Euglena gracilis, a photosynthetic protist, produces protein, unsaturated fatty acids, wax esters, and a unique β-1,3-glucan called paramylon, along with other valuable compounds. The cell composition of E. gracilis was investigated in this study to understand how light and organic carbon  affected growth and cell composition . Comparisons were primarily carried out in cultures grown at 23 °C, but the effect of growth at higher temperatures (27 or 30 °C) was also considered.E. gracilis was grown on glucose in either heterotrophic or mixotrophic conditions than when grown photoautotrophically, although the duration of exponential growth was longer. Temperature determined the rate of exponential growth in all cultures, but not the linear growth rate during light-limited growth in phototrophic conditions. Temperature had less effect on cell composition.Specific growth rates were slightly lower when E. gracilis was not expected to store large amounts of paramylon when grown phototrophically, we observed that phototrophic cells could contain up to 50% paramylon. These cells contained up to 33% protein and less than 20% lipophilic compounds, as expected. The biomass contained about 8% fatty acids (measured as fatty acid methyl esters), most of which were unsaturated. The fatty acid content of cells grown in mixotrophic conditions was similar to that observed in phototrophic cells, but was lower in cells grown heterotrophically. Heterotrophic cells contained less unsaturated fatty acids than phototrophic or mixotrophic cells. α-Linolenic acid was present at 5 to 18 mg g-1 dry biomass in cells grown in the presence of light, but at < 0.5 mg g-1 biomass in cells grown in the dark. Eicosapentaenoic and docosahexaenoic acids were detected at 1 to 5 mg g-1 biomass. Light was also important for the production of vitamin E and phytol.Although  Euglena gracilis, often considered with eukaryotic algae, contains compounds of commercial interest. E. gracilis accumulates paramylon  for energy and carbon storage, especially when grown on organic carbon (i.e. in mixo- or heterotrophic conditions) [E. gracilis [Microalgae, as a source of nutrients, bioactive compounds, biofuels and chemicals, have gained attention from both academia and industry –8. The uditions) ,9. Similditions)  and antiditions)  activityditions) . In anaegracilis . These egracilis ,14.E. gracilis features a high content  of E. gracilis is mostly composed of protein [E. gracilis biomass more digestible than other microalgae, which are protected by non-digestible polysaccharides [The fatty acid profile of al lipid ) of polyal lipid ,16. α-Toh amount . As a phindustry . Thus, t protein , making charides .E. gracilis grows in habitats, which vary widely in temperature and pH [E. gracilis can produce more than 3 g L-1 biomass (e.g. [E. gracilis in autotrophic cultures is around 1.1 d-1 in favourable growth conditions [-1 d-1 during the linear growth phase of E. gracilis in phototrophic conditions. Higher biomass concentrations and growth rates were achieved in heterotrophic and mixotrophic (photoheterotrophic) conditions [E. gracilis, so that mixotrophic growth continues to be of interest in E. gracilis studies. Some of the cell components are only formed in light, and the formation of some other components (e.g. PUFAs) is enhanced by light [e and pH . It can e and pH , and hasss (e.g. ). The spnditions ,22. Ogbonditions  reportednditions . As withnditions , 26, 27,by light . Recent by light  and unsaby light .E. gracilis is considered to be a promising candidate for development of a photosynthetic biorefinery [E. gracilis biomass and pure β-1,3-glucan for the food and feed industries.refinery ,29 and crefinery , Ltd andE. gracilis in photo-, mixo- and heterotrophic conditions, and analyse the E. gracilis cell composition of these cultures at 23 °C as a low temperature, in comparison with 27 or 30 °C as high temperatures. A detailed analysis of the fatty acid composition of E. gracilis lipids produced in photo-, mixo- and heterotrophic conditions was carried out for cultures grown at 23 °C, as well as for lipids produced at 30 °C in phototrophic conditions. Protein and paramylon content was determined only in phototrophic cultivations (23 and 30 °C), since previous studies have focused on their production in hetero- and mixotrophic conditions [Here we assess growth of nditions , 12. WaxEuglena gracilis (NIES-48) was purchased from the National Institute of Environmental Studies of Japan. Cells were maintained in chemically defined medium at room temperature (~22 °C) in non-agitated flasks with low light intensity illumination and sub-cultured every three weeks.Axenic 4)2SO4, 0.08 g (NH4)2HPO4, 0.16 g K2HPO4, 0.2 g MgSO4∙7H2O, 0.06 g CaCl2∙2H2O, 0.5 mg vitamin B1, 0.002 mg vitamin B12, 3.36 mg FeSO4∙7H2O, 2.88 mg H3BO3, 4 g Na2EDTA∙2H2O, 0.314 mg CuSO4∙5H2O, 1.765 mg ZnSO4∙7H2O, 0.236 mg MnCl2∙2H2O, 0.238 mg Na2MoO4∙2H2O and 0.108 mg CoCl2∙6H2O. For heterotrophic and mixotrophic cultivations, the same medium was modified to provide five-fold more vitamins and trace elements, and 2.5-fold more magnesium to ensure that growth would not be limited by these compounds with the higher biomass concentrations achieved in hetero- and mixotrophic conditions. Specifically, the heterotrophic and mixotrophic medium contained (per litre): 0.3 g (NH4)2SO4, 0.1 g (NH4)2HPO4, 0.2 g K2HPO4, 0.5 g MgSO4∙7H2O, 0.06 g CaCl2∙2H2O, 16.6 ± 0.7 g glucose, 2.5 mg vitamin B1, 0.01 mg vitamin B12, 16.8 mg FeSO4∙7H2O, 14.4 mg H3BO3, 20 g Na2EDTA∙2H2O, 1.57 mg CuSO4∙5H2O, 8.82 mg ZnSO4∙7H2O, 1.18 mg MnCl2∙2H2O, 1.19 mg Na2MoO4∙2H2O and 0.54 mg CoCl2∙6H2O.Chemically defined medium was adapted from Ogbonna, Tanaka et al.  for macr-2s-1 on culture surfaces. Light intensity was measured with a quantum sensor connected to a radiometer (Li-Cor. Inc. USA). Inocula were cultivated until the optical density