Uopt in m s−1 or body lengths s−1, BL s−1) were assessed and then applied to determine the effects of long-term forced and sustained swimming at Uopt on growth performance of juvenile yellowtail kingfish. Uopt was quantified in Blazka-type swim-tunnels for 145, 206, and 311 mm juveniles resulting in values of: (1) 0.70 m s−1 or 4.83 BL s−1, (2) 0.82 m s−1 or 3.25 BL s−1, and (3) 0.85 m s−1 or 2.73 BL s−1. Combined with literature data from larger fish, a relation of Uopt (BL s−1) = 234.07(BL)−0.779 (R2 = 0.9909) was established for this species. Yellowtail kingfish, either forced to perform sustained swimming exercise at an optimal speed of 2.46 BL s−1 (“swimmers”) or allowed to perform spontaneous activity at low water flow (“resters”) in a newly designed 3600 L oval flume (with flow created by an impeller driven by an electric motor), were then compared. At the start of the experiment, ten fish were sampled representing the initial condition. After 18 days, swimmers (n = 23) showed a 92% greater increase in BL and 46% greater increase in BW as compared to resters (n = 23). As both groups were fed equal rations, feed conversion ratio (FCR) for swimmers was 1.21 vs. 1.74 for resters. Doppler ultrasound imaging showed a statistically significant higher blood flow (31%) in the ventral aorta of swimmers vs. resters . Thus, growth performance can be rapidly improved by optimal swimming, without larger feed investments.Swimming exercise at optimal speed may optimize growth performance of yellowtail kingfish in a recirculating aquaculture system. Therefore, optimal swimming speeds ( Aquaculture is facing an increasing demand for sustainably produced fish. There is a great need to optimize the conditions for fish growth, but without compromising health and welfare. Currently, significant numbers of commercially produced species of fish suffer from impaired well-being and high mortality  where the cost of transport  is lowest and the energetic efficiency highest  at Uopt which explains that also other beneficial effects may occur at this speed.Exercise-enhanced growth is optimal at a particular swimming speed where a maximum of energy is diverted to the skeletal muscles and where a minimum is lost due to other processes. The swimming speed for optimal growth is most likely near optimal swimming speeds  is distributed circumglobally in subtropical seas, usually inhabiting deep pelagic waters  when exercised at 0.75 body lengths (BL) s−1 . Clearly, the potential of implementing exercise to improve growth rate in S. lalandi is indicated.The carangid yellowtail kingfish  of the Wageningen UR in Lelystad (The Netherlands) under numbers 2012012 and 2013162.n = 33; BL = 88 mm, BW = ~ 8 g) were obtained from the farm Silt BV . They were transported from the farm to the IMARES facilities in Yerseke by truck within 3 h. Fish were housed under similar conditions as in the hatchery: in natural seawater that was mixed with tap-water to 25.1 ± 0.1‰, at 23.4 ± 0.1°C and under a light regime of 16L:8D before and during the experimental periods. Fish were hand-fed three times per day at 7% BW day−1 with commercial feed . Water quality was monitored daily for O2, pH, NH4, NO2, and NO3. Oxygen levels were 7.54 ± 0.08 mg L−1, pH was 8.07 ± 0.07, NH4-N averaged 0.27 ± 0.05 mg L−1, NO2-N averaged 1.5 ± 0.2 mg L−1 and NO3-N averaged 8.9 ± 0.9 mg L−1 over the 2.5 months experimental period. During three periods in time, subsamples of this batch of fish were used for swim performance tests  or when the fish had swum at 1.00 m s−1 for 1 h. Oxygen data from fish that stopped swimming at speeds <0.60 m s−1 were not used. Also data of some smaller fish that were observed to benefit from reduced flow by continuously swimming very close to the walls of the swim-tunnels at the highest speeds were excluded for those speeds. The oxygen consumption data were plotted against the swimming speeds in m s−1.Before each swimming experiment, two fish were individually introduced in a swim-tunnel and were allowed to acclimatize for 1 h which did not appear to be stressful for the fish as they were spontaneously swimming around at low speeds. Then, oxygen consumption was measured while swimming at consecutive speeds of 0.20, 0.40, 0.60, 0.80, and 1.00 m sAfter swimming, each fish was anesthetized in clove oil (1:10 diluted in absolute ethanol and used as 2 mL in 10 L water). The anesthetized fish was measured for total length (TL) and fork length (FL).−1.The experiment was conducted in a 3600 L oval-shaped Brett-type swim-flume  continuously generated water circulation throughout the flume to safeguard water quality when the impeller was stationary.Water flow was generated at the start of the straight end opposite the two compartments by an impeller connected to an electric motor (KLEEdrive MS2 132M-4 B3 (7.5 kW), Brd. Klee A/S, Albertslund, Denmark; Figure u, v, and w in directions x, y, and z, respectively, as described above) for 10 s with a sampling rate of 10 Hz.Prior to the start of the experiment, water velocity in the swim compartment was measured using a downward-faced Vectrino acoustic Doppler velocimeter  with its focal point at the center of the water column, 25 cm from the upstream fence. When the impeller was stationary, flow in the swimming compartment was measured. Inverter frequencies were subsequently increased with 0.5 Hz increments from 2.5 to 8.5 Hz, and after each increment, water flow was left to stabilize for 5 min, after which water velocities were measured in three dimensions , three vertical transects  at three depths  which resulted in a total of 27 measurements. Using velocities u, v, and w, the dimensionless turbulence intensity (TI) was calculated according to Liao and Cotel  from the farm Silt BV  were randomly assigned to the rest-compartment  or the swimming compartment  of the flume and were acclimatized for 2 days.Juvenile yellowtail kingfish  were randomly selected and sampled (as “START” group). Individual fish were anesthetized in clove oil and total length (TL in mm) and body weight (BW in g) were measured from which Fulton's condition factor K was calculated, after which fish were euthanized by decapitation. The heart was dissected and heart weight (HW) was determined.n = 23) were forced to swim at Uopt in a sustained manner while resters (n = 23) were allowed to perform spontaneous activity at low water flow. Fish were fed commercial feed , three times per day on weekdays and twice per day during weekends. The water flow was stopped during feeding sessions. First, the resters were fed until apparent satiation and the amount of feed given was determined. Then, swimmers were pair-fed and received the same amount as the resters. After feeding, fish were left for 15 min and then the water velocity was gradually increased to Uopt. Fish were fed 2.65 ± 0.13 % BW d−1 and food conversion ratio (FCR) for both treatments was calculated as the ratio of biomass gain (wet weight in g) to total food intake (g).One day after the START group was sampled, the swimming trial commenced and the swimmers , NO2-N averaged 1.4 ± 0.2 mg L−1 (min 0.2 mg L−1 and max 3.1 mg L−1) and NO3-N averaged 14.8 ± 3.5 mg L−1  over 21 days. Nitrogenous waste-products were within safe limits for yellowtail aquaculture  with tap water. Mean water temperature was maintained at 23.6 ± 0.1°C. Oxygen gas was supplied through a ceramic diffuser downstream of the two compartments. Hundred percentage system volume was replaced daily to ensure high water quality. Water quality was monitored daily for On = 13 per treatment), blood flow was determined by Doppler ultrasound imaging, using an Esaote MyLabFive Vet ultrasonography unit  with a 18 MHz LA 435 ultrasound transducer (Easote). Blood flow was visualized using the brightness (B) mode, Colour Flow Mapping (CFM) and Pulse Wave (PW) with the following settings: Velocity 89%; Angle (θ) + 60°; Depth 6 cm, Gain,B 76%, Gain,CFM 70%, Gain,PW 52%; Frequency,B 18 MHz, FrequencyCFM, 8 MHz, and Frequency,PW 8 MHz. Anesthetized fish were positioned on a table with the right lateral side facing upwards. The ultrasound transducer was covered with Aquasonic ultrasound transmission gel  and held motionless against the ventral side of the fish near the head, in a longitudinal direction, allowing visualization of blood flow in the ventral artery, just downstream of the bulbus arteriosus , iW is the initial average weight of the START group (g) and t is time between measurements (d).where P < 0.05). K data were tested with Kruskal–Wallis tests.BW, TL, and FL data in part I (Swim performance tests and respirometry in swim-tunnels) were normally distributed and tested for differences occurring between the three size classes  with BW as the cofactor was performed on log transformed unpaired observations in search for group effects in the parameters HW and BF. Parameters were compared between resters and the START group and between swimmers and resters. In case there was no significant effect of the cofactor BW, ANOVA was used to determine whether there was a group effect.All data in part II (Swim training test in a swim-flume) showed normal distribution (Shapiro–Wilk tests). To test for background effects over time, resters were compared to the START group. To test for treatment effects, swimmers were compared to resters. TL and BW were compared using student's P < 0.05 were considered significant. All data are presented as mean ± standard error (SE).Differences with P < 0.05; Table Size parameters  significantly increased from one experimental group to the next  and one fish of group 3 (Ucrit = 0.60 m s−1 or 1.88 BL s−1) fatigued before swimming 1 h at 1 m s−1. One fish of group 1 was able to swim 1 h at 1 m s−1  without swimming close to the walls, as well as seven of group 2  and seven of group 3 .Seven fish of the smallest size group 1 at speeds of 0.80 m sU-shaped curves for each of the subsequent groups . For groups 2 and 3, most of the fish were still swimming at 1 m s−1 so that the minimal COT value determining Uopt may not have been reached. Polynomial curves  were plotted to calculate Uopt-values for both groups. The average COT was higher in group 1 as compared to the fish of groups 2 and 3  increased for each of the subsequent size groups from 0.70 m s−1 for group 1; to 0.82 m s−1 for group 2; to 0.85 m s−1 for group 3 (Table Uopt (in BL s−1) decreased: 4.83 BL s−1 for group 1, 3.25 BL s−1 for group 2, and 2.73 BL s−1 for group 3  values were plotted against the swimming velocities as polynomial  Figures . Only inFigures R = 0.9935Figures R = 0.9935Figures R = 0.9935Figures R = 0.9935−1 as a result of the circulation generated by the Speck pump. Horizontal water velocity (u) in the swimming compartment increased proportionally to the AC-frequency of the inverter . After 18 days at Uopt speeds, swimmers had grown an average 39 mm to 385 ± 4 mm whilst