Road dust as a by-product of exhaust and non-exhaust emissions can be a major cause of systemic oxidative stress and multiple disorders. Substantial amounts of road dust are repeatedly resuspended, in particular at traffic lights and junctions where more braking is involved, causing potential threat to pedestrians, especially children. In order to determine the degree of contamination in the heavily traffic-congested cities of Poland, a total of 148 samples of road dust (RD), sludge from storm drains (SL) and roadside soil (RS) were collected. Sixteen metals were analysed using inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectroscopy (ICP-OES) and atomic absorption spectroscopy (AAS) in all samples. Chemical evaluation followed by Principal Component Analysis (PCA) revealed that road environments have been severely contaminated with traffic-related elements. Concentration of copper in all road-environment samples is even higher, exceeding even up to 15 times its average concentrations established for the surrounding soils. Non-carcinogenic health risk assessment revealed that the hazard index (HI) for children in all road-environment samples exceeds the safe level of 1. Therefore, greater attention should be paid to potential health risks caused by the ingestion of traffic-related particles during outdoor activities. As reported by Garg et al. [10 and 60% is PM2.5. According to Harrison et al. [10 emissions and up to 21% to the total traffic-related PM10 emissions [10. In terms of ambient concentrations, this corresponds to values ranging from 0.05 to 11 μg·m−3 [Non-exhaust emission from traffic-related sources is considered the priority issue by the European Union within its sustainable transport strategy, since it greatly contributes to the overall problem of air pollution. However, despite the ongoing fight for better air quality, the vast majority of populations in urban areas is still exposed to air which does not comply with any World Health Organisation (WHO) Air Quality Guidelines and therefore causes a serious health threat. Kelly and Fussell  confirm g et al.  and Kukug et al. , over 50n et al. , in urbamissions ,8. Garg missions  estimatemissions , 0.1–10%1 μg·m−3 ,10,11,121 μg·m−3 . MoreoveAccording to Duong and Lee , the conThe main objective of the study was to evaluate metal contamination in various road-environment samples such as road dust, sludge from storm drains and roadside topsoil obtained from four of the biggest and highly congested cities in Poland, a country facing the worst air pollution problem in Europe. Werkenthin et al.  present The most traffic-congested cities of Poland  were selected as research sites. Over the last two decades, all four have faced significant decline in heavy industry. In each city, eight sampling areas were selected near roads with very high road traffic, junctions, and heavy pedestrian traffic. They were located as far as possible from industrial plants and far from residential areas to minimise the impact of contamination sources. Samples were taken monthly starting from May to November 2015 to avoid the impact of other non-traffic-related pollutants, such as those emitted from household furnaces. Research areas are depicted on Road dust, roadside topsoil and sludge from storm drains were digested with aqua regia according to 3050A protocol . The conThe concentrations of metals in road dust were then compared with median concentrations determined for the surrounding soils by Lis, Pasieczna  for KrakFurthermore, PI (pollution index) was calculated from Equation (1):asieczna  for Krakasieczna  for Warsasieczna  for Wrocasieczna ,44.−6 kg/mg. All parameters used in the calculation of ADD were found in U.S. EPA Exposure Handbook [In order to assess the non-carcinogenic risk for children and adults from road dust, sludge from storm drains and roadside topsoil, an average daily intake dose of deleterious substances and exposure through ingestion (ADD) of Ba, Cd, Co, Cu, Mn, Mo, Ni, Pb, Sb, Se, Sn, Zn and Zr was calculated from the following formula according to U.S. EPA .ADD = (kg), see ; IngR isHandbook .−3 mg/kg body weight per day according to [The potential non-carcinogenic risk of Ba, Cd, Co, Cu, Mn, Mo, Ni, Pb, Sb, Se, Sn, Zn and Zr was evaluated by the hazard quotient (HQ).c Tables . Since tc Tables , in thisrding to ,52,53,54If the HQ < 1, then non-carcinogenic toxic effects are unlikely. If HQ ≥ 1, then potential adverse health effects may occur. HQ > 10 suggests a high chronic risk . Though If HI < 1, then there is no significant risk. If HI > 1, there is a chance that non-carcinogenic toxic effects may be possible, with the increasing probability as HI increases ,55.p = 0.05) and the precisions of AAS and ICP-MS systems were satisfactory, which was verified by six different solution injections. Rh was used as an internal standard. Using ICP-MS, element correction equations were used for each element to minimise the impact of interferences.In order to obtain unambiguous and unbiased ICP-MS results, elements were additionally measured using inductively coupled plasma-optical emission spectroscopy (ICP-OES) , atomic absorption spectroscopy (AAS) , according to U.S. EPA method 7000 in the Laboratory of Trace Analyses at the Faculty of Geology, Geophysics and Environmental Protection, AGH University of Science and Technology. Analyses were performed according to standard certified analytical quality control procedure (PN-EN ISO 17294-1:2007). Moreover, reagent blanks and certified international reference material METRANAL™ 32  were used to ensure that the analytical results met the required criteria. Analyses of the reference material verified and confirmed the quality of the results. Analytical bias was statistically insignificant  and Warszawa (650 mg/kg). Its concentrations in road dust from those two cities were considerably lower, 345 mg/kg and 276 mg/kg, respectively. Concentrations of copper in all road-environment samples were even higher, exceeding 8 to 19 times the average concentration established for the surrounding soils. In Krakow, the concentration of Cu ranges from 27.2 mg/kg in roadside soil up to 499 mg/kg in sludge, and in Warszawa it varies from 45.3 mg/kg in roadside soil up to 339 mg/kg in road dust. The highest concentrations of Cd in all road-environment samples were found in Krakow and Wroclaw. Similarly, the concentrations of Pb were the highest in sludge from storm drains and road dust collected in those two cities.PI index calculated for road-environment samples in Warszawa was 9–13 for Cu, 3–7 for Cr, 1–4 for Pb, 7 for Ti, and, 3–10 for Zn. PI index determined for road-environment samples in Krakow was comparably high and equalled 10–15 for Cu, 3–9 for Cr, 3–4 for Pb, 10–20 for Ti and 3–6 for Zn. Additionally, high concentrations of Cd were also recorded in Krakow, especially in roadside soil (PI 5), indicating a high level of contamination. In Wroclaw, PI indicates an extremely high Cr and Ti contamination in all types of road-environment samples, ranging from 5–18 for Cr to 11–16 for Ti. PI indices for Cu and Zn are relatively high in Wroclaw, ranging between 2–4 for Cu and 4–6 for Zn. In Opole, PI indicates moderate to high contamination. Potential non-carcinogenic health risk (HQ) and the hazard index (HI) for children in heavily congested road urban environments are presented in −1) > Co (1 × 10−1) > Sb (1.32 × 10−1) > Se (8.33 × 10−2) > Pb (5.90 × 10−2) > Mo (5.57 × 10−2) > Zn (4.61 × 10−2) > Cu (1.23 × 10−2) > Cd (3.07 × 10−3) > Ba (2.77 × 10−3) > Sn (1.62 × 10−4). The results determined for sludge from drain storms were as follows: Zr (7.52 × 10−1) > Co (1.48 × 10−1) = Sb (1.48 × 10−1) > Se (1.21 × 10−1) > Zn (6.95 × 10−2) > Mo (6.83 × 10−2) > Pb (6.81 × 10−2) > Mn (5.58 × 10−2) > Cu (1.41 × 10−2) > Cd (5.42 × 10−3) > Ba (3.36 × 10−3) > Sn (2.05 × 10−4); for the roadside soil the results were the following: Zr (6.43 × 10−1) > (1.74 × 10−1) > Zn (1.52 × 10−1) > Co (1.25 × 10−1) > Sn (1.17 × 10−1) > Pb (9.89 × 10−2) > Se (8.34 × 10−2) > Mo (5.41 × 10−2) > Sb (5.07 × 10−2) > Cd (1.21 × 10−2) > Cu (7.20 × 10−3) > Ba (4.79 × 10−3). An average HQ index for children was calculated with respect to traffic-related elements in road dust samples collected in Krakow, Warszawa, Wroclaw, Opole; the results are presented in descending order: Zr (6.81 × 10−1) > Sb (8.17 × 10−2) > Co (6.95 × 10−2) > Se (4.54 × 10−2) > Mo (3.84 × 10−2) > Pb (3.44 × 10−2) > Mn (3.23 × 10−2) > Zn (2.54 × 10−2) > Mn (2.10 × 10−2) > Cu (7.42 × 10−3) > Cd (1.73 × 10−3) > Ba (1.73 × 10−3) > Sn (8.77 × 10−5). Results determined for samples of sludge from drain storm are as follows: Zr (1.08 × 10−1) > Co (9.52 × 10−2) > Sb (5.50 × 10−2) > Cu (3.78 × 10−2) > Se (1.49 × 10−1) > Mn (1.19 × 10−1) > Zn (9.63 × 10−3) > Pb (9.03 × 10−3) > Mo (2.79 × 10−2) > Cd (2.07 × 10−2) > Cu (1.41 × 10−2) > Cd (5.42 × 10−3) > Ba (3.36 × 10−3) > Sn (2.05 × 10−4); finally, for roadside soil the results were as follows: Zr (3.21 × 10−1) > Co (6.52 × 10−2) > Pb (5.98 × 10−2) > Se (4.68 × 10−2) > Mo (3.82 × 10−2) > Mn (2.92 × 10−2) Sb (2.79 × 10−2) > Zn (2.64 × 10−2) > Mn (1.82 × 10−2) > Cd (1.21 × 10−2) > Ba (3.73 × 10−4) > Sn (8.07 × 10−5).The results of calculations for road dust are presented in the descending order as follows: Zr  was also less than 1.0, therefore confirming that the exposure to environmental road samples was safe for adults. However, this was different for children, since HI index in that case exceeded the acceptable value of 1.0, signifying potential hazard. The HI values in road dust range from 1.08 (Krakow) up to 1.60 (Opole). In the sludge, they vary from 1.26 (Wroclaw) to 1.58 (Opole) and in roadside soil from 1.25 (Opole) to 1.83 (Krakow). Therefore, greater attention should be paid to adverse effects on children’s health caused by potential ingestion of traffic−related particles in road environments, e.g., at street crossings. It would also be important to determine the speciation of traffic−related metals in road environmental samples to better understand the health risks they pose for humans and the environment.The results of chemical analysis, followed by the calculation of pollution index (PI) for various components of road environment in Warszawa, Krakow, Wroclaw and Opole revealed that road dust, mixed sludge and sediment from storm drains as well as roadside soils were significantly contaminated with all of the investigated elements, in particular with Cu, Pb, Ti and Zn. The highest concentrations were found in samples of sludge from drain storms collected from all investigated cities. Sludge samples were approximately 20% more contaminated with metals than road dust and roadside soils. PCA has revealed that both road dust and sludge are strongly correlated with elements derived mostly from brake pads, tires and as a result of road surface abrasion due to frictional effects.HQ indices calculated for the analysed traffic−related elements were all lower than 1.0, potentially indicating non−carcinogenic effect. HI index for selected metals  for adults fell within the safe value. However, in the case of children, the HI values exceeded the safe level of 1.0 for road dust, sludge from storm drains and roadside topsoil in all investigated cities. Therefore, monitoring of road environments should be intensified, since road dust as a byproduct of exhaust and non−exhaust particulate emissions easily enters human airways and may have an adverse effect on health.