at 780 nm (OD780) was 0.3.Inocula for bioreactor cultivations were grown in 50 mL photoautotrophic growth medium in 250 mL Erlenmeyer flasks on a shaking platform (Infors AG Switzerland) at 90 rpm, 24 °C. One or two fluorescent tubes (8 W) were placed on the edge of the shaker to provide light intensities of 100 to 150 μmol photons m3PO4 or 1 M NaOH for titration), 200 rpm agitation, and 0.1 volume of air per volume culture per minute (vvm) aeration with CO2 enriched air . Cultures were maintained at pH 6.0 to prevent precipitation of salts in the medium, since this was within the optimal pH range (between pH 3 and 6) for E. gracilis [-2 s-1 at the inner surface of the reactor directly facing the lamps. Light intensity is thus described as either 2 or 3 x 400 μE m-2 s-1, to reflect the illumination provided to the surface of the cultures. The same bioreactors were wrapped in foil to eliminate stray light for heterotrophic cultures (at 23 and 27 °C).Sartorius BioStat B 2.5 L glass vessel bioreactors, with a water jacket,  were set up as described by Wang et al. . Two-litgracilis . Cultiva2 enriched gas stream and the exhaust gas  from the cultivations was analysed by a photoacoustic IR gas analyser , with air as reference or a Prima Pro Process mass spectrometer  calibrated with 3% CO2 in Ar, 5% CO2 with 0.99% Ar and 15% O2 in N2, 20% O2 plus 20% Ar in N2, and 0.04% ethanol in N2. The CO2 enriched gas stream composition was measured before inoculating each cultivation.The composition of the CO780 and dry weight. OD780 was used to monitor increase in biomass at low cell densities and was measured in triplicate against deionised water using an infrared spectrophotometer . Since phototrophic algal cultures only grew exponentially at low biomass concentrations, OD780 was used to calculate specific growth rates in these cultures. For dry weight determination, at least 3 mL of culture was centrifuged (3000 rpm 10 min), and the pellet was washed twice with deionised water. During the washing step, the cell biomass was transferred to a 2 mL pre-dried (105 °C) and weighed microcentrifuge tube. The tube containing the cell biomass was dried by lyophilisation  and re-weighed. Dry weight was determined for a minimum of 2 replicates (for low biomass concentrations which required large sample volumes), but usually for 4 and occasionally 6 replicates, depending on the volume required to obtain an accurate measurement. The dry algal biomass was used for lipid, protein and paramylon content analyses. Supernatant was retained for ammonium and glucose analysis.Cell biomass was measured as OD-1.Ammonium was measured using an ion selective ammonium electrode  with a detection limit of 0.3 mmol Let al. [et al. [Total lipid content was determined gravimetrically after chloroform-methanol (2:1) extraction adapted from Folch et al. , as desc [et al. .4 (1 ml) was added to acidify the samples, after which methyl esters and free fatty acids were extracted with petroleum ether. The separated petroleum ether layer was evaporated and the residue re-dissolved in 150 μl hexane. Fatty acid methyl esters were analysed on an Agilent 7890A GC equipped with an Agilent FFAP silica capillary column (25 m × 0.2 mm × 0.3 μm). Hydrogen was used as carrier gas with a split ratio of 20:1. The oven temperature was increased from 70 °C (2 min) to 235 °C at a rate of 10 °C/min, with a total run time of 30 min.Freeze-dried samples (2–25 mg) were homogenised with a Retsch Mixer Mill homogenizer  for 2 x 30 s  and extracted twice with 1 ml chloroform:methanol (2:1). The chloroform-methanol extracts, containing the lipids, were combined to obtain the total lipids and 30.45 μg C17:0 triacylglycerol  and 15.0 μg C17:0 free fatty acid (FFA) were added to a 300 μl aliquot before removal of the solvent under nitrogen flow. Lipids were re-dissolved in 1 ml petroleum ether (bp 40–60 °C). Transesterification was performed by adding 500 μl of 0.5 N sodium methoxide in methanol and two boiling stones to the solubilised lipids, then boiling at 45 °C for 5 min. Fifteen percent (w/v) NaHSON-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) for 20 min at 80 °C. The samples were analysed on an Agilent DB-5 silica capillary column (30 m x 0.25 mm x 0.25 μm). The split ratio was 20:1 and the oven temperature programme from 70 °C (1 min) to 240 °C at a rate of 10 °C/min, the total run time was 30 min. The samples (2 μl) were injected by a Gerstel MPS injection system and the data were collected in EI mode (70 eV) at a mass range of m/z 50–600.After quantifying fatty acid methyl esters (FAME) by GC, the same samples were trimethylsilylated to determine FFAs and other polar compounds by GC-MS (Agilent 7890A GC combined with 5975C MSD). The solvents were evaporated and samples re-dissolved in 50 μl dichloromethane and silylated with 25 μl et al. [E. gracilis biomass was weighed and re-suspended in 250 μl 10% (w/v) trichloroacetic acid (TCA). The suspension was ultrasonicated for 15 min  and then incubated in a 65 °C water bath for 30 min. The samples were vortexed several times during the incubation to ensure complete cell disruption. After cooling to room temperature, samples were centrifuged at 15,000 g, 4 °C, for 20 min. The pellets were resuspended in 0.5 ml alkaline solution containing 20 g L-1 Na2CO3 and 4 g L-1 NaOH to dissolve the proteins. The suspension was centrifuged to remove the non-soluble particles, including paramylon, from the protein extract. Protein was quantified using the DC protein assay kit (Bio-RAD), based on the Lowry method [Protein extraction and quantification was based on the methods developed by Slocombe et al. . Approxiy method .Paramylon extraction was adapted from Barsanti et al. [E. gracilis cells were washed with 3 ml ethanol, resuspended in 3 ml sodium dodecyl sulfate (SDS) solution (2 g L-1) and incubated at 37 °C for 24 h. Solids were collected