR levels, were explored via linear regression. We formally evaluated the dose proportionality of the patisiran component in PK parameters using a power model analysis. AEs were coded using the Medical Dictionary for Regulatory Activities coding system, version 15.0, and descriptive statistics provided for AEs, laboratory data, vital signs data, and ECG interval data. All statistical analyses were performed using SAS (version 9.3 or higher) and/or R (version 2.6 or higher).A total of 29 patients were enrolled across 7 countries: Brazil, France, Germany, Portugal, Spain, Sweden, and the USA. All patients were white, 69 % were male, and the mean (standard deviation [SD]) age was 56 15.6) years  discontinued following a protocol amendment  and 4 patients received patisiran 0.01 mg/kg; all other study cohorts included 3 patients as described. Of the cohorts treated with patisiran 0.3 mg/kg, 10 patients received their infusions over 60 min, and 9 patients  received the 70-min micro-dosing regimen. One patient in the 0.05 mg/kg Q4W cohort 2 did not meet the eligibility criteria because of liver function data, but was given a waiver to enter the study.Mean (SD) baseline serum TTR protein levels were similar across the dose cohorts: 272.9 (98.86), 226.5 (12.67), 276.1 (7.65), 242.6 (38.30), and 235.5 (44.45) μg/mL for the 0.01, 0.05, 0.15, 0.3 Q4W, and 0.3 mg/kg Q3W dosage groups, respectively.p < 0.001 by post hoc tests after ANCOVA) was observed after the first and second doses of patisiran in the 0.3 mg/kg Q4W and Q3W cohorts  and vitamin A    and area under the plasma concentration–time curve from zero to the last measurable time point (AUC0–last) increased in a dose-proportional manner over the dose range tested. Cmax and AUC0–last after doses 1 and 2 were comparable, with no accumulation. The median terminal half-life of patisiran at Day 0 and Days 21/28 was 39–59 h at doses > 0.01 mg/kg, and was relatively unchanged when comparing doses 1 and 2 for each dose cohort.Mean concentrations of the patisiran TTR siRNA component decreased after EOI , which occurred in 3/29 patients overall (10.3 %), all in the 0.3 mg/kg Q4W group (Table There were no dose-limiting toxicities or deaths due to TEAEs reported during the course of the study. The majority of TEAEs were of mild or moderate intensity. Four serious AEs (SAEs) were reported in 1 patient in the 0.3 mg/kg Q3W group , and the patient withdrew from the study because of the nausea and vomiting. An additional SAE (extravasation-related cellulitis) was recorded in 1 patient in the 0.3 mg/kg Q4W group. The SAEs of nausea and vomiting, and cellulitis were each recorded by the Investigator as related to the study drug.No clinically significant changes in liver function tests, renal function, or hematologic parameters were recorded. Transient increases in white blood cell count were observed approximately 24 h after each patisiran infusion, which were considered related to dexamethasone premedication. There were no substantial changes in serum G-CSF, IFN-α, IFN-γ, IL-1b, IL-12, and TNF-α cytokines. Values were below the lower limit of detection for most patients at the majority of time points. Transient increases in mean levels of IL-6, IL-1RA, and IP10 were observed after patisiran infusion in the 0.3 mg/kg Q4W group (and in the 0.3 mg/kg Q3W group for IL-6), although levels returned to baseline within 24 h. Transient increases in mean levels of complement factor Bb were also seen after infusion of patisiran doses 0.05–0.3 mg/kg. None of these elevations were associated with AEs.These phase II data demonstrate that administration of patisiran to patients with FAP led to robust, dose-dependent, and statistically significant knockdown of serum TTR protein levels. Mean sustained reduction in TTR of >80 % was achieved with 2 consecutive doses of patisiran 0.3 mg/kg dosed every 3–4 weeks, with a maximum knockdown of 96 % achieved in the Q3W group. These knockdown rates are consistent with the rates observed in the single ascending dose, placebo-controlled phase I study of patisiran . EvidencMultiple doses of patisiran were shown to be generally safe and well tolerated in this study. The majority of AEs were mild or moderate in severity and no dose-limiting toxicities were observed. While IRRs were the most common drug-related TEAE seen at 0.3 mg/kg, no IRRs were reported in the 0.3 mg/kg group that received the micro-dosing infusion regimen over 70 min. Premedication was used to reduce the risk of IRRs, and is thus considered in assessment of the tolerability of this regimen. Glucocorticoids such as dexamethasone can increase white blood cell levels , as obseThis multi-dose study defined patisiran 0.3 mg/kg Q3W delivered using the 70-min micro-dosing regimen as the formulation and dosage to administer in the ongoing open-label extension (OLE) phase of patients with FAP. Whilst both Q3W and Q4W achieved potent TTR knockdown, the level of TTR suppression was better maintained between doses with the Q3W schedule, supporting its future investigation. Preliminary data from the OLE study show sustained TTR knockdown of ~80 % after 168 days (9 doses), with no reported SAEs (n = 27) . The OLEn = 130) [n = 125) [Patient baseline demographics and disease characteristics in the present study were similar to those reported for other recent FAP studies. In particular, the mean age (55.6 years) was comparable to that reported for a large natural history study of patients with FAP   and for n = 130) , but wasn = 125) . Some 75n = 125) , 15, 32.The results of this study supported the initiation of a phase III study of patisiran. APOLLO is a randomized, placebo-controlled, phase III study of patisiran 0.3 mg/kg Q3W administered using the 70-min micro-dosing schedule in recruiting patients with Val30Met or non-Val30Met FAP who are not taking TTR stabilizers. The primary study endpoint is disease progression, measured as the change from baseline in mNIS + 7 score at 18 months . Secondary endpoints include measures of quality of life and disease burden, changes from baseline in motor and autonomic neuropathy measures, and safety.In conclusion, the results of this phase II study provide evidence that the investigational agent patisiran was generally well tolerated and effective in reducing both mutant and wild-type TTR levels in patients with FAP. The 0.3 mg/kg Q3W dosing schedule is under investigation in the phase III APOLLO trial.