resters only grew 21 mm to 367 ± 5 mm; a 92% greater increase in length  as compared to resters (1.5% BW d−1). Swimmers and resters had similar condition factors   showed any significant differences between groups Table . SwimmerIn this study we have established optimal swimming speeds for juvenile yellowtail kingfish allowing to apply this knowledge for an experimental swim-training trial of 18 days to investigate the effects of swimming exercise on growth performance and cardiac output. Exercise-enhanced growth was robust and not caused by increased feed intake. It may have been caused by increased feeding efficiency indicated by a lower FCR. Doppler ultrasound imaging showed a cardiac output which was significantly higher in exercised fish, no such data have been shown for fish before (to the best of our knowledge).Uopt  = 234.07(BL)−0.779 . We believe that our study shows exercise-enhanced growth for Seriola spp. of 46% because fish were forced to swim at their optimal swimming speed. This also agrees with the deduced highest weight gain in fingerling S. quinqueradiata as reported by Yogata and Oku  in several active teleosts  = 234.07(BL)−0.779 (R2 = 0.9909). The formula was used to execute an experimental scale training experiment of 18 days. Results show that forced sustained exercise at optimal swimming speeds leads to a 92% greater increase in BL and 46% greater increase in BW, an exercise-induced growth stimulation that is much more pronounced than the 10% growth stimulation shown earlier for S. lalandi by Brown et al. . One aspect that will be different is that not a swim-flume will be applied but tanks or perhaps raceways as part of RAS having very different hydraulics. It will be challenging to create the flows that are required to induce optimal swimming, especially for the larger sized fish (>500 mm). Flows could be created by using motor-driven impellers like in this study, pumps or gravity, but perhaps a good alternative may be a device that exploits the optomotor response to encourage exercise, like the Optoswim concept (Herbert et al., Conceived and designed the experiments: Arjan P. Palstra, Kees Kloet. Performed the experiments: Arjan P. Palstra, Daan Mes, Kasper Kusters, Jonathan A. C. Roques. Analyzed the data: Arjan P. Palstra, Daan Mes, Kasper Kusters, Jonathan A. C. Roques. Wrote the paper: Arjan P. Palstra, Daan Mes, Kasper Kusters, Gert Flik, Robbert J. W. Blonk.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The properties of the polymers were investigated by using UV–Vis absorption, fluorescence spectroscopy, cyclic voltammetry, and thermogravimetric analysis. The result shows that the band gaps of the PPV derivatives with a nitro substituent were decreased and the polymers had higher molecular weights (106), excellent solubility in common organic solvents, good film-forming ability, and better thermal stability. The polymers can be used as an efficient acceptor material in polymeric solar cells.In order to improve efficiency, processability, and stability, two groups of novel poly( The nitro moiety is a strong electron-withdrawing group, and inductive conjugation effects in nitrocellulose not only improve the nitro group’s electronic cloud density and increase the charge capacity, but also can enhance the electronic properties of conjugated polymers, increase the interaction between charges, and improve energy transfer and the quenching efficiency . In orde2 + B3” approach based on Gilch polycondensation is used for the polymer synthesis.Various modified Wessling reactions which originally use sulfur-based leaving groups have all been reported for the synthesis of a range of PPV derivatives –17. This1–P3 and P4–P6) with hyperbranched structure and containing a nitro substituent. Both groups of polymers exhibit a molecular weight of approximately 106, better solubility, a wide range of spectral absorption, and good thermal stability. These characteristics explain why the polymer is a good photoelectric material.In this publication, we report the synthesis and characterization of two groups of novel PPV derivatives  and aromatic proton peaks appeared in the 1H NMR spectra of the polymers, suggesting that the polymerization was complete.The detailed syntheses of the two groups of hyperbranched polymers are outlined in Scheme Mn) and the weight-average molecular weight (Mw) were determined by gel permeation chromatography (GPC) against standard polystyrene. The molecular weight values were merely estimates because of the differences in hydrodynamic radius between the hyperbranched polymers and the polystyrene standard. As shown in Table The number-average molecular weight . The polymers exhibited high thermal stability and onset of weight loss temperatures of 375, 377, 382 and 358, 379, 382 °C for P6) in anhydrous acetonitrile as supporting electrolyte, were measured at a scanning rate of 10 mV/s.Cyclic voltammetry (CV) was employed to investigate the electrochemical properties of the six copolymers. The oxidation and reduction potentials revealed in cyclic voltammograms indicate the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) levels, which correspond to ionization potentials (IP) and EA, respectively . The CV 1, P2, and P3), the onset potentials for oxidation (Eonsetox) were 1.39, 1.18, and 1.02 V and the onset potentials for reduction (Eonsetred) were −1.67, −1.84, and −1.99 V. Secondly, with a nitro substituent , the onset potentials for oxidation (Eonsetox) were 0.99, 0.97, and 0.91 V and the onset potentials for reduction (Eonsetred) were −1.80, −1.86, and −1.90 V.The HOMO and LUMO energy levels of polymers were calculated and the onset potentials for oxidation and reduction are shown in Table A in these polymers increases little by little. The result indicates that hole-injection and transporting properties have been improved. The result implies that the electron-rich alkoxy groups affect the electronic properties of the conjugated copolymer chains significantly. Comparison of the two groups of polymers shows that the introduction of a nitro group in the hyperbranched PPVs not only increased the HOMO energy level, but also made the band gap narrower.The HOMO energy levels of the two groups of polymer were gradually reduced. The reason is possibly that the ratio of monomer 1–P3 and P4–P6 in solution are shown in Figs. 1–P3 and P4–P6 in the film are shown in Figs. 1–P3 exhibit only major absorption at 503, 493, and 490 nm in solution. In Fig. 4–P6 show maximum absorptions at 497 nm in solution, and P4–P6 exhibit other major absorptions at 329, 333, and 335 nm in solution. Both groups of polymers absorb at around 500 nm, which is attributed to the transitions of main-chain π-systems of the polymer. The absorptions in the short wavelength region of the second group of polymers originate from the nitro substituent. The maximum absorption peak of polymers with a nitro substituent is sharper than that of polymers without this substituent. This is the reason why introducing the strong electron-withdrawing nitro group is good for the polymer π–π* interaction. The thin film absorption spectra show an obvious red shift relative to those in solution. According to the data given in Table 1–P6 is 38, 29.5, 27, 8, 8, and 13 nm, respectively. The PL spectra were recorded with an excitation wavelength corresponding to the absorption maximum wavelength of the polymer. Emission peaks of P1, P2, and P3 in chloroform solution were all 567 nm, and in thin film were 602, 596, and 596 nm, respectively. Meanwhile, the emission peaks of P4, P5, and P6 in THF solution were all 553 nm, and in thin film were 581, 586, and 583 nm, respectively. This red shift could be explained by the enhancement of the interaction in the solid states.The photophysical characteristics of the polymers were investigated in solution and the thin film. The UV–Vis absorption and photoluminescence (PL) spectra of P1–P3 and P4–P6) with hyperbranched structure and containing a nitro substituent via the Gilch reaction. The introduction of trisubstituted benzene resulted in the twisting of the polymer backbone and thus decreased coplanarity. By introducing trisubstituted benzene with a nitro substituent, it increased the absorption range and lowered the polymer band gap. Poly-benzene co 1,4-Bis(bromomethyl)-2-methoxy-5-(2’-ethyl)hexyloxybenzene phenylene vinylenene) (PTV-co-MEHPV) and Poly-benzene co 1,4-Bis(bromomethyl)-2-methoxy-5-(2’-ethyl)hexyloxybenzene phenylene vinylenene) (PTNV-co-MEHPV) have good solubility in common organic solvents. The number-average molecular weight and weight-average molecular weight are also large. All polymers we synthesized are red-light-emitting materials. The polymers can be used as an efficient acceptor material in PSC.We have successfully synthesized two groups of novel PPV derivatives  was slowly added over 1 h to a stirred solution of the two monomers at the chosen molar ratio (see below) in 25 cm3 dry THF that was cooled to 0 °C and under N2 atmosphere. The mixture was stirred at room temperature for 20 h. The polymerization solution was poured into 500 cm3 methanol. A crude polymer precipitated out, which was redissolved in chloroform and reprecipitated from methanol and then extracted in a Soxhlet apparatus with methanol to remove the impurities and oligomers. After drying filtration in vacuum, a bright red solid was obtained. In this paper, we adopt different ratios of starting monomers for the synthesis of the two groups of polymers. The proportions for P1–P3 are monomer A  and 4 ; those for P4–P6 are monomer A  and monomer B . 1H NMR of P1 and P4 : δ = 7.54–7.09 , 4.14–3.85 , 1.95–1.94 , 1.55–1.25 , 0.89  ppm; 13C NMR of P1 : δ = 10.09, 13.99, 22.42, 30.50, 38.12, 39.56, 55.79, 65.29, 109.01, 124.40, 134.56, 144.22 ppm; 13C NMR of P4 : δ = 8.85, 17.44, 24.45, 32.22, 40.01, 51.73, 61.73, 110.76, 122.41, 137.91, 143.84, 145.51, 148.69 ppm.A typical polymerization procedure for the preparation of the new polymers is described as follows. A solution of 8.4 cm


Dis. Model. Mech.6, 489-502.There was an error published in x-axis of Fig. 5 (the legend remains the same). The correct text and figure is shown below. This change does not alter any conclusions of the paper, which was a comparison of cells from patients and controls.Due to a technical error with the image analysis software, the published speed unit of peroxisome movement is incorrect. The stated peroxisome speed unit (µm/second) should be changed to µm/2 seconds in two different places in the paper: (1) twice in the Results section ‘Dynamics of peroxisome movement’, and (2) the t=9.19, d.f.= 24,398, P<0.0001).(1) The mean peroxisome speed in patient cells was 93% slower than in control cells (control, 0.172±0.001 μm/2 seconds; patient, 0.160±0.001 μm/2 seconds; The authors apologise to the readers for any confusion that this error might have caused.