Aedes albopictus is an extremely invasive, globally distributed and medically important vector of various human and veterinary pathogens. In Germany, where this species was recently introduced, its establishment may become modulated by interspecific competition from autochthonous mosquito species, especially Culex pipiens (s.l.). While competitive superiority of Ae. albopictus to Cx. pipiens (s.l.) has been described elsewhere, it has not been assessed in the epidemiological conditions of Germany. The present study aimed to determine if such superiority exists under the physicochemical and microclimatic conditions typical for container habitats in Germany.The Asian tiger mosquito Ae. albopictus and Cx. pipiens (s.l.)  to interspecific interaction  at (sub-)optimal temperatures  and differing food supply  were investigated using a randomized split-plot design. In addition to physicochemical measurements of the test media, natural physicochemical conditions were determined for comparative analyses in mosquito breeding sites across the Rhine-Main metropolitan region of Germany.In a replacement series experiment, the larval and pupal responses of Cx. pipiens (s.l.) to Ae. albopictus in terms of larval survival was more frequently observed than balanced coexistence. Food regime and multifactorial interactions, but not temperature alone, were controlling factors for interspecific competition. Larval food regime and the larval ratio of Ae. albopictus influenced the physicochemistry and algal growth at 15 °C, with increased Ae. albopictus mortality linked to a decreasing number of Scenedesmus, Oocystis and Anabaena algae.Under the physicochemical and microclimatic conditions similar to the breeding sites of the Rhine-Main region, competitive superiority of Ae. albopictus from isolated foci in Germany may generally be slowed by biotic interactions with the ubiquitous Cx. pipiens (s.l.)  and by limnic microalgae in microhabitats with high resource levels. Detailed knowledge of the context dependency in temperate mosquito ecology, and interrelations of physicochemistry and phycology may help to achieve a better understanding of the upcoming Ae. albopictus colonization processes in central and northern Europe.Under the present environmental conditions, the spread of The online version of this article (10.1186/s13071-018-2665-3) contains supplementary material, which is available to authorized users. Aedes (Stegomyia) albopictus Skuse 1894, a known vector of various human pathogens like dengue and chikungunya viruses, is gradually extending its distribution to colder climate regions   . After SCx. pipiens (s.l.), conductivity , temperature , oxygen concentration  and pH  were measured in situ. Also, 1 L plastic cups were filled with 600 ml water and placed at a sunny and a shaded site in Frankfurt am Main. The water temperature in the transparent containers was recorded at 20-min intervals  from mid-May to the end of July 2011. During this period, a minimum water volume of 600 ml was maintained.At a subset of sites positive for Ae. albopictus and Cx. pipiens molestus under different food regimes and temperatures was investigated in a replacement series experiment using a randomized split-plot design. The replacement series experiment with 4 replications was designed to test the impact of the whole plot factors ‘species’ (2 levels) × ‘larval ratio’ (5 levels) × ‘temperature’ (3 levels) and the subplot factor ‘larval food regime’ (2 levels) on survival, development time and growth of the aquatic life stages of the 2 species.The interspecific larval interaction of Ae. albopictus strain with origin Singapore, Cx. pipiens biotype molestus with origin Regensburg, Germany) to minimize unknown impacts of the field histories of the mosquito strains on their responses and better determine the fundamental nature of the context-dependency of interspecific interactions.The mosquitoes used in the experiments were purchased from Biogents AG  and reared in-house. We used 2 well-adapted long-term laboratory strains . In total, 3600 larvae were investigated.The eggs of hatching . First-iFirst-instar larvae of the two species, which had been synchronously released within 24–48 h, were allotted as pure or mixed cohorts of 30 larvae in 1 l test vessels (plastic cups) filled with 600 ml larval medium each.Ae. albopictus larvae (Ae: Cx30:00) and only Cx. pipiens molestus larvae (Ae: Cx00:30) and symmetrically and asymmetrically mixed treatments with a 20:10, 15:15 or 10:20 distribution of Ae. albopictus: Cx. pipiens molestus larvae  were prepared. The larval density of 50 larvae l-1 reproduced natural larval densities: for instance, 1.2–89.5 larvae l-1 had been reported from various containers in Italy /3} / {species A30:00/ species B30:00}. From these parameter-specific RCCs, the difference between species was calculated for each gender and experimental group to improve the comparability of parameter-specific competitiveness between 2 species .Treatment-specific relative crowding coefficient values (RCC) were calculated with the AL, BA, DW and PTy Harper  and modiy Harper  and Obery Harper : RCCspec species . Also, iP ≤ 0.01). Variance homogeneity of the data was tested using the Cochran’s test at the level of P ≤ 0.01. After confirmation of variance homogeneity for arcsin and log-transformed mortality [%], log-transformed PT50 data (gender-specific) and parameter-specific RCC values, four-factorial ANOVAs  and subsequent Tukey HSD tests were computed for these response variables. The heteroscedastic, but normally distributed dependent variables AL, BA and DW were analyzed with general linearized normal-log models with backward factor removal during model building and subsequent Wald statistics. The total average mortality of the two species  was compared with a t-test.The mosquito and physicochemical datasets were tested for Gaussian distribution using the Kolmogorov-Smirnov test and the D’Agostino Pearson omnibus normality test  were computed with mortality data against larval ratio. Also, first order regressions (least squares fit) were computed with AL, DW and BA data against the number of larvae of the other species, respectively. Extra-sum-of-squares-tests were applied to compare independent fits with global fits for mosquito responses to food regime within a temperature block  procedure with false discovery rate (FDR) of 0.05) and independent fits with global fits for each species and gender and within each temperature , respectively.P < 0.05) for their dependence on initial larval ratio and food. Water quality parameters were further tested for correlation with algal growth (Spearman). The dependence of algal growth at 15 °C from initial larval ratio or larval mortality and food regime and their interactions were tested with 2-way ANOVAs. The algal growth data at 15 °C were tested for Spearman's correlation with Ae. albopictus mortality at 15 °C and Cx. pipiens mortality at 15 °C, respectively. Also, non-parametric Spearman's correlation analyses with the initial larval quantity of Ae. albopictus or Cx. pipiens molestus and the cell number of specific algal groups were performed for both food-related subplots.Water quality parameters examined at 15 °C were tested by 2-way ANOVA (Cx. pipiens (s.l.)  were collected. The physicochemical parameters of a subset of Cx. pipiens (s.l.) positive microhabitats (n = 70) of mainly anthropogenic origin (88.6%) averaged to 15.0 ± 4.56 °C, 6.74 ± 4.02 mg l-1 dissolved oxygen, 390 ± 574 μS cm-1 electrical conductivity and pH 7.60 ± 0.84 . In contrast, the larval ratio in combination with species, food or temperature was significant in only 8 out of 27 sub-models. Three-factorial interactions of species × food × larval ratio strongly influenced the data , as did species × food × temperature (except BA). The triple interaction species × larval ratio × temperature had a significant influence on mortality, male PT50 and AL, whereas food × larval ratio × temperature had no significant influence on any effect variable. The four-factorial interaction was significant for female BA, female PT50 and male DW only  of Ae. albopictus  was significantly higher than the total average mortality of Cx. pipiens molestus  = 13.0, P < 0.0001). The highest mortality of Ae. albopictus (66.2 ± 6.04%) and Cx. pipiens molestus (39.6 ± 4.39%) was observed at 15 °C and 6 mg larva-1 food supply. The Cx. pipiens molestus larvae had an equally high mortality (39.6 ± 6.14%) at 25 °C and 3 mg larva-1 food supply. The lowest mortality of Ae. albopictus was 8.54 ± 2.46% and that of Cx. pipiens molestus 10.0 ± 2.17%, both in cohorts exposed to 20 °C and supplied with 6 mg larva-1 food.The split-plot design used in the 4-factorial replacement series experiment resulted in a larval mortality within the margins of expectation for intra- and interspecific competition with a larval density of 50 larvae l-1 Fig. . The tot-1 and exposed to 20 °C (F(3.34) = 1.87, BH-corrected P = 0.05), and those fed 3 mg larva-1 at 25 °C (F(3.34) = 3.78, BH-corrected P = 0.042). In the other 4 temperature-food treatments, the mortality in pure and mixed cohorts differed strongly between species  = 5.86, BH-corrected P = 0.025; 15 °C, 6 mg larva-1: F(3.34) = 3.78, BH-corrected P = 0.008; 20 °C, 3 mg larva-1: F(3.34) = 3.78, BH-corrected P = 0.033; 25 °C, 6 mg larva-1: F(3.34) = 3.78, BH-corrected P < 0.017; Fig. Mortality was mainly influenced by temperature. Further variance was produced by the factors food, species and larval ratio, by the 2-factorial interactions food × temperature, species × larval ratio, species × food, and the 3-factorial interactions species × food × temperature, species × food × larval ratio and species × larval ratio × temperature Table . BetweenCx. pipiens molestus larvae significantly increased the mortality of Ae. albopictus in the cohorts fed 6 mg larva-1 at 25 °C  = 0.55, P = 0.046) and 3 mg larva-1 at 20 °C  = 0.55, P = 0.023) and 25 °C  = 0.55, P = 0.009; Ae:Cx20:10vs Ae:Cx30:00, Tukey HSD MS(126) = 0.55, P = 0.002). The species × food × larval ratio interaction on mortality was demonstrated by several significant differences between food-related Ae. albopictus cohorts: The Ae:Cx15:15 cohort that had been fed 3 mg larva-1 differed from the cohorts Ae:Cx10:20, Ae:Cx20:10 and Ae:Cx30:00 provided with 6 mg food larva-1 at 20 °C (Tukey HSD MS(126) = 0.55, P = 0.004, P = 0.039, P = 0.001, respectively); the Ae:Cx15:15 cohort fed 3 mg larva-1 from the Ae:Cx 30:00 cohort fed 6 mg larva-1 at 25 °C (Tukey HSD MS(126) = 0.55, P = 0.039), and the Ae:Cx30:00 cohort fed 3 mg larva-1 from the Ae:Cx15:15 cohort fed 6 mg larva-1 at 25 °C (Tukey HSD MS(126) = 0.55, P = 0.010). In contrast, the presence of Ae. albopictus larvae did not influence the mortality of Cx. pipiens molestus (Tukey HSD MS(126) = 0.55, P > 0.05).The presence of 50 of male and female larvae . The PT50 of male and female larvae was additionally influenced by the 2-factorial interactions species × temperature, food × temperature and species × food and the 3-factorial interactions species × food × larval ratio and species × food × temperature. The triple interaction species × larval ratio × temperature had a significant effect on the PT50 of male, and the 4-factorial combination on that of female larvae only.Species and temperature were the major factors affecting the PTae Table . Also, a50 of both genders and species decreased as a function of temperature, with maxima at 15 °C and minima at 25 °C  and 0.7 days (Cx. pipiens molestus) and the mean PT50 of males by 2.1 days  and 0.5 days (Cx. pipiens molestus) if compared to the cohorts fed 6 mg larva-1 at 25 °C. At lower temperatures, the PT50 had an opposite trend with a higher average PT50 of females of 1.9 days  and 0.8 days (Cx. pipiens molestus) at 15 °C and 1.2 days  at 20 °C if larvae had been supplied with 3 mg food larva-1, although the average PT50 of Cx. pipiens molestus was similar in both food treatments at 20 °C.The PT °C Fig. . At 25 °50 of male larvae that had received 6 mg food larva-1, which was 0.3 days  and 1.6 days (Cx. pipiens molestus) shorter at 15 °C and 0.7 days  and 1.1 days (Cx. pipiens molestus) shorter at 20 °C than the average PT50 of males that had been fed with 3 mg larva-1. With regard to larval ratio, the PT50 of females decreased with an increasing larval ratio of the competing species, but this was only observed in the cohorts of Ae. albopictus provided with 3 mg food larva-1 and those of Cx. pipiens molestus given 6 mg larva-1.This general pattern of 2- and 3-factorial interactions of species × food × temperature equally applied to the average PTPupal growth  was mainly influenced by the single factors species, temperature and food and their 2- and 3-factorial interactions Table . The larAe. albopictus was generally greater (3.12 mm) than that of female Cx. pipiens molestus (2.89 mm), although it was equal at 20 °C and a food supply of 6 mg larva-1 (F(2.28) = 1.25, P = 0.30), and at 15 °C and 3 mg food larva-1 (F(2.27) = 3.14, P = 0.06)  = 16.01, P < 0.0001) and finally by both larval food regimes at 15 °C.The AL of female eater 3.1 mm than Cx. pipiens molestus differed significantly among the larval food regimes at every tested temperature (15 °C: F(2.27) = 17.26, P = 0.0001; 20 °C: F(2.28) = 12.25, P = 0.0002; 25 °C: F(2.27) = 23.39, P = 0.0002). The