by centrifugation and the supernatant discarded. SDS washing was repeated without incubation. The paramylon pellets were washed twice with 3 ml distilled water, then dissolved in 2 ml NaOH (1 M). The concentration of paramylon was determined using the phenol-sulfuric acid method described by Albalasmeh et al. [i et al. . Proteinh et al. .E. gracilis initially grew exponentially in photoautotrophic batch cultures  or less (2 x 400 μE m-2 s-1) light was made at 23 °C and demonstrated that the linear growth rate (0.35 g L-1d-1) was lower with less light provided than with 3 x 400 μE m-2 s-1  than at 30 °C (on average 18%). However, since the total lipid produced in a culture is a function of the lipid content of the biomass and the biomass concentration, the total concentration of lipids of the photoautotrophic cultures receiving the same light intensity (3 x 400 μE m-2s-1) were similar, but less lipid was produced when less light was provided , and their content remained constant throughout the cultivation. The maximum unsaturated fatty acid content (67 μg mg-1) was obtained in mixotrophic culture at the end of the exponential phase  and less than 5% free fatty acids; the remaining ca. 65% probably contained lipophilic compounds such as wax esters, pigments (including chlorophyll and carotenoids), phytol, and unidentified compounds. cultures .E. gracilis, the most abundant PUFA was ALA (see supporting information files -1) was obtained from the mixotrophic culture at 23 °C. ALA content was low (< 0.5 mg g-1) throughout the heterotrophic culture  and heterotrophically (maximum 3.2 mg g-1 cell dry weight) grown cells. The majority were esters of C14 and C16 fatty acids and fatty alcohols . In mixotrophic cultures the α-tocopherol content increased during exponential growth (from 0.04 to 0.18 mg g-1), whereas in the heterotrophic culture, it decreased to 0.01 mg g dry biomass-1 (E. gracilis cells (up to 14 mg g-1 biomass), but not in the heterotrophically grown ones (< 0.5 mg g-1 biomass). The phytol content was highest (1.4% of the cell dry weight) at the end of the exponential growth phase. Its content gradually decreased during the linear growth phase  was highest during the exponential growth phase  or 7.0% (30 °C) at the end of the exponential growth phase, and increased during the linear growth phase. After 9 days, 46.3% of the total cell dry biomass was paramylon, increasing to 53.2% at the end of the cultivation in the culture at 23 °C . The totE. gracilis culture was 45% higher at 30 °C than at 23 °C  when less light (2 x 400 μE m-2s-1) was provided than with 3 x 400 μE m-2 s-1  delayed growth on glucose (for 8 days). Light is also known to inhibit glucose transport in E. gracilis [E. gracilis, photoautotrophic and heterotrophic metabolic activities are reported to occur simultaneously only at very low light intensity [E. gracilis was grown in nitrogen-limited conditions with only 600 lux (~8 μmol m2 s-1) light and longer delays in its consumption in the presence of higher light intensity [2 s-1 and observed a delay of 8 days before glucose consumption started at 23 °C, reduced to only 4 days at 27 °C  conditi biomass ,43,45.E. gracilis accumulates much less lipid than oleogenic microalgal species like Chlorella (30–57% lipid) [Nannochloropsis (26–42% lipid) [E. gracilis, so much of the fatty acids extracted from E. gracilis are derived from the phospholipids of the cell and organelle membranes [E. gracilis are triacylglycerols [In general, % lipid)  and Nann% lipid) , but is % lipid) . Althoug% lipid) , the lip% lipid) ,44. Neutembranes . Only 11lycerols . Considelycerols ,50. In tE. gracilis are found in chloroplast membranes [E. gracilis contained up to five times more unsaturated fatty acids than the cells grown in the dark  [E. gracilis (23% of cell biomass) [cf. Figs E. gracilis cells can be harvested to provide either protein or paramylon, but that the cultivation process should be optimised for the specific target product, to maximise the yield and productivity.Unlike protein, most paramylon accumulated during the linear growth phase in photoautotrophic cultures . Up to ~biomass) ,12. Thisbiomass) . Accumulbiomass) , whereascf. Figs  did not E. gracilis grew well in photoautotrophic, mixotrophic and heterotrophic conditions at 23 °C, with mixo- and heterotrophic conditions primarily providing a benefit in producing high biomass concentrations. Providing light, whether in photoautotrophic or mixotrophic cultures, however, strongly influenced the cell composition. Total unsaturated fatty acids, omega-3 fatty acids, and α-tocopherol content in the cells was higher in the presence of light than in its absence. However, saturated fatty acids were still highest in cells grown in phototrophic conditions.Increasing the temperature provided only limited benefit to phototrophic cultures, since the temperature only affected the relatively short exponential growth phase. In phototrophic cultures, temperature also had little impact on the production of protein, paramylon or total lipid, although the production of some specific fatty acids, such as DHA were lower at high than at low temperature. In contrast, cultivation temperature was important when the cells grew on organic carbon, since the exponential phase was sustained until the organic carbon was consumed.E. gracilis cell components are all either essential nutrients or compounds that promote human and animal health. Thanks to their ability to produce this unique range of compounds, dried E. gracilis whole cells and E. gracilis extracts are already on the market Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