Cordyceps sensu lato, but it is hard to produce large amounts of this substance in industrial production. In this work, single factor design, Plackett-Burman design, and central composite design were employed to establish the key factors and identify optimal culture conditions which improved cordycepin production. Using these culture conditions, a maximum production of cordycepin was 2008.48 mg/L for 700 mL working volume in the 1000 mL glass jars and total content of cordycepin reached 1405.94 mg/bottle. This method provides an effective way for increasing the cordycepin production at a large scale. The strategies used in this study could have a wide application in other fermentation processes.Cordycepin is one of the most important bioactive compounds produced by species of Cordyceps militaris is an entomopathogenic fungus belonging to Ascomycota, Sordariomycetidae, Hypocreales, and Cordycipitaceae [ Cordyceps militaris is the type species of Cordyceps, which internally parasitizes larva or pupa of lepidopteran insects and forms fruiting bodies on their insect hosts. Cordyceps militaris has long been recognized as a desirable alternative for natural Ophiocordyceps sinensis [ Ophiocordyceps sinensis is causing substantial reductions in populations [ Cordyceps militaris produces many bioactive compounds, including polysaccharides, cordycepin, adenosine, amino acid, organic selenium, ergosterol, sterols, cordycepic acid, superoxide dismutase (SOD), and multivitamins [pitaceae  and is osinensis  as it haulations . Cordycevitamins , 5. C. militaris [http://www.ClinicalTrials.Gov verified by OncoVista, Inc., 2009).Cordycepin (3′-deoxyadenosine), a nucleoside analog, was first isolated fromilitaris  and is oilitaris , anticanilitaris , antifunilitaris , antivirilitaris , antileuilitaris , 12, andilitaris  activiti C. militaris [ Aspergillus nidulans [ C. militaris. The resulting mutants were higher cordycepin produces, than the wild strain [ C. militaris was substantially increased by the elicitor of Phytophthora sp. Research result showed that glucose and yeast extract were effective media components for improved cordycepin production by C. militaris [In previous work, cordycepin has been synthesized by chemical , 15 and ilitaris  or Aspernidulans , 17. Solnidulans , 19, subnidulans , 20–24, nidulans –27 have nidulans . Fermentnidulans , 19. Pronidulans , 29. Pronidulans  and the nidulans . New tecd strain , 31. Bu ilitaris , 33. Theilitaris , 32, culilitaris , 27 for  C. militaris (strain CGMCC2459) in 1000 mL glass jars. The results suggested that the optimization medium conditions were helpful for improved large scale cordycepin production.In this study, the effects of working volume, carbon sources, nitrogen sources, inorganic salts, growth factor, nucleoside analogue, and amino acid additions were studied in order to improve the cordycepin production by static liquid culture of C. militaris (strain CGMCC2459) used in the present study was collected from Mt. Qingcheng in Sichuan Province, China. The microorganism was maintained on potato dextrose agar (PDA) slants. Slants were incubated at 25°C for 7 days and then stored at 4°C. The seed culture was grown in a 250 mL flask containing 70 mL of basal medium  at 25°C on a rotary shaker incubator at 150 rev/min for 5 days [The isolate of2PO4 1 g/L; and MgSO4·7H2O 0.5 g/L. The pH was not adjusted, followed by autoclaving for 30 min on the 121°C. The static culture experiments were performed in 1000 mL glass jars  containing basal medium after inoculating with 10%  of the seed culture. The culture was incubated at 25°C without moving for 35 days, and samples were collected at the end of the fermentation from the glass jars for analyzing biomass dry weight and cordycepin production.The basal medium composition for the fermentation was as follows: sucrose 20 g/L; peptone 20 g/L; KH C. militaris were studied using a one-factor-at-a-time method for static culture. The effects of carbon sources on cordycepinproduction were studied by substituting carbon sources such as sucrose, lactose, soluble starch, and dextrin for glucose at 25°C for 35 days. Effects of nitrogen sources  and inorganic salts  were also studied using static culture. Growth factors (Vitamin B1 (VB1), Vitamin B6 (VB6), Vitamin B7 (VB7), Vitamin B11 (VB11), α-naphthylacetic acid (NAA), 3-Indoleacetlc acid (IAA), and 2,4-dichlorophenoxyacetic acid ) were supplemented for 10 mg/L in basal media. Nucleoside analogues (1 g/L) and amino acids (8 g/L) established in our previous study [The effects of factors affecting cell growth and the production of cordycepin byus study  as an inSamples collected at 35 days from the glass jars were centrifuged at 2810 ×g for 20 min. The mycelium at the bottom of tubes was washed sufficiently with a large amount of distilled water and dried to a constant dry weight at 55°C.μm membrane and the filtrate was analyzed by HPLC . Accurate quantities of cordycepin  were dissolved in distilled water, to give various concentrations for calibration. The mobile phase was 10 mmol/L KH2PO4, which was dissolved in methanol/distilled water (6 : 94). Elution was performed at a flow rate of 1.0 mL/min with column temperature at 45°C and UV wavelength of 259 nm. Mean values were computed from triplicate samples.For analysis of extracellular cordycepin, the resulting culture filtrate was obtained by centrifugation at 2810 ×g for 20 min. The supernatant was filtered through a 0.45 X1), peptone (X2), K2HPO4·3H2O (X4), MgSO4·7H2O (X5), and VB1 (X6) were investigated as key ingredients affecting cordycepin production. Based on the Plackett-Burman design, a 15-run was applied to evaluate eleven factors . Each factor was prepared in two levels: −1 for low level and +1 for high level. The Plackett-Burman design, an effective technique for medium-component optimization , 36, was C. militaris, for identifying the effects of process variables [2HPO4·3H2O, and MgSO4·7H2O) were denoted as X1, X4, and X6, respectively, and each of them was assessed at five different levels, combining factorial points , axial points , and central point (0), as shown in A, B, and C, respectively, and each of them was assessed at five different levels, combining factorial points , axial points , and central point (0), as shown in Response surface methodology using a central composite design was applied to batch cultures ofariables , 36. In P < 0.05. Statistical data analyses were performed in SPSS version 17.0 software packet. Design-Expert Version 8.0.5b software package  was used for designing experiments as well as for regression and graphical analysis of the experimental data obtained.Dry weight and cordycepin production are expressed as means ± SD. An analysis of variance (ANOVA) followed by Tukey's test was applied for multiple comparisons of significant analyses at  C. militaris by surface culture.Dissolved oxygen concentration is the key factor in the medium for cell growth and metabolite biosynthesis . Dissolv C. militaris were prepared at the working volumes of 100 to 900 mL (corresponding to a medium depth of 1.26 to 11.31 cm). As shown in In this study, we tried to establish the most efficient working volume of medium for improved cordycepin production. Cultures of C. militaris cordycepin production we added various carbon sources at a concentration of 20 g/L to the sugar-free basal medium. Glucose was previously found to be an excellent precursor of cordycepin production [To find a suitable carbon source foroduction . However N promoted a faster mycelial growth and consequently diverted the source of carbon toward energy and biomass production. The other effect was positive because a moderate input contributed to the maintenance of citric acid productive biomass [ C. militaris, various compounds containing nitrogen (inorganic and organic nitrogen) were added individually to nitrogen free basal medium at a concentration of 20 g/L. Among the 8 nitrogen sources tested, peptone, yeast extract, beef extract, casein, and NH4NO3 were favorable to the cordycepin production  of inorganic salts  were considered as significant for cordycepin production by static liquid culture methodology.The data  indicate factors  showed t2HPO4·3H2O, and MgSO4·7H2O should be examined. The levels of variables for central composite design experiments were selected according to the above results of Plackett-Burman design. Y1) as a function of sucrose (X1), K2HPO4·3H2O (X4), and MgSO4·7H2O (X6) concentrations:In order to evaluate the influence of medium component on cordycepin production, sucrose, KF-test analysis of variance (ANOVA) showed that the regression was statistically significant at 95% and 99% confidence levels . The Pred-R2 of 0.3183 was not as close to the Adj-R2 of 0.7954 as one might normally expect. The result suggested that some factors were not considered in the model. However, the “Adeq Precision” of 8.173 indicated that the model was adequate for prediction production of cordycepin.Results of e levels . The “F X1, sucrose, level 0.47; X4, K2HPO4·3H2O, level 0.21; X6, MgSO4·7H2O, level −0.20) when using canonical analysis of the Design-Expert Version 8.0.5b software package. The model predicted a maximum response of 1451.43 mg/L cordycepin production at levels of sucrose 24.7 g/L, K2HPO4·3H2O 1.11 g/L, and MgSO4·7H2O 0.90 g/L as optimized medium components.The response surface plot obtained from  is shown2. The –OH should be replaced in metabolic pathways. In addition, among 14 different amino acids were tested for 8 g/L. As shown in  C. militaris or component of cordycepin production. Hypoxanthine and L-alanine were the best additives to promote cordycepin production in this study.Chassy and Suhadolnik  reported C. militaris in 1000 mL glass jars after fermentation by static liquid fermentation. Y2) as a function of hypoxanthine (A), L-alanine (B), and culture time , concentrations:Similarly, central composite design was also applied to study the significant factors  and their optimal levels. F-test analysis of variance (ANOVA) showed that the regression was statistically significant at 95% and 99% confidence level . The Pred-R2 of 0.4162 was not as close to the Adj-R2 of 0.8378 as one might normally expect. The result suggested that some factors were not also considered in the model. However, the “Adeq Precision” of 11.222 indicated that the model was adequate for prediction production of cordycepin.Results of ce level . The “F A, hypoxanthine, level 0.11; B, L-alanine, level 0.06; C, time, level −0.23) by canonical analysis of the Design-Expert Version 8.0.5b software package. The model predicted a maximum response of 2008.48 mg/L cordycepin production at levels of hypoxanthine 5.45 g/L, L-alanine 12.23 g/L, and time 8.6 days  as optimized medium components.The response surface plot obtained from  is shown Cordyceps sp. These experimental designs have been successfully used to optimize medium for the mycelial growth and microbial metabolite production in liquid culture processes. In this study, static liquid culture conditions are optimized for the cordycepin production using response surface methodology and are an effective way to enhance the productivity of cordycepin and biomass in C. militaris.In previous work, the orthogonal design method –46, Box-2HPO4·3H2O 1.11 g/L; MgSO4·7H2O 0.90 g/L; VB1 10 mg/L; hypoxanthine 5.45 g/L; and L-alanine 12.23 g/L. Five experiments were performed to confirm the above optimal culture requirements. The data were 2011.15 mg/L, 2000.69 mg/L, 1989.22 mg/L, 1969.6 mg/L, and 2061.37 mg/L, respectively. The average cordycepin production was 2006.41 ± 34.37 mg/L. The experimental values were particularly close to the predicted values (2008.48 mg/L). The result confirmed the model suited the predictive of hyperproduction of cordycepin by C. militaris in static liquid culture. Batch culture was carried for cordycepin production under optimized culture conditions . This method provides an effective way for increasing the cordycepin production at a large scale. The strategies used in this study could have a wide application in other fermentation process.In this work, single factor design, Plackett-Burman design, and central composite design were employed to establish the key factors and identify optimal culture conditions which improved cordycepin production by