maximum AL of female Cx. pipiens molestus was measured in the cohort with 6 mg food larva-1 at 15 °C, followed by the groups fed 6 mg larva-1 and 3 mg larva-1 at 20 °C, the 3 mg larva-1 cohort at 15 °C, and finally by both larval food regimes at 25 °C.The AL of female Cx. pipiens molestus (0.80 mg) was higher than that of female Ae. albopictus (0.58 mg). In both species, the DW of females was always largest in the cohorts with 6 mg larva-1 food supply  and temperature (Table -1), demonstrating the strong impact of temperature on BA. The single factor food had a significant influence on females only, but food × species or food × temperature and the 3-factorial interaction species × food × temperature had significant effects on both males and females. The BA of both species was similar to 3 mg larva-1 food supply except Ae. albopictus at 25 °C, but the BA was lower in Ae. albopictus than Cx. pipiens molestus cohorts fed with 6 mg larva-1.The daily increase of weight (BA) was mainly influenced by the factors species , whereas RCC values ≥ 2.5 signifying high competition were never observed.Both species exhibited a weak to intermediate interspecific competitiveness with regard to growth and development as indicated by RCC values between 0.91 and 2.31  was balanced .The integration of RCCs revealed that differences from growth and development specific RCCs were almost balanced out given that the single factors species, gender, food and temperature and almost all interactions had no significant influence. Notably, the integrated RCC00:30 cohort provided with 6 food mg larva-1and the shortest standing time of 33 ± 2 days in the Ae:Cx00:30 cohort fed with 3 mg larva-1  and potentially toxic nitrite (2-fold) were lower than in the water of 3 mg larva-1 cohorts. In contrast, concentrations of ammonium (1.7 to 18-fold), silicon dioxide , oxygen (1.1 to 1.3-fold) and pH (1.2 to 1.3-fold) were higher than in the water of cohorts fed with 3 mg larva-1  = -0.588, P = 0.04), conductivity (Spearman's r(10) = -0.758, P = 0.006), pH (Spearman's r(9) = -0.720, P = 0.014), ammonium (Spearman's r(10) = -0.741, P = 0.007) and decreased nitrate (Spearman's r(10) = 0.842, P = 0.001) and nitrite (Spearman's r(10) = 0.801, P = 0.008), algal growth was significantly reduced in the water with cohorts fed 6 mg larva-1 (F(1.50) = 116.3,, P < 0.001, 46.6% total variation)  = -0.86, P = 0.011). Algal growth especially declined with an increasing number of initial Ae. albopictus larvae  = 13.17, P < 0.001, 21.1% total variation; food × larval ratio: F(4.50) = 4.58, P = 0.003, 7.3% total variation).In correlation to increased oxygen , but is rather controlled by food regime and species-food and species-temperature interactions (support of hypothesis ii). Furthermore, we discuss how the resource competition between Ae. albopictus and Cx. pipiens molestus is directly connected to species-specific foraging behaviour and physicochemical as well as phycological microhabitat parameters (support of hypothesis iii).In the present study, we show that the physicochemical and microclimatic conditions of potential and actual mosquito breeding sites in Germany offer suitable conditions for a balanced coexistence and frequent competitive superiority of Ae. albopictus and Cx. pipiens molestus had an almost identical population growth at 20 °C but a divergent one at a higher temperature  of female Additional file 3: Figure S3.Ae. albopictus and Cx. pipiens in pure and mixed cohorts in dependence of temperature and larval food regime. Line - linear regression. Dotted lines - 95% confidence interval. (PPTX 558 kb)Pupal weight. Dry weight  of female Additional file 4: Table S1.Ae. albopictus (RCCAe) and Cx. pipiens (RCCCx) based on biomass accumulation rate (BA), pupal dry weight (DW), pupal abdominal length (AL) and mean pupation time (PT50) are listed for the different experimental treatments. (DOCX 15 kb)Parameter-specific relative crowding coefficients. Relative crowding coefficient values for female and male


O-sorboyl-polydatin prodrug in biomass-derived 2-methyltetrahydrofuran (2-MeTHF) was achieved using Candida antarctica lipase B for the first time. Under the optimal conditions, the initial reaction rate, maximum substrate conversion, and 6′′-regioselectivity were as high as 8.65 mM/h, 100%, and 100%, respectively. Kinetic and operational stability investigations evidently demonstrated excellent enzyme compatibility of the 2-MeTHF compared to the traditional organic solvents. With respect to the antioxidant properties, three unsaturated ester derivatives showed slightly lower DPPH radical scavenging activities than the parent agent. Interestingly, further studies also revealed that the antiradical capacities of the acylates decreased with the elongation of the unsaturated aliphatic chain length from C4 to C11. The reason might be attributed to the increased steric hindrance derived from the acyl residues in derivatives.Efficient and highly regioselective synthesis of the potential 6′′- O-β-mono-D-glucoside) is the key pharmacodynamic constituent from Polygonum cuspidatum Sieb. et Zucc. which is an extensively used traditional Chinese medicinal plant in the clinical treatment of platelet aggregation, diabetes mellitus, ischemia–reperfusion injuries, and burn-generated cardiac dysfunction, and so forth [α- or β-unsaturated groups usually displayed the higher biological activity compared with the parent agents [Polydatin , a versatile enzyme immobilized on macroporous acrylic resin with a molecular weight of 33 kDa and approximate dimensions of 10 Å × 4 Å × 12 Å [Therefore, in this study, an effective enzymatic method in 2-MeTHF is explored for preparing potential dual prodrug of sorbic acid  ester of polydatin catalyzed byÅ × 12 Å , 18  was from Novozymes Co., Ltd., China. Vinyl sorbate, vinyl undecenoate, and vinyl crotonate were obtained from TCI. Polydatin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2-MeTHF, and t-amyl alcohol were purchased from Sigma-Aldrich. All other reagents were of analytical grade and were dried by 4 Å molecular sieves.CALB (μL) were withdrawn at specified time intervals from the reaction system, followed by a HPLC analysis. All experiments were conducted at least in triplicate and the errors did not exceed 5%. No chemical acylation was detectable as confirmed by the control experiments.The acylation was conducted with polydatin (0.03 mmol), vinyl sorbate (0.27 mmol), anhydrous solvent (3 mL), and 100 mg lipase while stirring at 200 rpm and at a certain temperature. To determine the concentrations of the polydatin and regioselectivities of the acylated products, aliquot fractions  containing 0.03 mmol polydatin, 0.45 mmol vinyl crotonate, and 100 mg CALB at 60°C and 200 rpm, followed by the assay of its activity and maximum polydatin conversion. The initial activity and maximum conversion obtained in the first batch were set to 100. The residual activity was defined as the ratio of the reused enzyme activity relative to the original enzyme activity in the same reaction system. t-butanol (2–18 mM). The reactions were carried out in 3 mL solvent containing different polydatin concentration, 9 equiv. of vinyl sorbate, and 100 mg CALB at 60°C and 200 rpm. The kinetic constants (Km and Vmax) were calculated from Hanes-Woolf plots.The kinetic constants of enzymatic sorboylation were investigated at different polydatin concentrations in 2-MeTHF (2–14 mM), THF (5–50 mM), acetone (4–16 mM), andEa) of enzymatic acylation in 2-MeTHF, THF, acetone, and t-butanol were investigated. Polydatin (0.03 mmol), CALB (100 mg), and vinyl sorbate (0.27 mmol) were added into the anhydrous solvent (3 mL) and incubated at different temperatures (in the range of 35–50°C) and 200 rpm. Ea value of enzymatic sorboylation was calculated according to the linear regression analysis of the Arrhenius plot.The apparent activation energy values (μm) Eclipse Plus-C18 column , a UV detector at 275 nm, and a solvent mixture system of water and methanol  at a flow rate of 1.0 mL/min. The retention times for polydatin and 6′′-ester were 2.59 and 5.27 min, respectively. The acylated derivative was purified by flash column chromatography using ethyl acetate (EA)/petroleum ether (PE) and its chemical structure was determined by 13C NMR (100 MHz) and 1H NMR (400 MHz)  in DMSO-d6. Qualitative analysis of the reaction mixtures was conducted by HPLC, using a 4.6 mm × 250 mm (5 6′′-O-Sorboyl-polydatin. 1H NMR (DMSO-d6) δ: 9.63 , 9.51 , 7.43 , 7.18–7.13 , 7.05 , 6.91 , 6.88 , 6.87 , 6.81 , 6.70 , 6.63 , 6.36 , 6.16–6.12 , 5.80 , 5.42 , 5.36 , 5.24 , 4.94 , 4.47 , 4.11 , 3.74–3.71 , 3.39–3.36 , 3.32–3.30 , 3.25–3.22 , 1.71 . 13C NMR (DMSO-d6) δ: 166.77 (C1′′′), 159.05 (C3), 158.86 (C5), 157.85 (C4′), 145.64 (C3′′′), 140.43 (C4′′′), 139.82 (C1), 129.86 (C2′ + C6′), 128.95 (C1′), 128.40 (Cvinyl-1), 125.76 (Cvinyl-2), 118.75 (C2′′′), 116.05 (C3′ + C5′), 107.62 (C6), 104.79 (C2), 103.37 (C1′′), 100.55 (C4), 76.89 (C2′′), 74.29 (C5′′), 73.62 (C3′′), 70.59 (C4′′), 64.17 (C6′′), 60.24 (C5′′′), 18.77 (C6′′′).μM DPPH. Each compound concentration was fixed at 0.1, 0.2, 0.4, 0.8, and 1.2 mg/mL, respectively. The absorbance at 517 nm was measured against a blank of pure ethanol in a light proof reaction vessel at room temperature for 30 min. DPPH scavenging capacity was estimated based on the difference in absorbance and expressed by the remaining radical DPPH percentage. Triplicate reactions were carried out for each antioxidant experiment and the results were based on the average values.The antioxidant activity of polydatin and its acylated derivatives was examined according to the previously reported procedure with a few modifications . BrieflyP, dielectric constant, viscosity, dissolving capacity, and molecular size unpredictably affect the behavior of the enzyme. Hence, eight organic solvents with different logP values (−1.10–1.43) were selected to further evaluate their effects on the CALB-mediated sorboylation of polydatin.The search for suitable solvent capable of maintaining enzyme activity, improving enzyme specificity, and accelerating the enzymatic reaction process has always been a central problem for biochemists , 21. So P, viscosity, and substrate solubility in this acylation. In those solvents such as THF, dioxane, cyclohexanone, and t-amyl alcohol, CALB displayed poor to good activities with 25.16–79.31% maximal conversions. By contrast, acetone and t-butanol seemed to be selectable media and gave 99.00% substrate conversions, but the initial rates were far from satisfactory (0.62–1.64 mM/h). However, when the reaction was carried out in ecofriendly 2-MeTHF, CALB showed highest reaction rate (3.04 mM/h) and excellent conversion (99.57%), indicating that the biomass-derived 2-MeTHF exhibited prominent biocompatibility with the lipase compared to the other conventional organic media. Furthermore, compared with the acetone and t-butanol, 2-MeTHF may be more beneficial to maintain enzyme activity owing to its more hydrophobic character [As shown in haracter .The influence of some crucial factors, such as the substrate molar ratio, temperature, and reaction time, was also investigated in detail. As can be seen in O-sorboyl-polydatin was followed under the optimal conditions for better understanding the enzymatic process. It was found in Gardossi et al. recently demonstrated that it is very necessary to monitor the procedure parameters  at a single timepoint . So, theThe successful incorporation of biocatalysts into fine chemicals production requires not only high catalytic activities but also excellent operational stabilities. The results for repeated uses of the enzyme in 2-MeTHF and THF are presented in Km value of the enzyme in 2-MeTHF (27.9 mM) is much lower than those attained in t-butanol, acetone, and THF . Simultaneously, the catalytic efficiency of CALB varied greatly, depending on the nature of media. For instance, CALB afforded the highest Vmax/Km value of 1.12 h−1 in 2-MeTHF system, which is 3.7- to 5.5-fold higher than that in other solvents studied. These promising observations indicated that the enzyme exerted an extremely higher affinity for the substrate and catalytic efficiency in this novel 2-MeTHF as compared to normal organic solvents.For better understanding the superiority of 2-MeTHF, measurement of kinetic constants of enzymatic sorboylation by using linear Hanes-Woolf plots was successfully explored. As illustrated in Ea) for the reaction is also determined by plotting the data in an Arrhenius plot  suggesting that the application of 2-MeTHF has proved to be more beneficial for overcoming the reaction energy barrier and rendering the process of enzymatic biotransformation.The apparent activation energy , apoptosis-inducing capability, and so forth of the acylated products are currently in process and will be reported in due course.In this paper, a practical enzymatic approach for acylating polydatin with vinyl sorbate in biomass-derived 2-MeTHF is described for the first time. CALB lipase was used successfully to acylate polydatin at the 6′′-position. Detailed investigations have revealed that the enzyme in 2-MeTHF gave the lowest The detailed informations  on the sorboyl-polydatin prodrug synthesized by Candida antarctica lipase B in biomass-derived 2-methyltetrahydrofuran are given in the supplementary material.