Schistosoma haematobium.Despite decades of experience with praziquantel treatment in school-aged children (SAC) and adults, we still face considerable knowledge gaps relevant to the successful treatment of preschool-aged children (PSAC). This study aimed to assess the efficacy and safety of escalating praziquantel dosages in PSAC infected with S. haematobium in Côte d’Ivoire. A total of 186 PSAC and 195 SAC were randomly assigned to 20, 40 or 60 mg/kg praziquantel or placebo. The nature of the dose-response relationship in terms of cure rate (CR) was the primary objective. Egg reduction rate (ERR) and tolerability were secondary outcomes. CRs and ERRs were assessed using triplicate urine filtration over 3 consecutive days. Available-case analysis was performed including all participants with primary endpoint data.We conducted a randomised, dose-finding trial in PSAC (2–5 years) and as comparator a cohort of SAC (6–15 years) infected with S. haematobium. Follow-up data were available for 157 PSAC and 166 SAC. In PSAC, CRs of praziquantel were 85.7% (30/35), 78.0% (32/41) and 68.3% (28/41) at 20, 40 and 60 mg/kg and 47.5% (19/40) for placebo. In SAC, CRs were 10.8% for placebo (4/37), 55.6% for 20 mg/kg (25/45), 68.3% for 40 mg/kg (28/41) and 60.5% for 60 mg/kg (26/43). ERRs based on geometric means ranged between 96.5% (60 mg/kg) and 98.3% (20 mg/kg) in PSAC and between 97.6% (20 mg/kg and 60 mg/kg) and 98.6% (40 mg/kg) in SAC. Adverse events were mild and transient.A total of 170 PSAC and 174 SAC received treatment. Almost 90% of PSAC and three quarters of SAC were lightly infected with S. haematobium. Over the dose range tested, praziquantel displayed a ceiling effect with the highest response for 20 mg/kg in PSAC. In SAC maximum efficacy was obtained with 40 mg/kg praziquantel. Further investigations are required in children with moderate to heavy infections.Praziquantel revealed dose-independent efficacy against light infections of ISRCTN15280205.This trial is registered with International Standard Randomised Controlled Trial Number The online version of this article (10.1186/s12916-018-1066-y) contains supplementary material, which is available to authorized users. Schistosoma, where infection with Schistosoma japonicum and S. mansoni causes mostly intestinal schistosomiasis, while S. haematobium is responsible for genitourinary schistosomiasis  × 100) was assessed. A bootstrap resampling method with 5000 replicates was used to estimate 95% CIs for ERRs.In order to calculate ERR, the AM and geometric mean (GM) of eggs per 10 mL of urine before and after treatment were assessed. Geometric mean egg counts were calculated as follows: emax models using the DoseFinding package (version 0∙9–14) of the statistical software environment R (v3.3.0) were implemented to predict the dose-response curves in terms of CRs and ERRs. Logistic regression was used to predict CR by infection intensity at baseline.ES. haematobium infection. Of these, 186 (29.6%) PSAC and 195 (45.7%) SAC had a detectable S. haematobium infection and were randomised for treatment. On the treatment day, 16 PSAC and 21 SAC were absent. PSAC received 20 mg/kg (n = 40), 40 mg/kg (n = 44) or 60 mg/kg (n = 44) praziquantel or placebo (n = 42). SAC were likewise allocated to 20 mg/kg (n = 46), 40 mg/kg (n = 46) or 60 mg/kg praziquantel (n = 44) or placebo (n = 38). Two PSAC and one SAC were not able to swallow the drug. One PSAC and one SAC were wrongly dosed .Overall, 1184 children were invited to participate in the study  in PSAC and SAC. Median haemoglobin values ranged between 10.5 and 11.0 g/dL in PSAC and between 11.0 and 11.7 g/dL in SAC.The median age, weight, height and sex of PSAC and SAC were balanced among the treatment groups Table . Three qmax model based on actual doses on the per protocol population is presented in Additional file The nature of the dose response based on CRs is depicted in Fig. S. haematobium eggs were not detected in the urine samples of 47.5% (19/40) and 10.8% (4/37) in PSAC and SAC, respectively , 78.0% (95% CI 62.4–89.4) and 68.3% 95% CI 51.9–81.9), respectively, whereas in SAC the respective CRs were 55.6% (95% CI 40.0–70.4), 68.3% (95% CI 51.9–81.9) and 60.5% (95% CI 44.4–75.0). In the placebo groups, .9–81.9, S. haematobium infection intensity. The CR in PSAC ranged from 73.0% (60 mg/kg) to 87.9% (20 mg/kg) in light infections and from 25.0% (60 mg/kg) to 66.7% (40 mg/kg) in heavy infections. In SAC, CRs were 70.6% (20 mg/kg) to 78.8% (40 mg/kg) for light S. haematobium infections and between 9.1% (20 mg/kg) and 27.3% (60 mg/kg) for heavy infections.ERRs are summarized in Table n = 12). At 24 h post-treatment, 25 (14.9%) and 47 (27.2%) adverse events were recorded in PSAC and SAC, respectively. For both age groups the number of adverse events was similar among the three praziquantel treatment arms, with fewer adverse events observed in the placebo-treated groups. The most common adverse events in PSAC and SAC 24 h post-treatment were diarrhoea (4.8 and 3.5%), stomach ache (3.6 and 9.8%), fever (6.0 and 13.3%), headache (3.0 and 15.6%) and nausea (2.4 and 6.9%).Adverse events data were available for 168 PSAC and 173 SAC , but it will take several more years until the drug is marketed and available to all PSAC. To be able to treat preschoolers safely and effectively, we studied ascending doses of praziquantel in PSAC and SAC infected with S. haematobium. Our results build on an earlier dose-finding study in S. mansoni-infected children [It was recommended in 2010 that PSAC should be included in preventive chemotherapy programmes  using anchildren .S. haematobium infection intensities compared to the two lower doses. For example, the CR in PSAC characterised by heavy infection intensities treated with 60 mg/kg praziquantel was as low as 25%. However, only a handful of PSAC suffered from moderate and high infection intensities; hence, no clear picture can be drawn for this age group. In SAC similar CRs were observed in children harbouring heavy infection intensities treated with 40 and 60 mg/kg. In summary, a high dose of praziquantel seems to have no additional