However, when the concentrations of nitrite nitrogen and nitrate nitrogen were above 4045 mg/L and 1427 mg/L, respectively, the removal of nitrite nitrogen, nitrate nitrogen, COD, voltage, and power density were decreased to be 86%, 88%, 77%, 180 mV, and 17 mW/m2 when nitrite nitrogen and nitrate nitrogen were increased to 4265 mg/L and 1661 mg/L. In addition, the composition of biogas generated in the anode chamber was analyzed by a gas chromatograph. Nitrogen gas, methane, and carbon dioxide were obtained. The results indicated that denitrification happened in anode chamber. In order to study the effect of nitrite and nitrate on the performance of microbial fuel cell, a system combining an anaerobic fluidized bed (AFB) and a microbial fuel cell (MFC) was employed for high-strength nitrogen-containing synthetic wastewater treatment. Before this study, the AFB-MFC had been used to treat high-strength organic wastewater for about one year in a continuous flow mode. The results showed that when the concentrations of nitrite nitrogen and nitrate nitrogen were increased from 1700 mg/L to 4045 mg/L and 545 mg/L to 1427 mg/L, respectively, the nitrite nitrogen and nitrate nitrogen removal efficiencies were both above 99%; the COD removal efficiency went up from 60.00% to 88.95%; the voltage was about 375 ± 15 mV while the power density was at 70 ± 5 mW/m Nitrogen is present in water as inorganic ions and organic compounds. Nitrogenous substances would benefit the growth of the plant. However, natural water will be seriously polluted and become eutrophication if nitrogenous materials are in excess. Eutrophication happens when the concentration of nitrogen, a major factor of eutrophication, exceeds 0.20 mg/L. The aquatic ecosystems will be damaged by the excess nitrogenous substances, which would seriously impact the survival of human and animals. Therefore, it is necessary to reduce nitrogen pollution loads from nonpoint source pollution or point source pollution.The nitrogenous substances in the wastewater are traditionally removed by nitrification and denitrification . In the 3−-N) L−1 day−1 [MFC technology was developed in the early 21st century. Carbon and nitrogen could be simultaneously removed as well as producing the electricity in MFC. Researchers discovered that the total Kjeldahl nitrogen and nitrate nitrogen could be removed by MFC , 13. The−1 day−1 , 23. In −1 day−1 . These s2(SO4)3·18H2O, MgSO4·7H2O, FeSO4·7H2O, CaCl2·6H2O, H3BO3, (NH4)2MoO4, CoSO4·7H2O, ZnSO4·7H2O, CuCl2·2H2O, NiCl2·6H2O, and MnSO4·H2O; their concentrations are 0.10 mg/L, 3.00 mg/L, 3.50 mg/L, 0.30 mg/L, 0.05 mg/L, 0.01 mg/L, 0.30 mg/L, 0.10 mg/L, 0.20 mg/L, 0.01 mg/L, and 0.50 mg/L, respectively [The wastewater was high-strength nitrogen-containing synthetic wastewater. The chemical components were glucose, sodium bicarbonate, potassium dihydrogen phosphate, ammonium chloride, sodium nitrite, sodium nitrate, and trace metal nutrient. The trace metal nutrient contains Alectively , 25. The2, DuPont, USA). The distance between the anode and cathode was 7.0 cm. The anode and cathode were made from carbon fiber paper . The total surface areas of the anode and cathode were 160 cm2 and 210 cm2, respectively. A copper wire was used to connect the electrodes to an adjustable resistor (0~99999.9 Ω). Ten percent of the anode chamber volume was added with porous polymer carriers (PPC). The PPC had a dry diameter of 0.32 mm, a wet diameter of 0.56 mm, a grain density of 1320 kg/m3, and a wet bulk density of 1010 kg/m3. Before this system was used for the present research, the reactor was cultured by anaerobic sludge. It had been successfully started up in a feed-batch mode at a temperature of 35 ± 2°C, pH of 6.8~7.5, aeration rate of cathode chamber of 16~24 L/h, and external resistance of 2000 Ω [The experimental apparatus was a two-chamber MFC system . It consf 2000 Ω . Then, if 2000 Ω . BiopartThe AFB-MFC reactor was operated in a continuous mode at 35 ± 2°C. The temperature was controlled by a temperature-controlled incubator. The reflux was 10.2 L/h. The aeration rate was 16~24 L/h in the cathode chamber and the dissolved oxygen was about 7.50 mg/L. The external resistance was 120 Ω. The hydraulic retention time (HRT) was twenty-two hours. In the untreated wastewater, the concentrations of glucose and ammonium chloride were 17257 mg/L and 5500 mg/L, respectively. The concentrations of sodium nitrite and sodium nitrate were increased with operation time. The concentrations of COD, ammonium, nitrite nitrogen, nitrate nitrogen, and total nitrogen were examined. Meanwhile, the voltage was recorded by computer. And the composition of biogas produced in anode chamber was analyzed.The COD concentrations of both influent and effluent were measured using a standard method . The pH The pollutants' concentrations of influent and effluent were examined in the experiment. The experimental results were shown in 2, and 1.73E − 5%, respectively. This indicated that the amount of electrons used to produce electricity was stable. Then, the voltage and power density decreased within the operation time. In other words, the voltage and power density decreased with the concentrations of nitrate and nitrite increasing. The voltage and power density decreased to 180 mV, 17 mW/m2, and 9.14E − 6%, respectively. It might be due to the excess of the electron accepters (nitrate and nitrite). The electrons were mainly used by electron accepters. This indicated that proper management of electron accepters was very crucial for obtaining high voltage/power density in a MFC [As shown in in a MFC .The results mentioned in In order to prove that nitrite and nitrate were removed by denitrification, the biogas produced in the anode chamber of AFB-MFC system was analyzed. Firstly, the biogas was collected when the influent concentrations of COD, ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen were 20821 mg/L, 1503 mg/L, 3917 mg/L, and 1427 mg/L, respectively. The corresponding removal rates were 87.50%, 14.41%, 97.59%, and 92.06%, respectively. The influent pH was 7.78, and the conductivities of the influent and catholyte were 18.01 mS/cm and 7.17 mS/cm, respectively. Then, the collected biogas was analyzed by a gas chromatograph, and the nitrogen, methane, and carbon dioxide were qualitatively analyzed. The results  showed tGlucose degradation equation in anode/anode chamber:Methanation equation in anode chamber:2−-N in anode chamber:Denitrification equation for NO3−-N in anode chamber:Denitrification equation for NOThe overall denitrification equation in anode chamber:The reaction equation in cathode:The results have demonstrated that nitrite and nitrate nitrogen could be removed by denitrification in anode chamber of AFB-MFC system. The synthetic wastewater with high COD, ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen could be efficiently treated by AFB-MFC system. Meanwhile, proper management of electron accepters was very important for obtaining high voltage/power density in a microbial fuel cell. However, ammonia nitrogen in anode chamber of AFB-MFC system could not be removed. Nitrogen, carbon dioxide, and methane were detected in the biogas which was collected from the anode chamber. These indicated that organic matter was transformed into carbon dioxide and methane, and nitrite and nitrate were transformed into nitrogen gas.


The Julianaceae bark and leaf extracts inhibited M. tuberculosis (>84.67%) and HIV-RT (<49.89%). The Clusiaceae leaves extracts also inhibited both targets (>58.3% and >67.6%), respectively. The IC50 values for six selected extracts and their cytotoxicity (50 µg/mL) to human macrophages were then determined. Amphipterygium glaucum, A. molle, and A. simplicifolium fairly inhibited M. tuberculosis with IC50 of 1.87–2.35 µg/mL; but their IC50 against HIV-RT was 59.25–97.83 µg/mL. Calophyllum brasiliense, Vismia baccifera, and Vismia mexicana effect on M. tuberculosis was noteworthy (IC50 3.02–3.64 µg/mL) and also inhibited RT-HIV (IC50 26.24–35.17 µg/mL). These 6 extracts (50 µg/mL) presented low toxicity to macrophages (<23.8%). The HPLC profiles of A. glaucum, A. molle, and A. simplicifolium indicated that their antimycobacterial activity cannot be related to masticadienonic, 3α, or 3β-hydromasticadienonic acids, suggesting that other compounds may be responsible for the observed activity or this might be a synergy result. The anti-HIV-RT and antimycobacterial activities induced by C. brasiliense can be attributed to the content of calanolides A, B, as well as soulatrolide.The extracts of 14 Julianaceae and 5 Clusiaceae species growing in Mexico were tested  Mycobacterium tuberculosis. In 1993, TB declared a global emergency by the World Health Organization [ Mycobacterium tuberculosis is facultative intracellular bacteria that have developed resistance to first and second line antituberculosis drugs. Antibiotic resistance and multidrug-resistant TB strains are a serious problem due to the lack of results in treatment design directed to disease control and eradication [Tuberculosis (TB) is an illness caused by the slow-growing acid-fast bacillusnization . Mycobacdication . Due to dication . Compoundication –8, as we Amphipterygium adstringens (Julianaceae) is a promising source of anti-TB compounds, since the stem bark extract inhibited in 95% the growth of M. tuberculosis at 50 μg/mL; this tree species is used in Mexican Traditional Medicine for the treatment of tuberculosis and other respiratory diseases [ M. tuberculosis or HIV. Julianaceae species are dioecious; that is, male and female trees are found; some morphological features are useful for sex differentiation; for instance, female specimens show flowers ordinarily in groups of four in a receptacle [ A. adstringens bark, an accumulation of masticadienonic and 3α-hydroxymasticadienonic acids has been found to be higher in female plants [Preliminary data indicate thatdiseases . Howeverceptacle . So far,e plants . M. tuberculosis. Among the 5 most active species against HIV-1 RT, the tropical tree Calophyllum brasiliense is remarkable [ M. tuberculosis [ C. brasiliense has also been proposed for developing a standardized phytodrug; however, to achieve this goal, there is a need to obtain biological material with a high content of active compounds [The leaf extracts of the 23 species of Clusiaceae distributed in Mexico have been examined against HIV-1 RT , but notmarkable : its leamarkable  and M. trculosis . The calrculosis ; this corculosis . The hexompounds , 17. The Mycobacterium tuberculosis H37Rv and HIV-RT. Plants were selected according to two criteria: Julianaceae species, based on their use to treat tuberculosis in Mexican Traditional Medicine [The aim of this study was to evaluate Mexican Julianaceae and Clusiaceae crude plant extracts againstMedicine , whereasClusiaceae and Julianaceae species were collected from different localities in Mexico . Voucher2Cl2–MeOH . The extracts were concentrated in vacuo to dryness and stored at room temperature until use.The leaves of Clusiaceae species were used for preparing the tested extracts, whereas, in the case of Julianaceae, the extracts were prepared from the bark and leaves of specimens of different genders . All plant materials (100 g) were dried at room temperature under darkness, ground, and macerated three times for 24 h with a mixture of CHμg/mL and sterilized by filtration throughout a 0.22 μm PTFE membrane. For M. tuberculosis susceptibility tests, extract solutions were prepared by diluting the stock extract in sterile 7H9 broth to obtain a 100 μg/mL concentration, whereas extract solutions for anti-RT tests were diluted in the buffer provided by the kit manufacturer to obtain a working concentration of 200 μg/mL .Stock solutions of all extracts were prepared in 100% dimethyl sulfoxide (DMSO) at a concentration of 2000 2 Falcon culture flasks under standard culture conditions of 5% CO2 at 37°C at an initial density of 1.0 × 106 cells/mL. The cultures were maintained by adding fresh medium with 10% fetal bovine serum every 2-3 days.To assess cytotoxicity, human monocytic leukemia THP-1 cells from ATCC were cultured in RPMI 1640 medium supplemented with nonheat-inactivated 20% fetal bovine serum, 1 mM HEPES. For all experiments, THP1 were cultured in 75 cm C. brasiliense leaves were analyzed by HPLC (Agilent 1100 series) according to previous reports [ 1, masticadienonic acid 2, 3α-hydroxymasticadienonic acid 3, and 3β-hydroxymasticadienonic acid 4 were quantified, whereas, for C. brasiliense, the concentrations of apetalic acid 5, calanolide B 6, and soulatrolide 7 were determined  in a proportion 18 : 52 : 30 v/v for 25 min with an isocratic flowrate of 