The MHC class I chain-related genes A and B (MICA and MICB), which are located on the human chromosome 6, encode proteins with 3 extracellular domains, a transmembrane segment, and a carboxy-terminal cytoplasmic tail. The extracellular domains consist of α1, α2, and α3 chains that flexibly function as a ligand for Natural killer group 2D (NKG2D), γδ T cells, and αβ CD8 T cells. MICA polymorphisms have been identified in over 100 alleles. Over 40 alleles have been sequenced for exons 2–6 of MICB . The role of MICA and MICB in human disease is mainly defined by its allele associations with cancer, autoimmune disorders, and success rates of hematopoietic stem cell transplantation (HSCT)5. In this study, we analyzed the possible association of MICA and MICB polymorphisms with ALL, AML, and MDS. Our results also reveal a correlation between amino acid variants in MICA external domains and ALL, AML, and MDS.Hematologic malignancies including acute lymphocytic (lymphoblastic) leukemia (ALL), acute myeloid leukemia (AML), and myelodysplastic syndrome (MDS) are clonal diseases, which commonly manifest as a result of dysregulation of hematopoiesis due to genetic damage6. The study was performed in accordance with the Declaration of Helsinki. The patients with ALL, AML, and MDS and 200 ethnically matched non-leukemic controls signed appropriate informed consent to participate. The use of the material was reviewed and approved by the local institutional review board (IRB) of The Catholic University of Korea, with written informed consent obtained for all samples collected (IRB Number: SCMC07BR131). DNA was isolated from peripheral blood. We applied sequence-based typing (PCR-SBT) as an authentication method to amplify the exons 2–5 of MICA7. Genotyping was performed by PCR-SBT4 for MICA-129 alleles (A to G) in exon 3 at position 454 of the MICA gene. MICB primers were designed based on exons 2–4 for genotyping of MICB-sequence alleles8. The χ2 test and Fisher’s exact test were used to determine statistical significance. P-values were corrected (Pc) using the Bonferroni’s method. The odds ratio (OR) was calculated using Haldane’s modification of Woolf’s method. Hardy-Weinberg equilibrium in controls was analyzed for each SNP using SNPStats on the website (http://bioinfo.iconcologia.net/snpstats/start.htm) (P > 0.05).We randomly selected a total of 324 patients diagnosed with ALL, AML, and MDS who were treated at the Department of Internal Medicine, Catholic Blood and Marrow Transplantation Center at Seoul St. Mary’s Hospital, between November 2010 and April 2013, and enrolled 324 patients with ALL, AML, and MDS  included 85 ALL , 172 AML , and 67 MDS . Diagnoses were based on the criteria specified by World Health OrganizationMICA*002:01 alleles and A9 alleles were higher than in controls . However, the A5.1  showed lower frequencies than in the controls. These significant associations were also found in the ALL, AML, and MDS groups. MICA*008:01 allele showed lower frequencies (for ALL (12 of 85 [14.1%] versus 74 of 200 [37.0%]): Pc = 0.002, OR = 0.3, 95% CI 0.1–0.6; for AML (72 of 172 [41.9%] versus 74 of 200 [37.0%]): Pc = 0.042, OR = 0.5, 95% CI 0.3–0.8; for MDS (11 of 67 [16.4%] versus 74 of 200 [37.0%]): Pc = 0.026, OR = 0.3, 95% CI 0.2–0.7). The frequencies of MICA-STR A9 alleles were higher in patients with ALL, AML, and MDS than in the controls (for ALL (34 of 85 [40.0%] versus 44 of 200 [22.0%]): Pc = 0.007, OR = 2.4, 95% CI 1.4–4.1; for AML (60 of 172 [34.9%] versus 44 of 200 [22.0%]): Pc = 0.023, OR = 1.9, 95% CI 1.2–3.0). MICA A5.1 alleles were less frequent than in the controls (for ALL (13 of 85 [15.3%] versus 76 of 200 [38.0%]): Pc < 0.001, OR = 0.3, 95% CI 0.2–0.6; for AML (42 of 172 [25.0%] versus 76 of 200 [38.0%]): Pc = 0.037, OR = 0.5, 95% CI 0.3–0.8; for MDS (12 of 67 [17.9%] versus 76 of 200 [38.0%]): Pc = 0.012, OR = 0.4, 95% CI 0.2–0.7). Interestingly, a methionine (ATG) to valine (GTG) change at position 129 of the α2-heavy chain domain categorized the MICA alleles into strong (MICA-129 met) and weak  binders of the NKG2D receptor5. We found significant data for the MICA external domain, especially that of the protein encoded by MICA-129. There are non-synonymous polymorphic sites covering exons 2–4, that encode non-conservative amino acid substitutions in MICA external domains2. Codon 14 of MICA protein including MICA*002:01 showed strong odds ratios (1.8 ~ 2.7) and corrected P-value (Pc < 0.048). The frequency of the MICB*005:03 of 10 MICB sequence alleles was lower in patients than in the controls : Pc = 0.002, OR = 0.2, 95% CI 0.1–0.5; for AML (5 of 172 [2.9%] versus 21 of 200 [10.5%]): Pc = 0.024, OR = 0.3, 95% CI 0.1–0.7) , regardless of the history of acute GVHD, the presence of MICA Abs before transplantation confers protection against cGVHD (P = 0.04)4. There is an inverse relationship between UC or cGVHD and total patients including ALL, suggesting that these genetic characteristics of MICA influence disease incidence.Although the MICA*002:01, MICA*008:01, and MICB*005:03 alleles associated with ALL, AML, and MDS using previously developed microarrays12, it will be possible to predict within more short time whether they are at risk or not. Furthermore, the investigation of MIC alleles in individual tumor samples, at the level of protein expression, should be of further interest. Our results illustrate that MICA-NKG2D played a role in disease pathogenesis in the majority of patients in our cohort of cases of ALL, AML, and MDS, and further investigation into this signaling axis may reveal potent therapeutic targets.With the typing of


Dear Editor,9/L for neutrophilic granulocytes. Azathioprine and prednisolone were stopped immediately, while human granulocyte colony-stimulating factor and recombinant human thrombopoietin were prescribed to increase blood cells. However, the patient still suffered a life-threatening progressive decrease of blood cells. The lowest level of neutrophilic granulocytes, reticulocytes, and white blood cells reached 0.01 × 109/L, 0.0040 × 1012/L, 0.25 × 109/L, respectively, on Sept 23rd, while the hemoglobin, platelets, and red blood cells dropped to the lowest level at 54 g/L, 22*10^9/L, and 1.66*10^12/L, respectively, on Oct 1st  and TPMT (rs1142345) of this patient and found that this patient has NUDT15risk/riskTPMTwt /risk genotype . Therefore, concurrence of rs1142345 and rs116855232 risk allele (at least homozygous for one SNP) is pretty rare , Hispanics (4.8%), and Africans (5.4%) but low in East Asians (1.3%), while that of rs116855232 is high in East Asians (10.4%) and Hispanics (7.1%) but rare in Caucasians (0.46%) and Africans (0.07%) (


Bacillus subtilis. After several rounds of mutagenesis and screening, two mutants 6E9 and 8E279 were obtained which exhibited 2.99 and 3.43 times higher catalytic efficiency, respectively. They also exhibited lower optimal reaction temperature and higher thermo-stability. D44V, Q268R and K285Q were identified as the three most beneficial amino acid substitutions introduced by site-directed mutagenesis. D44V/Q268R, which was obtained through random combination of the three mutants, displayed the highest catalytic activity. The Km,kcat/Km and enzyme activity of D44V/Q268R increased by 68%, 83% and 129% respectively, compared with that of wild-type urate oxidase. Structural modeling indicated that mutations far from the active site can have significant effects on activity. For many of them, the underlying mechanisms are still difficult to explain from the static structural model. We also compared the effects of the same set of single point mutations on the wild type and on the final mutant. The results indicate strong effects of epistasis, which may imply that the mutations affect catalysis through influences on protein dynamics besides equilibrium structures.Urate oxidase is a key enzyme in purine metabolism and catalyzes the oxidation of uric acid to allantoin. It is used to treat hyperuricemia and gout, and also in a diagnostic kit. In this study, error-prone polymerase chain reaction and staggered extension process was used to generate a mutant urate oxidase with improved enzyme activity from  Urate oxidase, which is also known as uricase, is an important enzyme in purine degradation. It catalyzes uric acid degradation and produces allantoin , which iUric acid exists in the human body mainly as free acid and urate salt, both of which are insoluble in water. The accumulation of uric acid in the body can result in hyperuricemia. In some cases, its crystallization and precipitation lead to gout . During Allopurinol is a drug used to treat gout. It inhibits xanthine oxidase and blocks the formation of uric acid. However, it increases the precursors of uric acid, hypoxanthine and xanthine, which increase the burden on the kidneys. Direct injection of urate oxidase can rapidly degrade uric acid without the accumulation of any additional intermediate products . Urate oAspergillus flavus  was used to cultivate E. coli. L-arabinose (0.2%) was added to the medium for inducing expression.B. subtilis ATCC 23857 was available in our lab. PCR amplification was performed using pBAD upstream primer and pBAD downstream primer (Nco I and Hind III sites of pBAD vector and transformed into E. coli BL21 (DE3) cells. The constructed pBAD vector containing urate oxidase gene was designated pBAD-UO.Gene of urate oxidase from m primer . The ampTaq DNA polymerase. The reaction mixture contained 0.2 mM each of dATP and dGTP, 1.0 mM each of dCTP and dTTP, 7 mM MgCl2 and 0.15 mM MnCl2. The plasmid pBAD-UO was used as template for the first round. The program was 30 cycles of 30 s at 94°C, 30 s at 50°C and 1 min at 72°C. The PCR product was digested with Nco I/ Hind III and ligated into plasmid pBAD/myc-His-A. The ligation products were transformed into E. coli BL21 (DE3), and around 6000 transformants in each cycle were recovered. All clones from the library were subjected to downstream screening assay. The best mutant was picked as the template for the next round screening (Random mutagenesis library was generated by error-prone PCR (EP-PCR) . The uracreening .Taq buffer, 25 mM Mg2+ and 0.5 U Taq polymerase. The program was 5 min at 94°C, 80 cycles of 30 s at 94°C and 5 s at 55°C. The resultant 1 kb DNA fragment was digested with Nco I/ Hind III and ligated into plasmid pBAD/myc-His-A. The ligation products were transformed into E. coli BL21 (DE3).Staggered extension process (StEP) was performed according to the previously described method . The mutg, 10 min, 4°C). The cells were subjected to three cycles of freezing at -80°C and thawing and then were resuspended in 300 μl lysis buffer , and incubated at 37°C for 20 min. After centrifugation , the cell debris was removed and the supernatant was used for subsequent enzyme activity assay.Single clones of strain BL21 (DE3) from the urate oxidase random mutagenesis library, harboring mutation were cultivated in 0.2ml LB medium supplemented with 100 μg/ml ampicillin in 96-well deep plates . The pla4. The absorbance at 293 nm was measured for 5 min at 30°C. Mutants with improved activity were selected for further analysis.During the screening process, urate oxidase activity was measured using 96-well plate. Eighty microliters of the crude enzyme of urate oxidase mutant was added into 120 μl of uric acid solution. Uric acid has strong absorption at 293 nm and the molar absorption coefficient is 1.15×10Prime STAR DNA polymerase, and 1μl of the primer pairs in a final volume of 50 μl. PCR products were digested with Dpn I at 37°C for 8 h and then transformed into competent E. coli BL21 (DE3) cells. The mutations were confirmed by sequencing.Site-directed mutagenesis was performed using a PCR-based method . PrimersE. coli BL21 (DE3) cells. A single clone was inoculated into 10 ml LB in a tube and cultured overnight at 37°C with shaking at 250 rpm. The culture was then transferred into a 250 ml conical flask containing 100 ml fresh LB medium containing 100 μg/ml ampicillin and induced with 0.2% L-ara. After 4 h of induction at 37°C, with shaking at 250 rpm, cells were harvested by centrifugation  and then disrupted by ultrasonication in 50 mM Tris-Cl buffer (pH 8.5). The urate oxidase enzyme with 6×His tag was purified by a HisTrapTM HP with AKTA purifier system . Purified proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein concentration was determined using the Bradford assay with bovine serum albumin as a standard [BSUO and mutants were expressed with a 6×His tag at the carboxyl terminus in standard .Urate oxidase enzyme activity was assayed in a 4 ml reaction system. Purified enzyme (400 μl) was added to reaction mixture consisting of 600 μl 0.4 mM uric acid (pH 8.5) and 3 ml 50 mM Tris-Cl (pH 8.5). The decrease of uric acid was determined by measuring the absorbance at 293 nm for 7 min at 28°C. One unit of enzyme activity was defined as the amount of enzyme necessary to transform 1μmol of uric acid into allantoin in 1 min at 30°C and pH 8.5.Km for the decomposition of uric acid, the enzyme activity was determined in a reaction mixture containing 50 mM Tris-Cl (pH 8.5), 400 μl purified enzyme, and a variable concentration  of uric acid at 28°C. Nonlinear regression was used for fitting the data to the Michaelis-Menten equation for determining the kinetic constants [To determine the apparent To determine optimal reaction temperature, enzyme activity was measured at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C and 50°C. The thermo-stability of the enzyme was evaluated by incubating the enzyme at 0°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C and 50°C for 15 min and assaying the residual activity under standard assay conditions [The optimal pH of the enzyme was determined by measuring relative activity at various pH values . The concentration of each buffer was 50 mM. The pH stability was also determined by incubating the enzymes at specified pH values at 4°C for 18 h. The residual activities were calculated based on the urate oxidase of non-incubated enzyme [Bacillus sp. TB-90 urate oxidase (PDB: 5ayj) which has been determined at 2.05 Å resolution without ligand was used as the modeling template. It has the highest sequence identity to BSUO (66.78%), so the resultant modeling structure was credible. PyMol was used to analyze the structure of the mutants. The structures of BSUO and all the mutants were constructed using these tools. The structure of the mutant enzyme was compared with that of wild type urate oxidase.Swiss-Model was used to construct homology models. The E. coli BL21 (DE3) cells. BSUO was purified by Ni-chelating affinity chromatography and analyzed by denaturing gel electrophoresis. The sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band of ~38 kDa which is in agreement with the molecular weight calculated from the amino acid sequence . After two rounds of EP-PCR, we obtained four mutants with similar activities and named them 2B2, 2B5, 2C2 and 2C3. Directed evolution was achieved through two different routes. In the first route, the four mutants were combined through staggered extension process and 3D1 was isolated through screening . We thenTo investigate the effects of temperature and pH on the catalytic efficiency, purified wild type BSUO and mutants 6E9 and 8E279 were characterized. The optimum temperature was found to be 40°C for wild type BSUO and 35°C for the mutants. The activity of 6E9 and 8E279 decreased to 81% and 84% respectively at 40°C . HoweverThe mutants had a different optimum pH compared to that of wild type BSUO. The highest activity for wild type BSUO was observed at pH 10.0, decreased to 62% at pH 8.0 and to 80% at pH 9.0. 6E9 and 8E279 exhibited highest activity at pH 9.0, which decreased to 85 to 95% at pH 8.0 and to 96 to 97% at pH 10.0. The pH stability of the wild type BSUO was same as the mutants. Enzymes were more stable in alkaline conditions .Site-directed mutagenesis was performed to understand the effect of the various mutations in 6E9 and 8E279. Seventeen single point mutations of BSUO, T4I, T4N, D44V, D44N, L100F, K127E, E128G, R137G, F182Y, S217T, D264V, Q268R, E279K, K285Q, K285I, H300R, and C319F were constructed. The mutants D44V, S217T, Q268R, K285I, K285Q, H300R and C319F showed higher activity demonstrating an improvement of 1.53, 1.32, 1.66, 1.51, 1.60, 1.45 and 1.39 times respectively compared to that of wild type BSUO . The actIn order to further understand the effect of single mutation on catalytic efficiency, we used 8E279 as template to do reverse mutagenesis. Mutations T4I, F182Y, K127E, D44V and K285Q which have different effect on catalytic efficiency were chosen . Fig 5B Km of 6E9 and 8E279 increased by 20% and 50% respectively. The kcat of 6E9 and 8E279 were 2 and 3.68 times higher than that of wild type BSUO. And the kcat/Km of 6E9 and 8E279 were 2.42 and 3.09 times greater than that of wild type BSUO . Therefore, it is helpful in examining the structure-function relationship.It is very important to choose the starting material for directed evolution. For expressing the recombinant enzyme in  enzymes . For clinditions . At the nditions , 26. BSUnditions . The theDirected evolution can improve the properties of an enzyme including catalytic activity and stability. The incomplete understanding of the catalytic mechanism of BSUO makes rational design difficult. Besides, in rational design, mutations are generally restricted to the amino acids around active site, ignoring other potential beneficial substitutions which are not in the active site. In this study, we conducted directed evolution of BSUO. After several rounds of random mutagenesis and screening, the two best mutants 6E9 and 8E279 were obtained. There were 17 amino acid substitutions in these mutants. Using site-directed mutagenesis, we constructed 17 mutants with single amino acid substitution. The activity and kinetic parameter of these mutants were measured. Among these mutants, D44V, Q268R and K285Q were the best three single point mutants.kcat/Km of them is 1.85, 1.62 and 1.42 folds higher of wild type BSUO  M: Protein marker. Lane 1: Crude BSUO. Lane 2: Purified BSUO. Lanes 3–15: purified enzyme of mutants T4I, T4N, D44V, D44N, L100F, K127E, E128G, R137G, F182Y, S217T, D264V, Q268R. (B) M: Protein maker. Lanes 1–9: purified enzyme of mutants E279K, K285Q, K285I, H300R, C319F, DK, DQ, QK, DQK.(EPS)Click here for additional data file.