benefit in the treatment of S. haematobium infections. This result is in contrast with our recent study, where we reported that in SAC infected with S. mansoni, CRs increased with higher doses of praziquantel [Several findings of our study are worth highlighting. First, the highest overall CRs among PSAC (85.7%) and SAC (68.3%) were obtained with 20 mg/kg and 40 mg/kg praziquantel, respectively and not with the highest dose administered, 60 mg/kg. For both age groups, CRs revealed even a slight inverse dose-rate effect. Similarly, ERRs increased very fast up to 98% and did not increase further regardless of the praziquantel dose administered. Interestingly, 60 mg/kg praziquantel also showed lower CRs in PSAC with moderate/high iquantel , while oS. haematobium-infected PSAC compared to 71.4% of SAC [S. haematobium, since reductions in egg output significantly correlated with decreased morbidity [Overall higher CRs were observed in PSAC (68–86%) when compared to SAC (56–68%), which mirrors a recent meta-analysis by Zwang et al., where 40 mg/kg praziquantel cured 87.3% of % of SAC . Nonethe% of SAC , 36. Oveorbidity , 38.Schistosoma species. However, our dose-finding study in S. mansoni-infected children showed that higher ERRs (based on GMs) were observed in children treated with 40 and 60 mg/kg compared to 20 mg/kg [S. haematobium, in contrast to preschoolers infected with S. mansoni [S. haematobium infections in this study, the use of two different doses, namely 20 mg/kg for S. haematobium and 40 mg/kg for S. mansoni in settings where Schistosoma species are overlapping, would raise logistical and operational challenges since control programmes are acting at large-scale levels such as district or country levels. Therefore, rigorous cost-effectiveness studies need to be implemented before a change of treatment guidelines could be considered. However, at a point-of-care level, using a test-and-treat approach, 20 mg/kg and 40 mg/kg could be recommended to treat PSAC for S. haematobium and S. mansoni infections, respectively.No dose-response relationship was observed for ERRs in both age groups above 20 mg/kg. This finding is in line with an earlier meta-analysis by Zwang et al.  which fo20 mg/kg . Recent 20 mg/kg . Regardl mansoni , yieldedS. mansoni study [Schistosoma drug efficacy trials—the overestimation of CRs due to potential false negatives in light infections is visible. More importantly, our observations emphasise the need for Schistosoma species-related standard operating procedures including reliable diagnostic tools, suitable for drug efficacy assessment for low infection intensities [In PSAC we observed a high CR in the placebo arm similar to what was observed in our ni study . The proni study , 41. Ourensities –42.With regard to safety outcomes, the main adverse events observed in both PSAC and SAC are in line with the adverse events reported in previous studies , 24, 34.S. haematobium, with high efficacy observed already at 20 mg/kg, particularly in light infections. No benefit was observed using higher praziquantel doses in the current study. However, to be able to provide ultimate dosing recommendations of praziquantel for PSAC, additional studies might be required to support our conclusions, including pharmacokinetic studies and studies in PSAC suffering from moderate and heavy S. haematobium infections.Praziquantel showed a high response rate in PSAC and SAC infected with Additional file 1:Table S1. Imputation on cure rates of individuals lost after treatment in 20, 40 and 60 mg/kg praziquantel and placebo treatment arms among Schistosoma haematobium-infected preschool-aged children and school-aged children (intention-to-treat). Figure S1. Emax model predicting cure rates (CRs) based on actual doses in preschool-aged children (blue symbols) and school-aged children (red symbols). Figure S2. Predicted probability of being cured by baseline infection intensity in preschool children (blue lines) and school-aged children (red lines). (DOCX 231 kb)


We firstly investigated the effect of dexmedetomidine, a highly selective α2-adrenergic receptor agonist, on mechanical and thermal hyperalgesia using a rat model of RIH. NMDA receptor subunits  expression and membrane trafficking as well as PKC and CaMKII expression in spinal cord L4–L5 segments were measured by Western blot analysis. The expression of NMDA receptor subunits  were also detected by immunohistochemistry. Further more, the effect of dexmedetomidine on NMDA receptor current amplitude and frequency in spinal cord slices were investigated by whole-cell patch-clamp recording. We found that remifentail infusion at 1.2 μg.kg−1.min−1 for 90 min caused mechanical and thermal hyperalgesia, up-regulated NMDA receptor subunits NR1 and NR2B expression in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Subcutaneously injection of dexmedetomidine at the dose of 50 μg/kg at 30 min before plantar incision significantly attenuated remifentanil-induced mechanical and thermal hyperalgesia from 2 h to 48 h after infusion, and this was associated with reversal of up-regulated NR1 and NR2B subunits in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Furthermore, remifentanil incubation increased amplitude and frequency of NMDA receptor-induced current in dorsal horn neurons, which was dose-dependently attenuated by dexmedetomidine. These results suggest that dexmedetomidine can significantly ameliorate RIH via modulating the expression, membrane trafficking and function of NMDA receptors as well as PKC and CaMKII level in spinal dorsal horn, which present useful insights into the mechanistic action of dexmedetomidine as a potential anti-hyperalgesic agents for treating RIH.Remifentanil-induced secondary hyperalgesia has been demonstrated in both animal experiments and clinical trials. Enhancement of N-methyl-D-aspartate (NMDA) receptor trafficking as