1.0 mL/min; the injection volume was 10 μL, and the elute was analyzed at 215 nm. Each analysis was followed by a 5 min washing with 100% acetonitrile and an equilibration period with the mobile phase for 15 min.The bark extracts of Julianaceae and reports , 19. In termined . The chr C. brasiliense extract were quantified using the chromatographic column Kromasil 100 C18, 5 μm, 250 × 4.6 mm. The isocratic system acetonitrile water (6 : 4 v/v) with the flowrate of 1 mL/min was used for 40 min; the injection volume was 10 μL and the detection wavelength 284 nm. Each analysis was followed by a 5 min washing with 100% acetonitrile, 2 min with water, and an equilibration period with the mobile phase for 3 min.The components ofμg/mL) whereas standards from C. brasilense, in six different concentrations . The linear regressions and their coefficients of determination (R2) were calculated for each compound as follows: oleanolic acid 1, y = 3.7085x − 17.043, 0.9987; masticadienonic acid 2, y = 10.766x + 4.3811, 0.9990; mixture of 3α and β-hydroxymasticadienonic acids (3 & 4), y = 11.466x + 22.14, 0.9993; apetalic acid 5, y = 19.547x + 135.64, 0.9933; calanolide B 6, y = 27.786x + 13.369, 0.9995 and soulatrolide 7, y = 35.075x + 209.85, 0.9934. Finally, the percentage of each compound in the extracts was calculated interpolating the linear regression equation. The results are reported as the percentage of extract (Identification of the compounds in the extracts was carried out by comparison with the retention times (RT) of pure compounds. The calibration graphs of standards were calculated and each compound was injected by triplicate over two different days; Julianaceae compounds were injected in seven different concentrations , was used as a positive control from 0.01 μM to 1 mM with increments of 1 logarithm.The extracts were evaluated by a nonradioactive immunocolorimetric assay  according to the protocol provided by the manufacturer. All extracts were first tested at 50 μL) to prevent dehydration in experimental wells. Colum 2 (B to G wells) was used to evaluate the reference drug rifampin; serial twofold dilutions in 100 μL of Middlebrook 7H9 medium were performed to obtain concentrations from 2.0 to 0.06 μg/mL. Wells 10 E and F were used for DMSO control, and wells 11 B to 11 E for the drug free control. One-hundred μL of supplemented 7H9 broth plus 100 μL the bacterial inoculum (1 × 106 ufc/mL) was added to each of these wells. Simultaneously a diluted control 1 : 100 was prepared from the bacterial suspension, representing 1% growth of the bacterial population tested. All other wells received 100 μL of the extract solution (100 μg/mL) and 100 μL bacterial inoculums. The final concentration of DMSO in well was <1.0% v/v, and all extracts were tested at 50 μg/mL. The IC50 values were calculated only for those extracts that inhibited ≥50% the mycobacterial growth; these extracts were tested at seven concentrations (3.125 to 200 μg/mL) with increments of 0.3 logarithms. Each microplate was incubated for 7–10 days at 37°C; after incubation, one growth control was developed with a mixture of 20 μL of Alamar blue solution (ABD Serotec) and 5 μL of sterile 20% Tween 80. The plates were reincubated at 37°C for 24 h. After this period, if the control well turned from blue (no growth) to pink (growth), the remaining wells were treated with Alamar-Tween, as previously described, and incubated for additional 24 h. Reduction of Alamar blue was calculated according to the manufacturer protocol. Optical density of the plate was measured at 540 and 600 nm with a spectrophotometer. The percentage of inhibition of the crude extracts was defined as 100 − percentage of reduction of Alamar blue.The activity of all extracts was tested using the microplate Alamar blue assay as previously described , 21. Out2 atmosphere. After the incubation, the plates were washed twice with RPMI supplemented medium and 100 μL of the extract solutions (50 μg/mL) was added to each well and reincubated for 24 h. After the reincubation time 10 μL of Alamar blue solution was added to each well, and the plates were reincubated for 24 h. The anthracycline doxorubicin was used as a positive control, and the data were interpreted as indicated by the manufacturer. Cytotoxicity was calculated as the ratio of the average OD (570 and 600 nm) obtained as compared with control wells (untreated macrophages).Crude extracts were evaluated against human macrophages THP1 cell line. The differentiation of THP1 cells was performed with PMA (phorbol 12-myristate 13 acetate) 50 nM . Twenty  A. adstringens, which inhibited 95% of mycobacterial activity at the same concentration [ C. brasiliense showed similar potency (82%) as compared with Julianaceae; the other Clusiaceae species inhibited the growth of M. tuberculosis H37Rv in the range of 58.3 to 70.3%. Concerning HIV-RT, the Clusiaceae extracts showed inhibition in a range of 27.3 to 67.6%, whereas the Julianaceae extracts inhibited this enzyme in the range of 7.9 to 49.8% . These results are consistent with previously published data forntration . Regardito 49.8% . Since mtrations .50 values were calculated for six species selected for their high activity in both targets. Three of them were Julianaceae (stem bark) and three Clusiaceae (leaves)  to 97.8 μg/mL (A. glaucum). Regarding HIV-RT inhibitory properties of plant extracts, several authors have pointed out that an IC50 ≤ 50 μg/mL may be considered potent [ M. tuberculosis. Assuming the same parameter, the extracts from C. brasiliense, V. baccifera, and V. mexicana displayed similar potency to both targets, while the extracts from A. glaucum, A. molle, and A. simplicifolium were potent only to M. tuberculosis (The IC(leaves) . The sixd potent , 24; howrculosis . 2 and α- and/or β-hydroxymasticadienonic acids (3 and 4). Under the chromatographic conditions used, quantifying individually the isomers 3 and 4 was not possible due to their similar retention times ; therefore, these compounds were quantified as the mixture of acids. Oleanolic acid 1, which has been previously reported as a constituent of A. adstringens bark, was not detected in the extracts studied ( 2 and mixture of 3 and 4 may not be related to M. tuberculosis activity since these three compounds from the most active extracts (IC50 < 2.35 μg/mL) were found in high , medium (A. simplicifolium), and low  content. In addition, the other Julianaceae bark extracts which also showed significant antimycobacterial activity (>84.6% at 50 μg/mL) showed no correlation with the concentrations of the analyzed compounds, as they include the species with the highest concentrations of 2, 3, and 4 , but also the species devoid of these compounds . The above findings suggest that the antimycobacterial active principle in the Julianaceae extracts is not compound 2, 3, or 4. The same can be stated for HIV-RT, since almost all of these extracts, with the exception of A. simplicifolium, showed poor activity (Tables The HPLC analysis of the three selected Julianaceae bark extracts showed the following metabolites: masticadienonic acid studied . The con 2 and 3α-hydroxymasticadienonic 3, as compared to extracts from female specimens ( 2 and 3 was higher in female plants [With regard to gender and production of secondary metabolites, male specimens showed the higher levels of masticadienonic acidpecimens . Our rese plants . C. brasiliense was analyzed by HPLC (In the case of Clusiaceae species, only by HPLC . ApetaliHIV infection decreases the number of CD4+ lymphocytes, so it is quite probable that an HIV+ patient can acquire or reactivate tuberculosis disease . During  Amphipterygium species had a promising activity against TB [ M. tuberculosis; however, these extracts showed moderate or poor activity to HIV-RT. No correlation with the content of triterpenoids as masticadienonic acid, 3α, and 3β-hydromasticadienonic acids was detected for anti-TB or anti-RT activities for the extracts of these species and deserves future investigations in order to identify the active compounds. According to our results Amphipterygium species are a source of potent anti-TB extracts with low cytotoxicity to macrophages.A previous report indicated that oneainst TB , and our M. tuberculosis. In particular, C. brasiliense organic extract from the leaves could be suitable for developing a phytodrug due to its content of active molecules to both targets and the calanolides A, B, C, and soulatrolide. Our results also evidence that biodiversity is a useful and valuable source for molecular leads aimed to M. tuberculosis and HIV. To date it has been described at least 84 natural compounds active against M. tuberculosis [ in vitro against HIV [A previous report indicated that the five Clusiaceae species here examined have moderate to high activity against HIV-RT , and ourrculosis . On the inst HIV . Only a  Amphipterygium simplicifolium, A. glaucum, and A. molle have been showed. These activities are not related to the triterpenes quantified in this study and suggest that other compounds are the active molecules. Our results provide sustenance to the use of species of Julianaceae plants in Mexican Traditional Medicine in the treatment of tuberculosis. Concerning Clusiaceae, the leaf extracts of the 5 species tested showed good activity against both targets. All the extracts showed low toxicity to human macrophages. Calophyllum brasiliense extract may be suitable for developing a phytodrug with dual activity against HIV-1 and M. tuberculosis due to its content of the active molecules calanolides and soulatrolide.In this study, the high antimycobacterial and moderate anti-HIV-RT activities of Julianaceae bark extracts, especially


The data presented here are the biochemical parameters of 156 patients with hyperphenylalaninemia. PKU patients, who, in order to maintain optimal serum Phe concentrations, receive dietary treatment consisting of a diet low in natural protein supplemented with special low protein foods and a Phe-free amino acid mixture, vitamins and minerals. The obtained data reflects a high percentage of patients with prealbumin and selenium deficiencies, as well as an increased level of folic acid. This data article is related to the research article entitled, “Vitamin and mineral status in patients with hyperphenylalaninemia”, by Crujeiras et al.  Patients were followed in their center from diagnosis to date. We included both patients diagnosed by newborn screening as well as those diagnosed later. Those who underwent sporadic monitoring were excluded, as well as those cases in which informed consent was not signed by parents and/or patients.12, folic acid, ferritine, 25-hydroxy vitamin D (25-OHD).The variables collected for each patient were: age, sex, phenotype (mild hyper-Phe (MHPA) 120–360 μmol/L; mild-moderate PKU 360–1200 μmol/L (MPKU); classic PKU>1200 μmol/L (CPKU) : Total protein: 6.3–8.5 g/dL; calcium (Ca): 8.7–10.8 mg/dL; phosphorus (P): 2,6-5.9 mg/dL, prealbumin: 21–41 mg/dL; vitamin B212In PKU patients, micronutrient deficiencies but no clinical symptoms have been reported, mainly for Zn, Se, Fe, folate and B12, ferritin, and Zn levels were in the normal range in almost all patients. Nevertheless, Palb was decreased in 34.6% of patients (74% from PKU) being the 94.4% of them below 18 years old and having the 96.3% an adequate adherence to treatment. Se was found to be diminished in 25% of patients, 76.9% of them with classic PKU but only in 2.56% of patients under BH4 treatment.Total protein, Ca, P, BAn adequate intake of Ca, P and 25-OHD is necessary for optimal bone accretion but it is not enough for bone normal development because several studies have showed that protein intake deficiency can influence the bone mass 12 with respect to non-treated . No other anthropometric parameters were found to be correlated with any variable in our study.Patients treated with BH4 presented lower levels of BWe noted in our study a high percentage of PKU patients with good adherence to diet with Prealbumin deficiency and total protein normal. Also a high percentage of PKU patients with Se deficiency had folic acid increase. On the contrary, only el 2.56% de patients under BH4 treatment showed Se deficiency.


DNA Research, 2005, 12(1), 39–51; doi: 10.1093/dnares/12.1.39x- and y-axes were not visible in the original published version. The authors would like to apologize for these errors.The authors did not fully acknowledge the original publication when they reused part of a figure in their Fig.