The difluorophenyl ring is disordered over two orientations; the final refined occupancy factors of the two components of disorder are 0.947 (4) and 0.053 (4). In the crystal, some Car—H⋯F interactions are present, which involve the most acidic H atom of the molecule.Molecules of the title compound, C In addition to their applications in drugs, bioactive and tautomeric compounds benzene-1,2:4,5-tetracarboximide, which is a fluorinated derivative of the simplest aromatic bis(imide).Following these issues, we report here the structural investigation of the title compound, Ci point-group symmetry. Thus, the asymmetric unit is formed by half a molecule, as shown in Fig. 1ortho-F atom, for which split positions were observed. The final refined occupancy factors of the two components of disorder are 0.947 (4) and 0.053 (4). Molecules adopt a nonplanar conformation, mainly because of a torsion around the O3—N1 bond. In particular, the pentaatomic ring (atoms C2/C3/C4/N1/C5) is planar within 0.0164 (14) Å, while the phenyl ring (C7/C8a/C9/C10/C11/C12a) is planar within 0.002 (2) Å. The dihedral angle between the mean planes of the two rings is 86.14 (8)°.Molecules of the title compound in the crystal lie on crystallographic inversion centres and have et al., 1987The C6—O3 bond length [1.402 (3) Å] is significantly longer than the mean value for esters of aromatic acids  and strong hydrogen-bond acceptors (carbonyl O atoms) in the title compound. Moreover, F atoms are present as well, whose low hydrogen-bonding-acceptor capability, if any, has been the subject of a long debate in the literature . 47 hits were found. Within this set, 40 hits are N-oxycarbonyl derivatives of succinimide and 6 hits are derivatives of phthalimide. Here follows the full list of refcodes for the CSD search: ADEFUL, AFUXEE, ALOPAU, AWUXOF, BANTOA, CILBUV, COZFOM, DOFTEZ, EABXIO, EABXIO01, FUPPEM, GULRAI, GUVCAB, ICEWIY, LOZFAH, MAMDOU, MILFOE, MIPHUP, MIZJEM, MOZYOQ, NANWUU, OQUPOG, PIGKIZ, SEZWIE, SOSDEK, SUDJIM, SUDWUL, SUDXAS, SUDXAS01, TEQDEY, TUJRIB, UJAFER, UJUBOS, UJUBUY, VALSUZ, WALPEH, WIDKEB, YAFMEY, YAFPOL, YAGBEP, YUJZIN, YUJZOT, ZEPSES, ZEPSIW, ZOQQOL, ZOQQUR, ZALKUV.A search of the Cambridge Structural Database  ranges between 1.375 and 1.408 Å, with an average value of 1.388 (6) Å. On the other hand, the distance O3—C6 (DIST2) is between 1.350 and 1.423 Å, with an average value of 1.393 (15) Å. The histogram of the distribution of DIST1 over the 47 hits found is shown in Fig. 3N,N′-Dihydroxybenzene-1,2:4,5-tetracarboximide  = 1.2Ueq(carrier). The difluorophenyl ring is disordered over two orientations, which differ by a rotation of 180° around the phenyl to carbonyl bond. Split positions were only observed for the ortho-F atom. The two split positions were refined by applying SADI restraints on bond lengths. The final refined occupancy factors of the two components of disorder are 0.947 (4) and 0.053 (4).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018001226/fy2124sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989018001226/fy2124Isup2.hklStructure factors: contains datablock(s) I. DOI: 1818213CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A nonfullerene acceptor based on a 3D tetraperylene diimide is developed for bulk heterojunction organic photovoltaics. The disruption of perylene diimide planarity with a 3D framework suppresses the self‐aggregation of perylene diimide and inhibits excimer formation. From planar monoperylene diimide to 3D tetraperylene diimide, a significant improvement of power conversion efficiency from 0.63% to 3.54% can be achieved. Organic photovoltaics (OPVs) made from electron donor–acceptor (D–A) bulk heterojunction (BHJ) based devices have attracted extensive attention due to their merits of light‐weight, low‐cost, flexibility, and solution processability. Fullerene derivatives with unique phase and multidimensional charge‐transporting capabilities have played a central role as electron acceptor in BHJ devices leading to ≈11% power conversion efficiency (PCE).FigureIn this work, we report a synthetic strategy to effectively transform planar subunits into a 3D nonfullerene acceptor with a compact tetrahedron core, of which four subunits are interlocked to reinforce structural rigidity of the molecule. The resulting molecule shows interesting correlations between its molecular dimensionality, material properties, and device performance. By coupling tetraphenyl‐silane with four planar PDIs to build a 3D nonfullerene acceptor  calculations were carried out initially to study the molecular structure of 3D tetra‐PDI Figure a,d and p60 with a diameter of 7.1 Å, Figure Tetra‐PDI gathers four PDI units into a crowded space  that will benefit the energy transfer and electron communication among PDI units. Actually, our initial attempt to install bulky PDI units onto tetra‐Ph‐methane failed due to the smaller core size of tetra‐Ph‐methane (C−C bond length ≈ 1.5 Å) compared to tetra‐Ph‐silane (C−Si bond length ≈ 1.9 Å) . The four PDI π‐planes (with a length of 14.3 Å and a width of 4.8 Å) in tetra‐PDI expose to the outside sphere of tetra‐Ph‐silane. DFT calculations indicate that tetra‐PDI can be seen as a molecule mimics fullerene derivatives in terms of π‐surface exposure and electron affinity ‐1‐bromo‐3,4:9,10‐perylene diimide (1) and tetrakisphenyl)silane (2) and phenylboronic acid, respectively Cl2 is found to have a much higher catalytic activity than the traditionally used Pd(PPh3)4, and tetrahydrofuran (THF) is a better solvent than toluene for the above Suzuki coupling reactions, especially for synthesizing highly steric‐hindered tetra‐PDI. Both tetra‐PDI and mono‐PDI are readily soluble in common organic solvents such as dichloromethane, THF and dichlorobenzene at room temperature due to the presence of solubilizing N‐substituted alkyl chains. The target molecules were fully characterized by 1H and 13C NMR, matrix‐assisted laser desorption ionization time of flight (MALDI–TOF) mass spectroscopy (MS). Interestingly, the results from 1H and 13C NMR spectra indicate that the chemical environments of the two N‐alkyl chains on tetra‐PDI are very different from those of the mono‐PDI subunits and previously reported bay‐substituted PDIs.[[qv: 5b–g]],Figures2– groups on mono‐PDI exhibit a set of dd peaks around 4.08 ppm (1H NMR), while the chemical shifts of the –NCH2– groups on tetra‐PDI are equally divided into two sections, one at 4.14 ppm and the other at 3.55 ppm. 1H NMR signals of the –(CH2)n– and –CH3 groups on tetra‐PDI also exhibit substantial splits. Splits were also abserved from the 13C NMR signals of tetra‐PDI Clly Scheme1. The Aside from the molecular structure study, to gain information of intermolecular interactions and crystallinity for both compounds, differential scanning calorimetry (DSC) was used to investigate their thermal behaviors. As shown by Figure 2Cl2 solution (0.8 ×10 −7m), as well as in thin solid films. Tetra‐PDI and mono‐PDI in CH2Cl2 solution exhibit absorption peaks at 539 and 536 nm, respectively, with the maximum extinction coefficients of 1.4 × 105 and 9 × 104m−1 cm−1, accordingly. Compared with the absorption in solution, the thin film of tetra‐PDI shows a similar profile with a very small redshift of absorption peak from 539 to 541 nm. However, mono‐PDI shows absorption peaks at 536 nm for solution while 563 nm for film. The large redshift of film absorption (27 nm) indicates that mono‐PDI has a strong tendency to aggregate. The UV–vis results of tetra‐PDI and mono‐PDI support the observations from DSC. Time‐resolved fluorescence spectroscopy was also applied to test the photoluminescence (PL) lifetime of tetra‐PDI and mono‐PDI in both solutions and films . It was found that tetra‐PDI had a faster PL decay (t = 6.38 ns in solution and 10.96 ns in film) than mono‐PDI (t = 8.60 ns in solution and 15.07 ns in film). The shorter PL lifetime of tetra‐PDI may ascribe to the faster intramolecular excitation energy transfer between PDI units of 3D tetra‐PDI, which will favor charge separation and transport in BHJ.Optical and electronchemical properties were subsequently investigated. Figure 2Cl2 solution. Both PDIs undergo two quasi‐reversible reduction events and neither shows any oxidative activity in the scan range  in CHFigure3/Ag  were fabricated with ortho‐dichorobenzene (DCB) as the processing solvent to investigate the performance of solution‐processed BHJ OPVs.[[qv: 5a]] After optimizing the blend ratio of the active layer to be 1:1.2 between PBDTT‐F‐TT and tetra‐PDI, the device showed a PCE of 3.21%  of 7.66 mA cm−2, and a fill factor (FF) of 0.48) oxy]benzodithiophene‐2,6‐diyl}{3‐fluoro‐2‐[(2‐ethylhexyl)‐carbonyl]‐thienothiophenediyl})  was chosen as the donor, which has complementary absorption spectrum and matched frontier energy levels  with tetra‐PDI. As shown in JSC = 2.18 mA cm−2, a VOC = 0.86 V, and a FF = 34%. Although the VOC of mono‐PDI solar cell is similar to that of tetra‐PDI, as expected from their similar LUMO energy levels, the photocurrent and PCE of tetra‐PDI‐based devices are almost five times higher than that of mono‐PDI. The rigid 3D architecture of tetra‐PDI lead to more efficient charge separation and transport, which increase the JSC and FF. Processing additives such as 1,8‐diiodooctane (DIO) and 1‐chloronaphthalene (CN) have been widely used for nonfullerene‐based OPVs to improve PCEs.[[qv: 4c–f]],[[qv: 5a–h]],VOC of 0.86 V, JSC of 8.39 mA cm−2 and FF of 49%)  of both the PBDTT‐F‐TT and tetra‐PDI emission, which suggests efficient charge transfer driven by both PBDTT‐F‐TT and tetra‐PDI Figure c. AlthouFigure−3 cm2 V−1 s−1) than that of mono‐PDI pristine film (1.2 × 10−4 cm2 V−1 s−1). This suggests the introduction of Si‐cored 3D framework improves charge‐transport, which in turn, improves the performance of tetra‐PDI based devices. We noted that previous mono‐PDIs with bay‐substitution  exhibited very low electron mobility, ranging from ≈10−6 to 10−4 cm2 V−1 s−1.JSC and PCE .Atomic force microscopy (AFM) was also employed to investigate the surface morphology of the active layers. AFM scans were carried out for 1:1.2 ratio blend films spin‐coated on ITO substrates using the same fabrication conditions for devices. 3 hybrid silane framework. The Si‐cored tetra‐PDI is small and compact to constrain the free rotation of PDI‐subunits. As shown by NMR, DSC, UV–vis, and PL lifetime analyses, this 3D yet interlocked structure can suppress the commonly observed aggregation in planar PDI to facilitate charge transport and energy transfer among PDI subunits. From planar mono‐PDI to 3D tetra‐PDI, improved performance could be achieved for a proof‐of‐concept device without using any posttreatments or additives. This represents an important development for solution‐processed BHJ OPVs, and demonstrates a new strategy for designing 3D electron‐accepting materials with favorable properties for OPV applications.In summary, we have developed a novel PDI‐based 3D nonfullerene electron acceptor with an interlocked architecture, which originates from the sp1 and 2Synthesis of : Details for the synthesis of intermediates N,N′‐di(2‐hexyldecyl)‐1‐bromo‐3,4:9,10‐perylene diimide (1) and tetrakisphenyl)silane (2) can be found in Scheme S1 and S2, Supporting Information.Synthesis of Tetra‐PDI: Compound 1 , compound 2 , THF (6 mL), and K3PO4 aqueous solution  were mixed together in a two‐necked round bottom flask. The mixture was degassed by argon, then 10 mg Pd(dppf)Cl2·CH2Cl2 (0.0125 mmol) was added. The mixture was stirred and refluxed under argon at 80 °C for 24 h. After cooling to room temperature, the mixture was poured into 100 mL NaCl aqueous solution and extracted with CH2Cl2 several times. The organic layers were combined, dried over MgSO4 and filtered. Removal of solvent by rotary evaporator afforded the crude product, which was then purified by silica gel column chromatography using CH2Cl2 as eluent. Red powder was obtained . 1H NMR : 8.83–8.50 , 8.08 , 7.92 , 7.60 , 4.14 , 3.55 , 2.21 – 0.41 ; 13C NMR : 163.82, 163.70, 163.18, 162.91, 144.75, 141.49, 138.59, 135.60, 134.92, 134.52, 134.39, 132.98, 131.52, 131.05, 129.78, 128.60, 128.56, 128.04, 127.27, 123.30, 123.41, 122.82, 122.70, 122.41, 121.59, 44.74, 44.45, 36.70, 36.14, 31.87, 31.80, 31.72, 31.53, 31.50, 30.06, 29.83, 29.73, 29.59, 29.47, 29.29, 29.24, 26.49, 26.29, 26.26, 22.64, 22.55, 14.08, 14.01; MALDI–TOF MS m/z: [M + Na]+ calcd for C248H308N8O16Si, 3685.32; found, 3687.97.Synthesis of Mono‐PDI: The procedure for the synthesis of mono‐PDI was similar to tetra‐PDI, using THF as solvent, K3PO4 as base, and Pd(dppf)Cl2·CH2Cl2 as catalyst for the Suzuki coupling of compound 1 (110 mg) with phenylboronic acid (15.9 mg). The target mono‐PDI was obtained . 1H NMR : 8.58–8.31 , 7.99 , 7.65 , 7.57–7.38 , 4.08 , 1.95 , 1.28 , 0.85 ; 13C NMR : 163.83, 163.68, 163.53, 142.46, 141.69, 136.03, 134.71, 134.61, 134.31, 132.42, 130.98, 130.82, 130.38, 130.09, 130.02, 128.84, 128.67, 128.41, 128.21, 127.91, 127.37, 123.42, 123.13, 123.03, 122.58, 122.59, 122.17, 44.69, 44.61, 36.66, 36.62, 31.89, 31.87, 31.75, 31.71, 31.66, 30.05, 29.74, 29.59, 29.31, 26.50, 22.66, 14.11; MALDI–TOF MS m/z: [M + Na]+ calcd for C62H78N2O4, 915.32; found, 915.89.Device Fabrication: ITO‐coated glass substrates (15 Ω sq−1) were cleaned ultrasonically with detergent, deionized water, acetone, and isopropyl alcohol in sequence for 10 min each. Subsequently, they were treated with oxygen plasma for 1 min. A ZnO precursor solution was spin‐coated onto precleaned ITO glass substrates at 4000 rpm for 60 s and then annealed at 200 °C for 1 h in air to complete the thin layer of ZnO (≈30 nm). The ZnO precursor solution was prepared by dissolving zinc acetate dehydrate C4H6O4Zn·2(H2O)  and monoethanolamine  in 2‐methoxyethanol  under stirring for 8 h for hydrolysis reaction and aging. The active layers (≈85 nm) were obtained by spin‐coating the solution of PBDTT‐F‐TT:PDI acceptors (tetra‐PDI or mono‐PDI) that was filtered with a polytetrafluoroethylene filter (0.45 μm) atop the thin layer of ZnO. The D–A blends were dissolved in DCB with or without 3% DIO or CN and stirred overnight in a nitrogen‐filled glove box. The anode of each device, MoO3/Ag (8/150 nm), was thermally evaporated at a pressure of about 10−7 torr. The area of each device was 3.14 mm2 defined by a shadow mask.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