well as protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaMKII) have been reported to be involved in the induction and maintenance of central sensitization. In the current study, it was demonstrated that dexmedetomidine could prevent remifentanil-induced hyperalgesia (RIH) via regulating spinal NMDAR-PKC-Ca As potent analgesics, opioids are available for relieving moderate to severe pain.Meanwhile, their prolonged usage may paradoxically be related to the development of opioid-induced hyperalgesia (OIH) –3. OIH i2+/calmodulin-dependent protein kinase II (CaMKII) pathway , Remifentanil group , Dexmedetomidine group , Dexmedetomidine group 1 , Dexmedetomidine group 2 , Dexmedetomidine group 3 . After incubation, the NMDA receptor-mediated miniature excitatory postsynaptic current (mEPSC) was detected to evaluate the function of NMDA receptor.The incisional pain rat model was constructed as described previously . A 1-cm Paw withdrawal threshold (PWT) was recorded by electronic von Frey filaments  to measure mechanical hyperalgesia.After adult rats were placed individually in mental cages (20 cm×20 cm×15 cm) with a wire grid bottom for 10 min, Von Frey filaments were exerted vertically to the plantar side of the right hindpaw. Lifting of the hindpaw off the cage surface or flinching was regarded as a positive response. A critical value of 60 g was used to prevent paw damage, and each trial was repeated 3 times at 10-min intervals. The calculated average value was PWT.Paw withdrawal latency (PWL) was recorded by a 55°C hotplate  to assess thermal hyperalgesia. A critical value of 60 seconds was used to prevent paw damage, and each trial was repeated 3 times at 10-min intervals. The calculated average value was PWL.After completing the last behavioral tests, rats were sacrificed under sevoflurane anesthesia. The chest was cut along the midline to expose the heart, then an infusion needle was inserted into the apical portion of the aorta, the right atrial appendage was cut and normal saline was infused rapidly until transparent liquid flowed out. Then a longitudinal incision along the dorsal midline was made and L4-L5 spinal cord segments were extracted rapidly and kept in liquid nitrogen. The segments were homogenized in ice-cold sodium dodecyl sulfate lysis buffer containing protease inhibitors . Then the lysate was centrifuged at 14,000r for 10 min at 4°C, and the supernatant was removed as total protein. A membrane compartment protein extraction kit  was applied to extract the membrane protein. Eventually after the total protein was boiled at 95°C for 10min and membrane protein was boiled at 65°C for 10min, they were store at -20°C. Samples were separated on 8%-10% SDS-PAGE gel electrophoresis, and then transferred onto nitrocellulose membranes . The membranes were blocked with 5% skim milk in TBST for 2 hour and subsequently incubated overnight at 4°C with mouse anti-rat epidermal growth factor receptor , monoclonal mouse anti-β-actin antibody , polyclonal rabbit antibodies against rat NR1, NR2A, NR2B, PKCγ, CaMKIIα, and polyclonal mouse antibodies against rat pCaMKIIα  respectively. And then the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse IgG antibodies  for 1 h. The density of each band was detected by a chemiluminescence imaging system and measured by Gene Tools Match software . The percentage of endogenous control (EGFR or β-actin) immunoreactivity was expressed as the results.The L4-L5 spinal cord segments were extracted rapidly after the last behavioral test. 4% paraformaldehyde in phosphate buffered saline (PBS) was applied to fix the samples for 6 h, and then paraffin was used to embed the samples. The samples were cut into 7-μm-thick sections and placed onto glass slides. After deparaffinization and rehydration in descending concentrations of ethanol, the sections were treated with avidin-biotin peroxidase for 15 min. To make the results more intuitive, citrate solution was used for antigen retrieval. 5% normal goat serum was selected for treatment to block non-specific reactions for 1h at room temperature. Then the sections were incubated with primary antibody of rabbit polyclonal anti-NR1, NR2A, NR2B  overnight at 4°C respectively. After rewarming for 45min, the sections were incubated with biotinylated secondary antibody  for 30 min at room temperature and stained by diaminobenzidine . Sections were counterstained with hematoxylin, differentiated by ethanol hydrochloride, dehydrated in ascending series of ethanol, cleared in xylene and coverslipped with neutral gum. Images were obtained from an Olympus eclipse 80i microscope .2 and 5% CO2 at pH 7.4 for 1 h. ASCF is composed of (in mM): 126 NaCl, 3.5 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3 and 10 D-glucose. The slices were then transferred to a recording chamber in succession. The chamber was perfused with oxygenated ACSF continuously and placed on an upright microscope  and continuously perfused with oxygenated ACSF. Individual neurons were identified by a television monitor connected to the low light-sensitive CCD camera . Borosilicate glass microelectrodes with tip openings of 1–2 μm and electric resistance of 3–5 MΩ were drawn by vertical electrode puller  and applied to whole-cell patch-clamp recording. Microelectrodes were filled with an intracellular solution composing of (in mM) 130 KCl, 10 HEPES, 0.5 CaCl2, 10 EGTA, 2 MgCl2, 2 Mg-ATP and 0.3 Na-GTP at pH 7.3. CNQX (20 μM), tetrodotoxin (TTX 10 μM) and bicuculline (BIM 20 μM) were added into perfusion solution before recording to accurately verify the NMDAR miniature excitatory postsynaptic currents (mEPSC). In previous trials, AP-5  was added after NMDA receptor- mEPSC was detected, and mEPSC fade away a few minutes later. Therefore, the mEPSC was mediated by NMDA receptors. ACSF without Mg2+ is used to observe NMDA receptor-mediated synaptic transmission. All