Trichoderma obtained from samples of wood collected in the forests of the Gorce Mountains (location A), Karkonosze Mountains (location B) and Tatra Mountains (location C) in Central Europe and to examine the cellulolytic and xylanolytic activity of these species as an expression of their probable role in wood decay processes. The present study has led to the identification of the following species and species complex: Trichoderma atroviride P. Karst., Trichoderma citrinoviride Bissett, Trichoderma cremeum P. Chaverri & Samuels, Trichoderma gamsii Samuels & Druzhin., Trichoderma harzianum complex, Trichoderma koningii Oudem., Trichoderma koningiopsis Samuels, C. Suárez & H.C. Evans, Trichoderma longibrachiatum Rifai, Trichoderma longipile Bissett, Trichoderma sp. , Trichoderma viride Schumach. and Trichoderma viridescens complex. Among them, T. viride was observed as the most abundant species  in all the investigated locations. The Shannon’s biodiversity index (H), evenness (E), and the Simpson’s biodiversity index (D) calculations for each location showed that the highest species diversity and evenness were recorded for location A—Gorce Mountains . The preliminary screening of 119 Trichoderma strains for cellulolytic and xylanolytic activity showed the real potential of all Trichoderma species originating from wood with decay symptoms to produce cellulases and xylanases—the key enzymes in plant cell wall degradation.The aim of this study was to explore the species diversity of The online version of this article (doi:10.1007/s13353-015-0326-1) contains supplementary material, which is available to authorized users. Trichoderma (teleomorph Hypocrea) are found in many ecosystems, but the most common and natural habitat of these fungi is known to be soil  Bainier, Trichoderma atoviride P. Karst., Trichoderma virens  Arx, Trichoderma lonibrachiatum Rifai, Trichoderma gamsii Samuels & Druzhin., Trichoderma citinoviride Bissett, Trichoderma koningiopsis Samuels, C. Suárez & H.C. Evans, Trichoderma spirale Bissett and Trichoderma koningii Oudem., which are known to generally have outstandingly high opportunistic potential. These species succeed in various heterotrophic interactions exhibiting saprotrophic and mycoparasitic  lifestyles , hemicellulose  and lignin (a complex polyphenolic structure), which together with proteins, fats, waxes, terpenes, phenols, alcohols and alkanes form the complex and rigid structure of the plant cell walls. Degradation of plant cell wall biopolymers is a complex process, which requires the synergistic action of a large number of extracellular enzymes. Several Trichoderma species, especially Trichoderma reesei, are known to be producers of cellulolytic and hemicellulolytic enzymes  lifestyle of Trichoderma  and associated Trichoderma parareesei nom. prov. have been found worldwide in pantropical climatic zones , five of the above-mentioned species  were also isolated, mostly from pieces of decaying wood , Karkonosze Mountains (location B) and Tatra Mountains (location C) in Central Europe and to examine the cellulolytic and xylanolytic activities of these species as an expression of their probable role in wood decay processes.As saprotrophs, Trichoderma strains investigated in this study were obtained from samples of decaying wood collected in the forests of the Tatra Mountains, Gorce Mountains and Karkonosze Mountains, located in Central Europe in Poland , Gorce Mountains  and Karkonosze Mountains  located in Central Europe  and incubated as above. Pure cultures were subsequently transferred to tubes containing SNA and stored at 4 °C for further study.The pieces of wood with white or brown rot were packed in paper bags, dried at room temperature if wet, and stored until isolation. In the laboratory, small sterile samples (0.5 × 0.2 cm) were taken from each piece of wood, plated on Synthetischer Nährstoffarmer Agar  gene. Mycelium for DNA extraction was obtained as described previously  as well as TrichOKEY and TrichoBLAST  obtained in this work as well as on the sequences retrieved from NCBI GenBank (http://www.ncbi.nlm.nih.gov) as the closest matches: DQ109573, EU280135, DQ677646, DQ677647, DQ677648, DQ841735, DQ323428, DQ323431, DQ841745, FJ860726, EU871028, FJ860763, FJ442236, KP115288, FJ442276, KP115287, FJ442273, AY737760, EU280074, FJ860799, EU401564, X93963, DQ672607, DQ672606, DQ672605, EU871012, FJ860611, DQ841725, DQ841721, FJ860642, DQ109549, FJ179578, EU401613, AY737736, KP115273, FJ463360, KJ665699, FJ463289, KP115274, FJ463395, JN175595, EU280051. Fusarium graminearum  was chosen as outgroup. DNA sequences were initially aligned with CLUSTAL W , Brno, Czech Republic was used as the reference strain. The Trichoderma collection of 119 isolates were screened for cellulase and xylanase activity under the same conditions, specifically optimized for cellulase/xylanase production with T. reseei QM 9414 strain. Trichoderma isolates were first cultured on PDA medium in Petri dishes for 3 days at 25 °C. Following incubation, the agar discs  were cut from the sporulated cultures and transferred into 30 mL of autoclaved induction medium (pH 5.5) consisting of (per liter of distilled water) 10 g carboxymethyl cellulose , 2 g ammonium phosphate, 2 g potassium orthophosphate, 0.3 g magnesium sulfate, 0.3 g calcium chloride, 1 g bacteriological peptone , 2 g yeast extract , 30 mg libremix  and 0.001 g cobaltous chloride. The incubation was carried out in 100 ml Erlenmeyer flasks without shaking at 25 °C and optimally for 5 days (in the case of cellulase production) and 7 days (in the case of xylanase production). All analyses were done in triplicate.g). Total cellulase activity was determined using the filter paper activity (FPA) assay  birch wood xylan  in 0.5 mL of 50 mM citrate buffer (pH 4.8), supplemented with 0.5 mL enzyme solution (supernatant). The reaction mixture was incubated at 50 °C for 15 min. Released reducing sugars were determined by dinitrosalicylic acid reagent (DNS) method of Miller  was calculated according to the formula: H = −Σpi * lnpi, where pi is the proportion of ith species Shannon . The Shaα = 0.05).The statistical analysis was performed using the programs Statistica version 10.0 (StatSoft Inc.) and Microsoft Excel version 2010. Inference about the significance of differences between enzyme activity of isolates was conducted using one-way ANOVA and was determined using Tukey’s test (Trichoderma isolates obtained from samples of decaying wood collected in the forests of the Tatra Mountains (location C), Gorce Mountains (location A) and Karkonosze Mountains (location B) in Poland were identified at the species level based on morphological as well as ITS1, ITS2 and tef1 sequencing data. Finally, 12 species or species complex were found: T. atroviride, T. citrinoviride, T. cremeum P. Chaverri & Samuels, T. gamsii, T. harzianum complex, T. koningii, T. koningiopsis, T. longibrachiatum, T. longipile Bissett, Trichoderma sp. , T. viride Schumach. and T. viridescens complex. Furthermore, the tef1 sequences analysis of the isolates belonging to the T. harzianum species complex and T. viridescens species complex revealed five isolates as Trichoderma atrobrunneum F.B. Rocha, P. Chaverri & W. Jaklitsch, 11 isolates as Trichoderma harzianum Rifai, one isolate as Trichoderma viridarium Jaklitsch, Samuels & Voglmayr, three isolates as Trichoderma trixiae Samuels & Jaklitsch and ten isolates as Trichoderma paraviridescens Jaklitsch, Samuels & Voglmayr. The identification, origin and NCBI GenBank accession numbers of all isolates are given in Table Trichoderma strains originated from decayed wood collected in a mountain forest in Poland and selected from NCBI GenBank as close relatives, the phylogenetic analysis were performed. The results were presented as a consensus ITS tree and tef1 tree  only from location C. On the other hand, four species  were found in all the locations. Among the 12 species described in this study, T. viride was the most abundant species . It was also observed as the most dominant fungus in all the locations investigated. Another very common species complex  occurring in the three locations was T. viridescens. After T. viride and T. viridescens complex, T. harzianum was commonly isolated  from samples of decaying wood. However, the presence of this species complex was mainly ascertained in location C. Whereas, the other species  were represented by several strains and accounted for the minority of isolates .The analysis were based on the 119 ń et al. . GroupinE = 0.82, D = 0.79). The Shannon and Simpson diversity indexes, estimated for the other two sites showed, that location C had higher species diversity  than location B , while the species evenness measure resulted in E = 0.67 for location B and E = 0.64 for location C. Consequently, location C was characterized by the least homogenous distribution of species. The overall diversity values for Trichoderma species originating from decaying wood in Polish mountains were H′ = 1.53, E = 0.62 and D = 0.67.The obtained data were used to calculate the Shannon’s biodiversity index (H), evenness (E) and the Simpson’s biodiversity index (D) for each location and the total population. The highest species diversity and evenness were recorded for location A  and between species , and T. longibrachiatum species were found to be efficient producers of cellulolytic activity. Trichoderma harzianum complex, T. koningii and T. citrinoviride species displayed moderate cellullase activity. A lower cellulose-decomposing potential was recorded for T. koningiopsis, T viride, T. atroviride, T. longipile, Trichoderma sp. (H. parapilulifera) and T. viridescens complex. Considering the diversity within T. atroviride species, cellulase activity ranged from 0.135 ± 0.001 (isolate AN705) to 0.201 ± 0.035 (isolate AN240) FPU/mL and within T. citrinoviride species, this activity ranged from 0.135 ± 0.002 (isolate AN710) to 0.281 ± 0.009 (isolate AN303b) FPU/mL. The activity produced by two isolates of T. koningii (AN398 and AN399) was 0.267 ± 0.001 and 0.200 ± 0.028 FPU/mL, respectively. In the set of 16 isolates of T. harzianum complex there were isolates  identified as T. atrobrunneum, that showed weak cellulose-decomposing potential, but there were also five isolates of T. harzianum  with high decomposition activity towards cellulose (over 0.3 FPU/mL). Cellulase activity displayed by isolates belonging to T. viridescens species complex ranged from 0.074 ± 0.003 (T. paraviridescens AN328) to 0.271 ± 0.002 (T. paraviridescens AN702) FPU/mL. However, the variation in cellulase activity differed significantly among the largest group of T. viride isolates, the lowest value being 0.075 ± 0.004 (isolate AN340), whereas the highest was as high as 0.283 ± 0.000 FPU/mL for isolate AN701.A total of 119 ń et al. , belongiń et al. . Howeveres Figs.  and 3. TT. gamsii was found to be the best producer of xylanolytic activity (over 2.4 U/mL), in contrast to T. citrinoviride, which was characterized as the least effective decomposer of xylan , whereas the highest was 2.396 ± 0.011 U/mL (AN499). Similarly, xylanolytic activity varied among isolates of T. atroviride from 1.808 ± 0.003 (AN 705) to 2.519 ± 0.006 (AN497) U/mL. Within T. harzianum species complex, the highest xylanase activity was observed for the T. harzianum AN369 isolate (2.887 ± 0.022 U/mL) and the lowest for the T. harzianum AN480 isolate (1.634 ± 0.001 U/mL). Xylanase activities of two T. koningii isolates, AN399 and AN398, were 2.001 ± 0.0077 and 2.052 ± 0.095 U/mL, respectively. Among the 63 isolates of T. viride, the most effective in xylan degradation were three isolates: AN813 (3.100 ± 0.016 U/mL), AN810 (3.076 ± 0.008 U/mL) and AN827 (3.070 ± 0.008 U/mL), whereas AN242 was observed to be a weak decomposer of cellulose (1.227 ± 0.004 U/mL). In the set of 20 isolates of T. viridescens species complex, the highest levels of xylanase activity were achieved by the T. paraviridescens AN702 (3.099 ± 0.004 U/mL) and AN609 (3.070 ± 0.005 U/mL) and the lowest was presented by the T. paraviridescens AN709 isolate (0.946 ± 0.001 U/mL).However, the variation in xylanolytic activity differed significantly among the Trichoderma. Eight Trichoderma species and species complex, namely T. atroviride, T. citrinoviride, T. harzianum complex, T. koningii, T. viride, T. viridescens complex, T. koningiopsis and T. gamsii, have also been isolated from decaying wood collected in parks and forests of a lowland region of Poland in our preliminary investigations  from samples of decaying wood. Recently, the taxonomic of T. harzianum species complex was revised to include at least 14 species . However, this species has been isolated from soil, decaying wood and other fungi  obtained in the present research from decaying wood in the Gorce Mountains (location A). Previously, only its teleomorph (Hypocrea cremea) had been found by Chaverri et al.  H. Karst. (Hess Picea abies (L.) H. Karst, Fagus sylvatica L., and Abies alba Mill., while admixture species include larch, elm, ash and grey alder (Loch and Armatys Picea abies (L.) H. Karst with low occurrence Abies alba Mill. and Fagus sylvatica L. and isolated individuals of Pinus cembra L. and Larix decidua Mill.  was found as the least homogenous. It is worth recalling that in November 2004, spruce stands in the Tatra National Park on the area of 12,000 hectares were affected by a catastrophic windthrow and 1 year later (in July 2005) wildfire broke out on a part (220 ha) of this area , and the lowest levels of species richness at location B (Karkonosze Mountains). We attribute this phenomenon to differences in the biodiversity of trees, and consequently, the type of wood at the different locations. The majority of the Karkonosze National Park consists of forests dominated by st. Hess . The vegst. Hess . The mosTrichoderma strains investigated in this study were isolated from pieces of wood with decay symptoms. Consequently, the question arose as to whether all these strains are able to participate in decomposition of particularly xylan and cellulose containing organic matter. The cellulolytic and xylanolytic activity assay is one of the approaches to the preliminary determination of the potential of the strains for enzymes production and has been widely used in many studies for screening of a broad spectrum of fungal isolates and environmental samples  and birch wood xylan under assay conditions used. The possibility of cellulase and hemicellulase complex formation by T. atroviride, T. citrinoviride, T. gamsii, T. harzianum, T. koningii, T. longibrachiatum, T. viride and T. viridescens complex, has already been stated in previous studies  and immuno-compromised human pathogens, such as T. longibrachiatum  has evolved at least twice  and then reversed to mycoparasitism possibly twice . Chaverri and Samuels  and 22 sequences retrieved from GenBank. Fusarium graminearum was used as an outgroup in the analysis. The ITS sequences analysis involved 42 nucleotide sequences corresponding to a total of 444 characters in the final dataset, of which 318 were conserved, 126 were variable and 86 parsimony informative. Maximum parsimony analysis led to 434 equally parsimonious trees, with similar clade topologies and tree length 107, consistency index CI = 0.850, retention index RI = 0.962 and rescaled consistency index RCI = 0.818. The sequence accession numbers obtained from NCBI GenBank (http://www.ncbi.nlm.nih.gov) are shown in parentheses. Bootstrap values higher than 50 % (1000 replicates) are shown at each branch. (GIF 83 kb)The consensus tree of maximum parsimony analyses based on ITS sequences dataset of 19 selected High resolution (TIF 36716 kb)Online resource 2tef1 sequences dataset of 19 selected Trichoderma strains investigated in present study  and 22 sequences retrieved from GenBank. Fusarium graminearum was used as an outgroup in the analysis. The tef1 sequences analysis involved 42 nucleotide sequences and there were a total of 463 positions in the final dataset, of which 178 were conserved, 284 were variable and 266 parsimony informative. Maximum parsimony analysis generated 26 equally parsimonious trees of 436 steps length with consistency index CI = 0.566, retention index RI = 0.853 and rescaled consistency index RCI = 0.484. The sequence accession numbers obtained from NCBI GenBank (http://www.ncbi.nlm.nih.gov) are shown in parentheses. Bootstrap values higher than 50 % (1000 replicates) are shown at each branch. (GIF 88 kb)The consensus tree of maximum parsimony analyses based on High resolution (TIF 36764 kb)Online resource 3Trichoderma species. Values represent mean ± standard error of three independent analyses (assay of cellulase/xylanase activity) for each strain. (DOC 126 kb)Cellulolytic and xylanolytic activity of 120 isolates belonging to different Below is the link to the electronic supplementary material.