HE3286 increased brain-derived neurotrophic factor (BDNF) in the optic nerve head and retina, while decreasing inflammatory and pathogenic proteins associated with elevated IOP compared to vehicle treatment. Treatment with HE3286 also increased nuclear localization of the transcription factor NFκB in collicular and retinal neurons, but decreased NFκB in glial nuclei in the optic nerve head. Thus, HE3286 may have a neuroprotective influence in glaucoma, as well as other chronic neurodegenerations.Glaucoma is a group of optic neuropathies associated with aging and sensitivity to intraocular pressure (IOP). The disease is the leading cause of irreversible blindness worldwide. Early progression in glaucoma involves dysfunction of retinal ganglion cell (RGC) axons, which comprise the optic nerve. Deficits in anterograde transport along RGC axons to central visual structures precede outright degeneration, and preventing these deficits is efficacious at abating subsequent progression. HE3286 is a synthetic sterol derivative that has shown therapeutic promise in models of inflammatory disease and neurodegenerative disease. We examined the efficacy of HE3286 oral delivery in preventing loss of anterograde transport in an inducible model of glaucoma (microbead occlusion). Adult rats received HE3286 (20 or 100 mg/kg) or vehicle daily via oral gavage for 4 weeks. Microbead occlusion elevated IOP ~30% in all treatment groups, and elevation was not affected by HE3286 treatment. In the vehicle group, elevated IOP reduced anterograde axonal transport to the superior colliculus, the most distal site in the optic projection, by 43% ( Glaucoma is the leading cause of irreversible blindness worldwide  is a synthetic derivative of androst-5-ene-3β,7β,17β-triol (AET), a non-glucocorticoid anti-inflammatory metabolite of the adrenal steroid, dehydroepiandrosterone  were randomly assigned to one of three treatment groups: vehicle, 20 mg/kg HE3286 or 100 mg/kg HE3286. We measured IOP bilaterally in awake rats using a TonoPen XL rebound tonometer  as previously described  were obtained from Charles River Laboratories  and maintained in a 12-h light-dark cycle with standard rodent chow available Forty-eight hours prior to perfusion, rats were anesthetized with 2.5% isoflurane and injected intravitreally with 2 μl of 0.5 mg cholera toxin subunit B (CTB) conjugated to Alexa Fluor-488  as previously described . Immunohistochemistry of whole eyes and brain was performed as previously described and at identical conditions between cohorts . Comparisons between multiple groups were made using one way analysis of variance (ANOVA) followed by the Holm-Sidak Pairwise Multiple Comparison test . Comparisons of a sample mean to a hypothesized or predicted value were made using a one-sided t-test. Actual p-values of significance are indicated where appropriate in the results section or figure legends. All comparisons for which significance is reported achieved or exceeded a post-hoc calculation of power of 0.80.All data are expressed as mean ± standard error unless indicated otherwise. The number of samples used in each experiment is provided in the appropriate methods description or figure legend. Statistical comparisons between two independent measurements were made using two-sided p = 0.523). Weights on day 28 (last day of treatment) ranged from 290 to 440 g, with no significant difference in mean weight between the three treatment groups (p = 0.130). However, rats that received vehicle gained 14.3 g during the experimental period, while rats that received 20 mg/kg HE3286 lost 21.2 g and rats that received 100 mg/kg HE3286 lost 38.0 g. Comparing percent change in weight over the course of treatment for each animal showed that HE3286 decreased weight in both the 20 mg/kg group  and 100 mg/kg group .Two rats, one from each HE3286 group, were euthanized before completion of the study due to complications from oral gavage. Treatment with HE3286 for 28 days had few directly observable adverse effects, as rats were active, responsive and showed no overt signs of distress. HE3286 did result in weight loss in both the 20 and 100 mg/kg groups (Table p = 0.638). Microbead injection elevated IOP in all eyes for the full 28 day period regardless of treatment  and for the 100 mg/kg HE3286-treated group , respectively. Mean IOP of saline eyes did not significantly differ for any treatment group (p = 0.997); the same was true for microbead eyes (p = 0.997).Baseline IOP measurements ranged from 19.15 ± 0.50 to 20.29 ± 0.55 mm Hg and were similar for all treatment groups . Elevated IOP due to microbead injection decreased intact transport to the SC in vehicle-treated rats to 57.4 ± 6.0% intact transport . Treatment with HE3286 attenuated transport deficits dramatically, as intact transport to the SC from microbead-injected eyes was 71.9 ± 9.8 and 83.0 ± 7.1% for the 20 and 100 mg/kg HE3286 treatment groups, respectively . Compared to SC from microbead-injected eyes in the vehicle group, the improvement was significant for the 100 mg/kg HE3286 cohort (p = 0.025). No difference in SC volume was detected for any groups or any treatments (p = 0.082).Deficits in anterograde axonal transport from retina to SC are an early sign of pathogenesis in rodent models of glaucoma, including microbead occlusion . This ratio was diminished by 70% compared to vehicle in the 20 mg/kg HE3286 group  and by 43% in the 100 mg/kg group . In the ONH . The 34% increase in this ratio for the 100 mg/kg dose was not significant compared to vehicle . Finally, in the retina, treatment with HE3286 increased the microbead:saline ratio of BDNF by 282% in the 20 mg/kg group (0.64 ± 0.05) and by 501% in the 100 mg/kg group (1.00 ± 0.05) when compared to vehicle ; the 100 mg/kg HE3286 ratio was significantly below an expected ratio of unity (p = 0.02). Thus, as microbead-induced elevations in IOP degrade anterograde transport in the optic projection, HE3286 treatment generally has the effect of opposing the change in BDNF levels induced by elevated IOP.When quantified Figure , the ratH Figure , middle,2) and microbead (7.017 ± 0.003 mm2) eyes or vehicle (7.019 ± 0.002 mm2) and HE3286 groups (7.012 ± 0.005 mm2) did not vary (p ≥ 0.088). Similarly, total area quantified in the nerve head did not vary between saline (7.019 ± 0.002 mm2) and microbead (7.022 ± 0.002 mm2) eyes or between vehicle (7.018 ± 0.003 mm2) and HE3286 groups . Finally, total area quantified in the retina was similar between saline (2.756 ± 0.203 mm2) and microbead (2.712 ± 0.198 mm2) eyes or between vehicle (2.996 ± 0.204 mm2) and HE3286  groups.Note that for BDNF and all other immunolabeled proteins we quantified, the total SC area for saline (7.013 ± 0.006 mmp = 0.037). The 100 mg/kg dose had a similar influence . The microbead:saline ratio of Iba1 in ONH for vehicle rats was significantly greater than an expected ratio of unity: 1.56 ± 0.12 (p = 0.045). This ratio was diminished by 83% in the 20 mg/kg HE3286 group ; 100 mg/kg HE3286 had far less effect . For vehicle retina, the Iba1 microbead to saline ratio was significantly more than 1 . Treatment with both 20 mg/kg and 100 mg/kg HE3286 decreased this ratio compared to vehicle: 1.09 ± 0.08 for the 20 mg/kg group (p = 0.018) and 0.99 ± 0.07 (p = 0.009) for the 100 mg/kg group.Given HE3286's proposed anti-inflammatory action and the contribution of microglia as neuroinflammatory mediators in glaucoma, we examined how treatment influences levels of ionized calcium-binding adapter molecule 1 (Iba1), a microglia-specific marker  is a pro-inflammatory cytokine that can promote RGC survival . This trend was reversed by 100 mg/kg HE3286, which increased the IL6 ratio by 219% compared to vehicle: 1.75 ± 0.51 (p = 0.047). In the ONH . Treatment with both 20 mg/kg and 100 mg/kg HE3286 prevented the increase with elevated IOP, reducing the ratio by 58%  and 66% , respectively, compared to vehicle. For APP in ONH . Similarly, 100 mg/kg HE3286 elicited a 54% decrease to 5.63 ± 0.84 (p = 0.019). In the retina . Treatment with HE3286 significantly decreased the ratio by 97 and 94% for the 20 mg/kg and 100 mg/kg doses, respectively, compared to vehicle (p < 0.001). Both the ratio for 20 mg/kg HE3286 (0.25 ± 0.05) and 100 mg/kg HE3286 (0.48 ± 0.14) were significantly lower than unity (p ≤ 0.035).When quantified Figure , the ratH Figure , the micH Figure , treatmea Figure , elevateAs with BDNF Figure  and Iba1Next we examined how HE3286 treatment influences localization of common markers for neurodegeneration in glaucomatous retina. Ceruloplasmin (Cp) is a positive acute phase protein upregulated during inflammation Denko, . In vehip = 0.018; Figure p = 0.003) and by 96% in the 100 mg/kg group . For IL1β . Treatment with 20 mg/kg HE3286 further reduced IL1β by 76% . However, 100 mg/kg HE3286 had the opposite effect, increasing levels by 440% compared to vehicle . Elevated IOP in vehicle rats significantly increased levels of C1q compared to saline retina: 1.225 ± 0.061 . As expected, levels of ChAT were the same in saline and microbead retinas in all treatment groups .When quantified, the ratio of Cp in microbead to saline retina in the vehicle group was well above an expected value of unity: 4.61 ± 0.49 , consistent with our qualitative observation . In the ONH . Treatment with 100 mg/kg HE3286 decreased the ratio of microbead to saline levels by 35% (0.97 ± 0.12), which was significant compared to vehicle (p = 0.038). Treatment with 20 mg/kg HE3286 had little effect compared to vehicle . Similar to the SC, nuclear NFκB in the retina increased with IOP elevation for the HE3286 treatment groups ; this was also significantly different from an expected ratio of 1 (p = 0.031). The 20 mg/kg HE3286 dose also increased NFκB nuclear labeling in microbead retina (1.368 ± 0.185), but the 51% increase was not significant both compared to vehicle (p = 0.087) and to an expected ratio of unity (p = 0.117).Using an established algorithm . In contrast, rats treated with 100 mg/kg HE3286 had significantly thinner retinas in saline-injected eyes (87.70 ± 0.59 μm) compared to microbead-injected eyes . Comparing saline-injected eyes across the groups showed that treatment with HE3286 decreased retinal thickness 12–34% compared to treatment with vehicle (p < 0.005). Similarly, retinal thickness decreased 15–26% in microbead eyes treated with HE3286 compared to vehicle (p < 0.002). Retinal thinning was dependent on dosage, as a 13–25% decrease was observed in eyes that received 100 mg/kg HE3286 compared to 20 mg/kg (p < 0.003). To determine if retinal thinning involved neuronal loss, we counted RGCs that were positive for CTB uptake and DAPI nuclear labeling and measured the thickness of the inner nuclear and outer nuclear layers of the retinas  or 100 mg/kg (3.16 × 10−4 ± 7.48 × 10−6 RGCs/μm2) HE3296-treated retinas compared to vehicle-treated retinas . In contrast, HE3286 treatment reduced INL thickness 16% for the 20 mg/kg dose (14.1 ± 0.2 μm) and 21% for the 100 mg/kg dose (13.2 ± 0.1 μm) compared to vehicle . This response was dose-dependent, as 100 mg/kg HE3286 reduced INL thickness a further 6% compared to 20 mg/kg (p = 0.029). A similar reduction in ONL thickness was observed at the higher dose of HE3286 (27.1 ± 0.2 μm) compared to vehicle (33.6 ± 0.4 μm) or the 20 mg/kg dose While examining vertical retinal sections following immunohistochemistry, we noted what appeared to be a decrease in retinal thickness within the mid-peripheral region of the retina in rats treated with HE3286 compared to vehicle Figure . To exams Figure . TreatmeIn this study, we examined the therapeutic potential of a sterol derivative, HE3286, in an inducible model of glaucoma. Daily oral gavage with either 20 mg/kg or 100 mg/kg HE3286 did not affect IOP following microbead or saline injection. Even so, treatment with HE3286 did have tremendous influence on outcomes typically associated with IOP-related axonopathy. We found that HE3286 (1) preserved anterograde axonal transport of CTB from the retina to the superior colliculus in microbead-injected eyes, (2) countered the influence of elevated IOP throughout the optic projection with respect to levels of BDNF, Iba1 and other proteins associated with neuroinflammation and neurodegeneration, (3) modulated pathogenic markers of glaucoma within the retina, (4) increased NFκB localization to neuronal nuclei in the superior colliculus and retina, and (5) decreased NFκB localization to glial nuclei in the optic nerve head.HE3286 treatment also resulted in weight loss and retinal thinning. In a safety and pharmacokinetic study, Ahlem et al. found that HE3286 had no adverse effects in chronic toxicity studies and was not immunosuppressive . We also acknowledge support from the Vanderbilt Vision Research Center .The authors declare that this research was supported by a grant from Harbor Therapeutics, Inc . The reviewer MH and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Trichoderma reesei are largely unknown. Elucidating these processes is the key for unveiling how external carbon sources regulate cellulase formation, and ultimately for the improvement of cellulase production and biofuel production from lignocellulose.Despite being the most important cellulase producer, the cellulase-regulating carbon source signal transduction processes in FUS3-like tmk1 in T. reesei leads to improved growth and significantly improved cellulase formation. However, tmk1 deletion has no effect on the transcription of cellulase-coding genes. The involvement of the cell wall integrity maintenance governing yeast Slt2-like Tmk2 in cellulase formation was investigated by overexpressing tmk3 in T. reesei Δtmk2 to restore cell wall integrity. Transcriptional analysis found little changes in cellulase-coding genes between T. reesei parent, Δtmk2, and Δtmk2::OEtmk3 strains. Cell wall integrity decreased in T. reesei Δtmk2 over the parent strain and restored in Δtmk2::OEtmk3. Meanwhile, cellulase formation is increased in T. reesei Δtmk2 and then decreased in T. reesei Δtmk2::OEtmk3.In this work, the role of the mitogen-activated protein kinase (MAPK) signal transduction pathways on cellulase formation was investigated. The deletion of yeast These investigations elucidate the role of Tmk1 and Tmk2 on cellulase formation: they repress cellulase formation, respectively, by repressing growth and maintaining cell wall integrity, while neither MAPK regulates the transcription of cellulase-coding genes. This work, together with the previous investigations, suggests that all MAPKs are involved in cellulase formation, while Tmk3 is the only MAPK involved in signal transduction for the regulation of cellulase expression on the transcriptional level.The online version of this article (doi:10.1186/s13068-017-0789-x) contains supplementary material, which is available to authorized users. Trichoderma reesei (syn. Hypocrea jecorina) is the most prominent industrial cellulase producer that was demonstrated capable of producing as much as 20 g L−1 cellulases [The economical and environment-friendly utilization of earth’s largest biomass reserve lignocellulose for the purpose of biofuel production requires the abundant production of cellulase, a multicomponent synergistic enzyme cocktail secreted by microorganisms . Trichodllulases .The formation of cellulases has been shown to be controlled on both transcriptional and secretion levels in filamentous fungi , 5. TranIn the past decade, the most significant breakthrough on the understanding of cellulase-regulating signal transduction pathways is the elucidation of the light regulation pathway . This siSaccharomyces cerevisiae, the homologues of three of which have been widely found in filamentous fungi: Hog1-like MAPK pathway, Slt2-like MAPK pathway, and Fus3-like MAPK pathway [S. cerevisiae, the Hog1 pathway responds to high osmolarity signals and functions in combating this environmental stress, the Slt2 pathway primarily functions in cell wall integrity maintenance, and the Fus3 pathway functions in the pheromone-induced mating type differentiation process [Mitogen-activated protein kinases are ubiquitous protein kinases in eukaryotes that play the role of signal transduction and amplification . It is a pathway , 22. In  process , 23.S. cerevisiae [S. cerevisiae, Slt2 homologues in filamentous fungi are also primarily involved in cell wall integrity maintenance with a few exceptions [Trichoderma species were shown to participate in the production of mycoparasitism-related proteins such as chitinases and N-acetylglucosaminidases [T. reesei.Mitogen-activated protein kinases are involved in a variety of physiological functions in filamentous fungi. Hog1 homologues in filamentous fungi function primarily in high osmolarity resistance similarly with revisiae –29. Invorevisiae , 27, strrevisiae , 30, andceptions –42. Prevceptions , 32, 41,ceptions , 37, 42,ceptions , hyphal ceptions , 43, surceptions , 41, strceptions  and circceptions . The funceptions –50, sporceptions , 51, hypceptions –55, secoinidases , 56–58, inidases . This fuT. reesei, namely, the Fus3-like Tmk1, the Slt2-like Tmk2, and the Hog1-like Tmk3 [tmk3 leads to a strong reduction of cellulase formation and transcription of cellulase-coding genes, suggesting the role of Tmk3 in positively regulating the expression of cellulase-coding genes [tmk2 has little effect on the transcriptional level of cellulase-coding genes but leads to a higher cellulase formation level [T. reesei was still unknown.Three MAPKs have been identified from in silico analysis in ike Tmk3 . Previouike Tmk3 , 59. Ourng genes . On the T. reesei. This was done by constructing and characterizing T. reesei Δtmk1, thereby finding out whether Tmk1 is involved in the regulation of cellulase formation, as well as the overexpression of tmk3 in a T. reesei Δtmk2 strain, which led to the identification of the mechanism on how Tmk2 influences cellulase formation. Based on this functional characterization, previously identified roles of Tmk2 and Tmk3 in T. reesei, as well as the mechanistic investigations carried out in this work on how MAPKs influence cellulase formation in T. reesei, this picture of the involvement of MAPKs in cellulase production can be obtained.In this work, we aim to generate a full picture on the role of MAPKs in cellulase formation in tmk1 in T. reesei was successfully carried out with homologous recombination  were grown on potato dextrose agar (PDA) plates, minimal media plates containing glycerol, glucose, or lactose as the substrate, and Avicel double layer plates . Sensitivity assays to CR and CFW were carried out for T. reesei TU-6, T. reesei Δtmk2, and T. reesei Δtmk2::OEtmk3  Δku70 strain was used as the parent strain in this work, and hereafter abbreviated as T. reesei TU-6 (or TU-6) strain. T. reesei Δtmk2 strain is the tmk2 deletion strain using T. reesei TU-6 as the parent strain [The non-homologous end joining pathway-deficient, uridine auxotrophic defective t strain .T. reesei Δtmk1 and T. reesei Δtmk2::OEtmk3 strains was carried out by homologous recombination essentially as previously described [T. reesei Δtmk1, transformed DNA fragment contains 1.8 kb upstream homologous arm, pyr4 that restores uridine biosynthesis, and 1.8 kb downstream homologous arm , Pgpd from Aspergillus nidulans, ptrA gene from Aspergillus oryzae that confers pyrithiamine resistance and tmk3 sequence as the downstream homologous arm (2.2 kb)  solution used for media preparation was prepared following the previously reported recipe . For groT. reesei strains in MM solution containing 2% Avicel and 2% wheat bran for 48 h (1 × 107 spore inoculation). Fermentation was carried out in 3-L fermenters  containing 2 L of media . Two hundred milliliters of seed cultures were inoculated to initiate fermentation. Growth took place for 120 h at 30 °C. Agitation rate was adjusted to allow dissolved oxygen levels above 30%.For fermentation experiment, seed cultures were prepared by growing T. reesei Δtmk1 strains was carried out by inoculating 1 × 105 spores onto plates and grown for 3 days (6 days for double layer Avicel agar plates) prior to colony diameter determination. Three individual replicates were carried out.Phenotypic analysis of 260, FPase (Filter Paperase) activities, pNPCase (p-nitrophenyl-β-d-cellobiosidase) activities, CMCase (carboxymethyl cellulase) activities, and protein concentration determination with the Lowry assay were carried out following the previously described protocols [The analysis of Arotocols , 66, 67.260 and dry mycelial weight was determined by first cultivating T. reesei TU-6 and T. reesei TU-6 Δtmk3 in potato dextrose broth (PDA minus agar) and MM media with glucose or lactose as the carbon sources in 3-L fermenters. For dry mycelial weight determination, approximately 11 mL of the cultures were extracted from fermenters, washed, filtered, and dried in an oven at 80 °C for over 12 h. The mycelia were subsequently weighed. The determination of A260 was carried out as previously described [The correlation between Aescribed . Four re5 spores of T. reesei strains were inoculated on CR- and CFW-containing plates and were grown at 30 °C for 3 days prior to colony diameter determination. Three replicates were carried out.For analysis of CR and CFW resistance, approximately 1 × 106 spores of T. reesei strains were inoculated to media containing 2% Avicel and 2% wheat bran and grown for 3 days prior to extraction of total RNA for transcription quantification. The qPCR procedures and data manipulation were carried out essentially as previously described [For qPCR analysis, approximately 1 × 10escribed . Three bFor determination of transcriptional abundance of cellulase-coding genes at different time points after inoculation to media containing Avicel + wheat bran, spores were first inoculated to MM media +2% glucose and grew for 2 days, after which mycelia were extracted, washed, and inoculated to MM media +2% glycerol for growth for 1 day. The mycelia were further washed and inoculated to MM media containing 2% wheat bran and 2% Avicel. Cells were extracted for transcriptional analysis upon transfer, as well as 4, 12, and 24 h after the transfer.t-tests were carried out. p < 0.05 was considered statistically significant. A minimum of three replicates were carried out for each experiment.To test if two sets of data are statistically different, two-tailed Student’s Additional file 1. Southern blotting analysis of T. reesei TU-6, Δtmk1-1 and Δtmk1-2. Panel A: Schematic drawing of strain construction and southern blotting analysis. Panel B: southern blotting of T. reesei strains. M: DNA marker; TU-6: T. reesei TU-6; Δtmk1-1: T. reesei Δtmk1-1; Δtmk1-2: T. reesei Δtmk1-2.Additional file 2. The sensitivity of T. reesei TU-6, Δtmk1-1 and Δtmk1-2 to CR and CFW. Panel A: growth of T. reesei TU-6, T. reesei Δtmk1-1 and T. reesei Δtmk1-2 on CR-containing agar plates; Panel B: growth of T. reesei TU-6, T. reesei Δtmk1-1 and T. reesei Δtmk1-2 on CFW-containing agar plates. TU-6, T. reesei TU-6; Δtmk1-1, T. reesei Δtmk1-1; Δtmk1-2, T. reesei Δtmk1-2.Additional file 3. Southern blotting analysis of T. reesei TU-6, Δtmk2 and Δtmk2::OEtmk3. Panel A: Schematic drawing of strain construction and southern blotting analysis. Panel B: southern blotting of T. reesei strains. M: DNA marker; Δtmk2: T. reesei Δtmk2; Δtmk2::OEtmk3: T. reesei Δtmk2::OEtmk3.