responses were recorded by an EPC 10 amplifier and Pulse 8.52 software . Current data was filtered with an eight-pole, low-pass Bessel filter at 2.9 kHz and digitized at 10 kHz for off-line analysis. mEPSC was then analyzed by Clampfit 9.0 . Total charge transfer by all mEPSC was determined for each condition in 5 min and repeated 8 times.Rat spinal cord slices were prepared as described previously. The postnatal (14–21 days old) rats were anesthetized with sevoflurane . The L4–L5 spinal cord segments were separated rapidly with the same method preciously, sliced into transverse slices (400 μm) with a vibratome , then incubated in ACSF at room temperature (22°C-25°C) and aerated with 95% Ot test when appropriate. The results of Western blot, immunohistochemistry, amplitude and interevent intervals of NMDAR-mediated mEPSC in different groups, one-way ANOVA followed by Tukey HSD test were performed. Statistical analysis was performed with GraphPad Prism 5.0 . A P value<0.05 was considered statistically significant.Statistical analysis was performed by SPSS 19.0 . All data were expressed as mean±standard deviation(SD). Time course data for both the thermal and mechanical hyperalgesia were analyzed by two-way ANOVA with repeated measures to detect interactions between treatments. ANOVAs with statistically significant interactions between treatments (P < 0.05) were followed by post hoc comparisons using Bonferroni’s P >0.05). Compared with group C, PWT and PWL were significantly decreased at 2, 6, 24, 48 h in other groups except group D (P<0.01). Remifentanil infusion at the rate of 1.2 μg.kg−1.min−1 for 90 min or plantar incision significantly decreased PWT and PWL at 2, 6, 24 and 48 h after plantar incision  and membrane (m) expression of NR1, NR2B and the ratio of m/tNR1, m/tNR2B was significantly increased after incision or remifentanil infusion  and PWT (B) were evaluated at −24 h, 2 h, 6 h, 24 h and 48 h after infusion. Data were analyzed by repeated measures ANOVA and expressed as mean±SD. Compared with the group C, *P < 0.01; compared with the group R, #P < 0.01; compared with the group I, $P < 0.01; compared with the group R+I, &P < 0.01; compared with the group D+R+I, §P < 0.01; N = 8, analysis of variance.Remifentanil was infused intravenously at the rate of 1.2 μg.kg(TIF)Click here for additional data file.S2 Fig#P < 0.01; compared with the group I, $P < 0.01; compared with the group R+I, &P < 0.01; compared with the group D+R+I, §P < 0.01; N = 8, analysis of variance.Membrane trafficking of NR1 and NR2B subunits was increased after remifentanil administration and incision, while dexmedetomidine might produce a preventive effect. The spinal cord L4–L5 segments were removed after the last behavioral test for western blot. EGFR and β-actin were internal control. The band intensity of group C was assigned a value of 1. Bands of membrane and total NR1, NR2A, NR2B protein were detected by Western blot . (B) Bar chart of the ratios of mNR1/EGFR, tNR1/β-actin and m/tNR1. (D) The ratios of mNR2A/EGFR, tNR2A/β-actin and m/tNR2A. (F) The ratios of mNR2B/EGFR, tNR2B/β-actin and m/tNR2B. Data were analyzed by ANOVA and expressed as mean±SD. Compared with the group C, *P < 0.01; compared with the group R, (TIF)Click here for additional data file.S3 FigThe L4-L5 segments of spinal cords were collected after the last behavioral testing for immunohistochemistry. When compared with group C, NR1 and NR2B expression is dramatically increased after remifentanil infusion with incision; while the process was significantly suppressed by pretreatment with dexmedetomidine(A). No considerable changes were observed in NR2A subunit (A). Representative photomicrographs of the L4-6 spinal cord are shown here .(TIF)Click here for additional data file.S4 Fig#P < 0.01; compared with the group I, $P < 0.01; compared with the group R+I, &P < 0.01; compared with the group D+R+I, §P < 0.01; N = 8, analysis of variance.Mean IOD of NR1, NR2A and NR2B subunits were calculated by IPP software . Data were expressed as mean±SD. Compared with the group C, *P < 0.01; compared with the group R, (TIF)Click here for additional data file.S5 Fig#P < 0.01; compared with the group I, $P < 0.01; compared with the group R+I, &P < 0.01; compared with the group D+R+I, §P < 0.01; N = 8, analysis of variance.The expression of PKCγ, CaMKIIα and pCaMKIIα were significantly increased after remifentanil infusion and incision, while the phenomenon was prevented by dexmedetomidine. The L4–L5 segments of spinal cords were removed after the last behavioral test for western blot. β-actin was internal control. The band intensity of group C was assigned a value of 1. Bands of PKCγ, CaMKIIα and pCaMKIIα protein by Western blot. (B) Bar chart of the ratios of PKCγ/β-actin. (D) Bar chart of the ratios of CaMKIIα/β-actin. (E) Bar chart of the ratios of pCaMKIIα/β-actin. Data were analyzed by ANOVA and expressed as mean±SD. Compared with the group C, *P < 0.01; compared with the group R, (TIF)Click here for additional data file.S6 Fig#P < 0.01; compared with the D1 group, $P < 0.01; compared with the D2 group, &P < 0.01; compared with the D3 group, §P < 0.01; N = 8, analysis of variance.In the presence of TTX (10 μM), GABAR antagonist bicuculline  and AMPAR antagonist CNQX (20 μM), NMDAR-mediated mEPSCs were recorded at the holding potential of −70 mV. Representative traces of mEPSCs under blank control (group C), remifentainil 4 nM(group R), dexmedetomine 4 nM(group D), dexmedetomine 2 nM + remifentainil 4 nM(group D1), dexmedetomine 4nM + remifentainil 4 nM(group D2), dexmedetomine 6 nM + remifentainil 4 nM(group D3) showed in graph A. Scale bar, 100 pA, 30 s. The Bar chart of mEPSCs frequency and mEPSCs amplitude were showed in graph B and C. Data were expressed as mean±SD. Compared with the C group, *P < 0.01; compared with the R group, (TIF)Click here for additional data file.