The risk of cardiovascular diseases (CVD) is increased tremendously among menopausal women, and there is an increasing demand for alternative therapies for managing factors like dyslipidemia that contribute to CVD development.Nigella sativa was evaluated for its hypolipidemic effects among menopausal women. In a randomised trial, hyperlipidemic menopausal women were assigned to treatment (n = 19) or placebo groups (n = 18), and given N. sativa or placebo for two months after their informed consents were sought. At baseline, blood samples were taken and at one month intervals thereafter until one month after the end of the study.In this study, N. sativa significantly improved lipid profiles of menopausal women  more than the placebo treatment over 2 months of intervention. One month after cessation of treatment, the lipid profiles in the N. sativa-treated group tended to change towards the pretreatment levels.The results showed that N. sativa is thought to have multiple mechanisms of action and is cost-effective. Therefore, it could be used by menopausal women to remedy hypercholesterolemia, with likely more benefits than with single pharmacological agents that may cause side effects. The use of N. sativa as an alternative therapy for hypercholesterolemia could have profound impact on the management of CVD among menopausal women especially in countries where it is readily available. Also, CVD continues to cause significant morbidity and mortality in those women,2. In faponsible. Particuponsible. The useponsible.Nigella sativa commonly known as black seed has been widely used in many Asian, Middle Eastern and Far Eastern countries as a spice and food preservative as well as a health remedy in traditional medicine for the treatment of numerous disorders. This plant has been a great focus of research and has several traditional uses and consequently has been studied extensively for its therapeutic properties[N. Sativa, which contribute to its cardioprotective effects[N. sativa on lipid parameters of menopausal women. Thus, we studied the effects of N. sativa seed powder on serum cholesterol, HDL-and LDL-cholesterol and TG in menopausal women for possible hypolipidemic activity.Although it is a physiological transition in a woman’s life, menopause is accompanied by problems, and hence women at this age must be managed appropriately for any health risks or problems, to ensure a fulfilling life. Nowadays, there is an increasing demand for plant bioresources in the management of chronic diseases like CVD, instead of synthetic drugs, to avoid side effects. Moreoveoperties. Its medoperties. Further effects-16. HoweN. sativa were imported from Iran through a local company named Sari Tani Desa SDN BHD located in Shah Alam, Malaysia. Voucher specimens of seeds were kept at the Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia and the seed was identified and authenticated by Professor Dr. Maznah Ismail, Head of the Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Malaysia. Further, N. sativa seeds were processed into pharmaceutical grade capsules containing N. sativa powder and bottled according to Good Manufacturing Practices (GMP No.: MALLP20121468). Each capsule was prepared containing 500 mg powder of N. Sativa, and each bottle contained 60 capsules, stock for a month period to help with compliance of the respondents. While for the placebo, we used wheat germ which was bought from the same company, Sari Tani Desa Sdn. Bhd. and the entire process of wheat germ capsulation was same as N. sativa seeds, however with lesser dose of 100 mg per capsule.Seeds of All chemicals used in this study were of analytical grade, and procured from Fisher Scientific  and Merck . Reagents and diagnostics kits used for TC, LDL, HDL and TG were supplied by Randox Laboratories Limited .N. sativa in menopausal women. Thirty seven women (n = 37), who were menopausal for at least ≥ 12 months, within the age range of 45–60 years and who presented with moderate risk of hyperlipidemia were chosen as respondents in this study. Respondents were non-smokers, did not consume alcohol and did not have a history of any other chronic disease or drug/herb ingestion. Those with any of the features above were excluded from the study. These subjects were recruited among the university’s staff or ex-staff based on a health screening. Each qualified subject was informed about the study verbally and in writing according to Good Clinical Practice. These selected subjects were randomized into 2 groups (treatment and placebo) as follows: treatment group received capsulated N. sativa powder (n = 19), while the placebo group (n = 18) received the placebo capsules. The physical and pathological histories of these subjects were recorded, and their baseline anthropometric parameters measured  was drawn from the subjects at baseline and at intervals during (1 month of treatment) and after the treatment period (at the end of 2 months of treatment and one month after the end of study) for further analyses. Furthermore, at each follow up interval, blood pressure measurements were taken , Universiti Putra Malaysia (UPM). Ethical clearance was obtained from the Faculty of Medicine and Health Sciences Medical Research Ethics Committee to conduct this study , which aimed to evaluate the hypolipidemic effects of Selectra XL chemical analyzer . Fasting blood glucose was determined using a glucometer .Whole blood was collected in plain tubes and further centrifuged at 2500 rpm for 15 min under 25°C. Serum was collected for the analyses of TC, TG, HDL and LDL levels using commercial diagnostic kits  on All experimental values are presented as means ± standard deviation (SD). Statistical analysis was performed using SPSS windows program version 19 . The One-way Analysis of Variance (ANOVA) with Bonferroni correction was used for analysis of data. Difference was considered to be significant if p < 0.05.N. sativa seeds powder on lipid profiles in humans especially among those with an increased risk of dyslipidemia . The baseline characteristic of the respondents in both treatment and placebo groups are presented in Table This study was an attempt at determining the effects of N. sativa and placebo groups at baseline, 1st and 2nd months of treatment and one month after treatment ended (follow-up) were 6.09 ± 1.04, 5.69 ± 0.99, 5.11 ± 0.91, and 5.61 ± 0.79; placebo was 6.06 ± 1.02, 5.88 ± 0.77, 5.79 ± 0.68 and 5.84 ± 0.49 mmol/L, respectively. Mean TC of the respondents in the N. sativa group improved significantly, p < 0.05, by 16.09% at two months of treatment compared to baseline and significantly greater, p < 0.05, than that in placebo group.Mean TC of the N. sativa and placebo groups at baseline, 1st and 2nd months of treatment and follow-up were 1.76 ± 0.35, 1.54 ± 0.31, 1.37 ± 0.24, and 1.29 ± 0.35; 1.72 ± 0.53, 1.68 ± 0.46, 1.56 ± 0.24 and 1.47 ± 0.51 mmol/L, respectively. Similar trend of reduction in TG levels (22.16%) as TC levels was observed with two months treatment with N. sativa seeds powder which was significantly greater, p < 0.05, than the placebo group.Mean TG values of the st and 2nd months of treatment and follow-up were 4.65 ± 0.89, 3.89 ± 0.83, 3.41 ± 0.76 and 3.78 ± 0.58; 4.59 ± 0.48 and 4.42 ± 0.55, 4.34 ± 0.61 and 4.39 ± 0.28 mmol/L, respectively. Reduction of LDL-C was 26.67% with significant changes, p < 0.05 with two months of treatment of N. sativa seeds powder in comparison to baseline. Again, the improvements in LDL-C throughout the treatment period were significantly greater, p < 0.05 in the N. sativa group compared to the placebo group.While, mean changes of LDL-C in the treatment groups at baseline, 1st and 2nd months of treatment and follow-up were 1.31 ± 0.22, 1.33 ± 0.30, 1.41 ± 0.25 and 1.47 ± 0.26; 1.32 ± 0.25, 1.34 ± 0.19, 1.35 ± 0.25 and 1.38 ± 0.27 mmol/L, respectively. HDL-C was found to have improved slightly in both groups, but no significant changes (p > 0.05) were observed between the two groups. Overall, improvements of lipid profile  throughout the treatment period were significantly greater, p < 0.05 in the N. sativa group compared to the placebo group except for HDL-C levels, which was similar to that produced by placebo.Finally, HDL-C levels in both respective treatments at baseline, 1N. sativa-treated group and the control over the period of intervention, N. sativa produced better results and significant changes were observed in fasting blood glucose. The changes in these parameters also showed similar patterns to those of lipid profile; over the 2 months of intervention, the changes showed a downward trend but increased 1 month after cessation of intervention.Although the differences in blood pressures Table  were notMenopause begins 12 months after a woman's final menstrual period and is associated with a dramatic drop in estrogen levels. This change in hormone levels often contributes to adverse metabolic changes that occur during the transitional pre- and post-menopausal periods, which make women prone to diseases. It has been proposed that estrogen hormone may be responsible for the protective effects seen amongst pre-menopausal women. EstrogeN. sativa in menopausal women as part of the increasing drive to provide better insights into the effects of plant bioresources on health, and their potential use for therapy of chronic diseases. Also, increasing numbers of women are thought to use complementary medicines for disorders associated with their menopause, and studies like these could provide evidence-based rationale for their choice of plant bioresources. Specifically, about 75% of women were reported to be using herbal and complementary medicines in their menopausal period in place of hormone replacement therapy (HRT) due to considerations on its side effects[N. sativa proved to have many beneficial health effects, particularly as hypolipidemic agent in various clinical trials[N. sativa seed powder on hyperlipidemic menopausal women, while only a few have assessed the effect of N. sativa in overriectomized rats[N. sativa in menopausal women, this study was necessitated to determine the protective effects of N. sativa on hyperlipidemic menopause women. The importance of this study is underlined by the fact that the overall cause of hyperlipidemia and the mechanisms leading up to its development in menopausal women differ considerable from in premenopausal women or men in general.In this study, we determined the hypolipidemic effects of  effects. Further effects. Moreovel trials,26. To ozed rats. With thN. Sativa, compared to the placebo group. The results also indicated a tendency for N. sativa to improve glycemia and blood pressure better than the placebo. Our results are in keeping with those of Inayat et al. (2009), who reported that oral treatment with N. sativa powder in hypercholesterolemic patients at the dose of 1 g daily for two months was found to reduce TC, LDL and TG, and increase HDL to highly significant extents[N. sativa powder (500 mg) and statin in cardiac patients visiting Ch. Pervaiz Elahi Institute of Cardiology, Multan, Pakistan compared to placebo group receiving statin only[N. sativa seed extract in male patients with mild hyperlipidemia and hypertension caused a significant dose-dependent decline in the levels of TC, TG and LDL relative to baseline data[The results of the present study showed significant decrease in hyperlipidemia among menopausal women following treatment with  extents. Furthertin only. In anotine data.N. sativa seeds powder does not seem to be due only to one component, but rather likely due to synergistic action of its different constituents including thymoquinone (TQ), migellamine, soluble fiber (mucilage), sterols, flavanoids and high content of polyunsaturated fatty acids (PUFAs)[N. sativa. Earlier, we demonstrated that N. sativa was able to regulate cholesterol synthesis through regulation of HMG-CoA reductase, Apo-A1, Apo-B100 and LDL-receptor genes, an effect mediated by TQ and other N. sativa constituents[N. sativa[N. sativa, have been proposed. TQ was shown to inhibit non-enzymatic lipid peroxidation in liposome and works as a scavenger of various reactive oxygen species including superoxide anion and hydroxyl radicals[N. Sativa. Particularly, antioxidants (flavonoids) have been proposed to decrease cholesterol synthesis and suppress reactive oxygen species, nitrogen species formation and protect the antioxidant defence system[N. sativa could also contribute to decreased dietary cholesterol absorption, increased primary bile synthesis and its fecal losses. Besides, high content of PUFAs in N. sativa may also result in decreased serum TC[The hypolipidemic effects of  (PUFAs). Additio (PUFAs)-34. Multtituents-34. In fN. sativa,34. In aradicals. Antioxie system. Flavanoe system. Lipid le system and stere system in N. saserum TC,40.N. sativa caused significant decrease in TC, LDL and TG, and slight increase in HDL among menopausal women receiving N. sativa powder at a dose of 1 g daily for two months compared to placebo group. The study also demonstrated that N. sativa may improve other biochemical indices like glycemia and blood pressure in menopausal women. With an increasing demand for more alternative therapies for menopausal women due to concerns of side effects with pharmacological therapies, and considering that multiple factors play a role in the development and complication of hyperlipidemia among menopausal women, N. sativa may be used by hyperlipidemic menopausal women as a good complementary therapy. N. sativa is an easily available and cost-effective remedy to treat dyslipidemia, and its proposed multiple mechanisms of action may produce better hypolipidemic effects compared to standard pharmacological agents or other medicinal plants with a single mechanism of action. Studies like these contribute to better understanding of N. sativa effectiveness as a therapeutic agent, and could lead to its widespread use all over the world.The present study showed that CVD: Cardiovascular diseases; HDL: High density lipoprotein; LDL: Low density lipoprotein; N.S: Nigella sativa; TC: Total cholesterol; TG: Triglyceride; TQ: Thymoquinone.The authors declared no conflict of interests.RMI, NSH, MI and RM designed the study and together with MUI and SMS conducted the study, and biochemical and data analyses. RMI, MUI and SAAG drafted the manuscript, while RM, MI and LAL reviewed the manuscript and approved the final version for submission.


Lymnaea acuminata in clear glass aquaria having diameter of 30 cm. Snail attractant containing bait formulations was prepared from different binary combination (1 : 1 ratio) of carbohydrates  and amino acid  in 100 ml of 2% agar solution + sublethal (20% and 60% of 24 h and 96 h LC50) doses of different molluscicides . Snails fed on bait containing sub-lethal concentration of different molluscicides and the snail attractant, causing a significant inhibition in alkaline phosphatase (ALP) and acetylcholinesterase (AChE) activity in the nervous tissue of the vector snail L. acuminata. Maximum inhibition in ALP (20% of control) and AChE (49.49% of control) activity was observed in the nervous tissue of the L. acuminata exposed to 60% of 96 h LC50 of eugenol in the bait pellets containing starch + histidine, starch + methionine, respectively.Fascioliasis, a snail-borne parasitic zoonosis, has been recognized for a long time because of its major veterinary and human impact. Different Bait formulations were fed to the snail  Fasciola hepatica and F. gigantica in different parts of the world [Fasciola, and more than 90 million people are at risk of fascioliasis [Lymnaea acuminata is the intermediate host of the Fasciola species [L. acuminata. Fascioliasis is an important cattle and human disease caused by two major species of he world , 2. Worlioliasis . Normallioliasis , 5. In n species , 6–9. On species –16. Bait species  is an ef species  in bait L. acuminata (2.25 ± 0.20 cm in length) were collected locally from lakes and low-lying submerged fields. These snails were acclimatized for 72 hours in dechlorinated tap water at 25 ± 1°C. The pH of the water was 7.2–7.3, and dissolved oxygen, free carbon dioxide, and bicarbonate alkalinity were 6.5–7.2 mg/L, 5.2–6.3 mg/L, and 102.0–105.0 mg/L, respectively.Adult Agar-agar, carbohydrates , amino acids , and different active molluscicides such as eugenol, ferulic acid, umbelliferone, and limonene were used in bait formulations. The pure active components, eugenol (2-Methoxy-4-(2-propenyl) phenol), ferulic acid (4-Hydroxy-3-methoxycinnamic), umbelliferone , and limonene ((R)-4-Isopropenyl-1-methyl-1-cyclohexene), were purchased from Sigma chemical Co., USA. 50) concentration of molluscicides  were prepared in 100 mL of 2% agar solution by the method of Madsen, [Bait formulations containing binary combination (1 : 1 ratio) of different carbohydrates , amino acids , and sublethal . Six sets of experiments have been designed with ten snails, each for all molluscicides used in this study.The bioassay was performed by the method of Tiwari and Singh , 20. TheAfter 24 h of bait feeding, the snails were washed with water, and the nervous tissue was dissected out from snail brain and used for the measurement of enzyme activities. Alkaline phosphatase (ALP) and acetylcholinesterase (AChE) activity were measured in treated as well as control groups of snails.50 of bait for the next 72 h in fresh water. In the withdrawal experiment, ALP and AChE activities in the nervous tissue of snail fed on bait formulations were measured in withdrawn snails after 96 h feeding of 60% of 96 h LC2·6H2O and 165 mg p-nitrophenyl phosphate sodium salt in 42 mL of 0.1 N NaOH, and, mixture was made up to 100 mL with double distilled water). The mixture was mixed thoroughly and incubated for 30 min. at 37°C. 10 mL of 0.02 N NaOH was added to the incubation mixture. The reaction was stopped by the addition of an excess of NaOH. The alkaline phosphatase activity was measured colorimetrically at 420 nm which is a measure of the yellow colour of nitrophenol produced by the hydrolysis of p-nitrophenyl phosphate buffer. The enzyme activity was expressed in moles substrate hydrolyzed/30 min/mg protein.The alkaline phosphatase activity was measured by the method of Bergmeyer,  as modifL. acuminata was homogenized (50 mg/mL) in 0.1 M phosphate buffer (pH 8.0) for 5 minutes in an ice bath and centrifuged at 1000 xg for 30 minutes at 4°C. The clear supernatant was taken as an enzyme source. The enzyme activity was measured in a 10 mm path-length cuvette using an incubation mixture consisting of 0.1 mL of enzyme source, 2.9 mL of 0.1 M phosphate buffer (pH 8.0), 0.1 mL of chromogenic agent DTNB ) and 0.2 mL of freshly prepared acetylthiocholine iodide. The change in optical density at 412 nm was continuously observed on spectrophotometer for 3 minutes at 25°C. Enzyme activity was expressed as moles SH hydrolyzed/minute/mg protein.Acetylcholinesterase activity was measured by the method of Ellman et al.,  as modift-test was applied to determine the significant (P < 0.05) difference between treated and control animals [Each experiment was six times replicated estimation (measurement in six different pool of nervous tissue). The values were expressed as mean ± SE. Student's  animals . 50 of eugenol, ferulic acid, umbelliferone, and limonene in bait formulations caused a significant (P < 0.05) inhibition in alkaline phosphatase activity in the nervous tissue of snail L. acuminata (L. acuminata fed on 60% of 96 h LC50 of eugenol (P < 0.05) recovery in alkaline phosphatase activity was observed in the nervous tissue of L. acuminata earlier fed on 60% of 96 h LC50 of eugenol bait (20% of control),when discontinued for the next 72 h (34.66% of control). The sub-lethal feeding on 20% and 60% of 24 h and 96 h LC50 of eugenol, ferulic acid, umbelliferone, and limonene caused a significant inhibition in the AChE activity in the nervous tissue of the snail L. acuminata (50 of eugenol containing bait (P < 0.05) recovery in the AChE activity in the nervous tissue of the 72 h withdrawn (56.70%) snails with respect to snails fed on 60% of 96 h LC50 of eugenol bait.Sub-lethal feeding to 20% and 60% of 24 h and 96 h LCcuminata . Maximum eugenol . Significuminata . Maximuming bait . There w50 of the active components, eugenol, ferulic acid, umbelliferone, and limonene, with attractant carbohydrates and amino acids was more effective in killing the L. acuminata. Earlier, it has been reported that direct release of eugenol, ferulic acid, umbelliferone, and limonene in aquarium water has significant molluscicidal activity against L. acuminata [L. acuminata exposed to 60% of 96 h LC50 of ferulic acid and umbelliferone, respectively. In the present study, the mode of entry of molluscicides into the snail body is through the digestive system. In an earlier study, it was through the body surface, when molluscicides were released directly in water. Although the entry of molluscicide inside the body is different, both methods are equally effective in killing the snails. Snails fed with a sub-lethal dose, that is, 20% and 60% of 24 h and 96 h LC50 of different molluscicides, caused a significant inhibition in ALP and AChE activity in the nervous tissue of snail L. acuminata. The inhibition in ALP and AChE activities may be due to the direct interference of these active molluscicidal with enzyme. Kumar et al., [L. acuminata when these active molluscicides were fed to snails in bait formulations. Alkaline phosphatase plays a critical role in protein synthesis [ L. acuminata. Nagababu et al. [L. acuminata when used directly in aquarium water [The result of the present study indicates that the sub-lethal feeding on 20% and 60% of 24 h and 96 h LCcuminata . Kumar ecuminata  have demcuminata  have rep et al.,  reportedynthesis , shell fynthesis , and othynthesis  and its ynthesis , 31 in gynthesis . The AChynthesis , 33–38. ynthesis . The enzynthesis . The aceynthesis  which inu et al.  reportedu et al. . Eugenolu et al. . Althougum water , yet it um water . This co