Pseudomonas aeruginosa biofilm in endotracheal tubes (ETTs).Despite the several strategies available for the management of biofilm-associated ventilator-associated pneumonia, data regarding the efficacy of applying antibiotics to the subglottic space (SS) are scarce. We created an in vitro model to assess the efficacy of antibiotic lock therapy (ALT) applied in the SS for eradication of P. aeruginosa biofilm in ETTs using a single dose (SD) and a 5-day therapy model (5D). We used sterile saline lock therapy (SLT) as the positive control. We compared colony count and the percentage of live cells between both models.We applied 2 h of ALT to a 5 (9.7 × 104-0) vs. 8.16 × 107 (7.0 × 107-0) (p = 0.05) and 53.2% (50.9%-57.2%) vs. 91.5% (87.3%-93.9%) (p < 0.001). The median (IQR) cfu counts/ml and percentage of live cells in the 5D-ALT and 5D-SLT groups were, respectively, 0 (0-0) vs. 3.2 × 107 (2.32 × 107-0) (p = 0.03) and 40.6% (36.6%-60.0%) vs. 90.3% (84.8%-93.9%) (p < 0.001).The median (IQR) cfu counts/ml and percentage of live cells in the SD-ALT and SD-SLT groups were, respectively, 3.12 × 10P. aeruginosa biofilm after application of 5D-ALT in the SS. Future clinical studies to assess ALT in patients under mechanical ventilation are needed.We demonstrated a statistically significant decrease in the viability of  Ventilator-associated pneumonia (VAP) is one of the most common nosocomial infections, with significant morbidity and mortality. Between 9% and 27% of intubated patients develop VAP, although the incidence can increase to 46% in patients who require mechanical ventilation for more than 48 h after major heart surgery (MHS) , 2.6 bacterial cells/cm recovered from the endotracheal tube (ETT) . The. The7]. ate Fig. .Fig. 1aLock therapy consisted of a 2-h application of 3 ml of either SDD solution  or sterile saline in the subglottic space. In the case of 5D-ALT and 5D-SLT, we used BHI between lock therapy periods. We also assessed whether SDD solution or sterile saline leakage occurred during lock therapy.Once lock therapy had finished, the solution was removed, and the ETTs were washed with sterile saline. The subglottic area of the ETT was then cut into 3 segments, each measuring 0.5 cm Fig. . One segSonication was performed in 2 ml of buffer solution for 1 min at 50 Hz. For culture, the solution was serially diluted and cultured on blood agar plates, which were incubated for 24 h at 37 °C. Colony counts were expressed on a logarithmic scale as the number of cfu/ml.BacLight kit™  for 15 min protected from the light. Staining was performed using 0.5 μl of SYTO® 9 (stock 3.34 mM DMSO) and 0.5 μl propidium iodide (stock 20 mM DMSO) in 20 μl of sample mounted on coverslips and imaged using confocal laser scanning microscopy (CLSM) in an inverted confocal fluorescence microscope  equipped with ACS APO 10×/0.30 and ACS APO 63X/1.30 objectives. Samples were imaged using an ACS APO 63X/1.30 objective. During imaging, SYTO 9 emits green fluorescence and is used to identify living microorganisms with an intact membrane, whereas propidium iodide emits red fluorescence and stains dead bacteria with a damaged membrane. We analyzed 3 CLSM images of each sample. FIJI software  was used for image quantification. The percentage of live bacteria was calculated as the ratio between the number of live cells and the total number of cells ×100.For the analysis of the live/dead cells, the remaining sonicate was centrifuged (after culture), and the pellet was resuspended in 50 μl of sterile saline and stained using the Live/Dead® BacLight kit™ for 15 min protected from the light. Staining was performed using 1.5 μl of SYTO® 9 (stock 3.34 mM DMSO) and 1.5 μl propidium iodide (stock 20 mM DMSO) in 1 ml phosphate-buffered saline. The samples were mounted on coverslips and imaged using CLSM at ACS APO 10X/0.30 objective [For visualization of the biofilm biomass in the ETT, we inactivated the ETT segments by freezing at −80 °C for 72 h. Then, after thawing for 30 min, the segments were stained with the Live/Dead® bjective , 29. We Biofilm structure was imaged using a 0.5-cm half section of the ETT segment fixed in 2.5% glutaraldehyde and then in osmium tetroxide (1%) and potassium ferricyanide (0.8%). The samples were then dehydrated in graded alcohol and sputter-coated with gold atoms . Samples were imaged via a scanning electron microscope . We displayed 6 SEM images for each sample.. Quantitative variables are expressed as the median and interquartile range (IQR). Non-normally distributed continuous variables were compared using the Kruskal-Wallis and Mann-Whitney tests. The chi-square or Fisher exact test was used to compare categorical variables.Qualitative variables appear with their frequency distributionp < 0.05 for all the tests. The statistical analysis was performed with SPSS 21.0.All statistical tests were 2-tailed. Statistical significance was set at 5 (9.7 × 104-0.0) cfu/ml in the SD-ALT group and 8.16 × 107 (7.0 × 107-0.0) cfu/ml in the SD-SLT group (p = 0.05). The median (IQR) percentage of live cells detected by CLSM was 53.2% (50.9%-57.2%) in the SD-ALT group and 91.5% (87.3%-93.9%) in the SD-SLT group (p < 0.001)  of 2.15 × 1001) Fig. .Fig. 2aP. aeruginosa biofilm for both SD-ALT and SD-SLT, as also shown by SEM. This finding correlated with the median (IQR) number of total cells: SD-ALT, 915.0 (279.0-1074.5) vs. SD-SLT, 246.0 (168.5-1130.5) (p = 0.35)  Fig. . Howeveroup Fig. .7 (2.32 × 107-0.0) in the 5D-SLT group (p = 0.03). The median (IQR) percentage of live cells in each group was 40.6% (36.6%-60.0%) for 5D-ALT and 90.3% (84.8%-93.9%) for 5D-SLT (p < 0.001)  cfu/ml from the sonicate of the ETT segments of 0.0 (0.0-0.0) in the 5D-ALT group and 3.2 × 1001) Fig. .p = 0.01). This finding can be observed in the CLSM images of the ETT segments, in which the thickness of the biomass was lower for 5D-ALT  number of total cells: SD-ALT, 32.0 (28.0-35.5) vs. SD-SLT, 50.0 (36.5-57.5)  (p = 0.04). Although there were no statistically significant differences in the median (IQR) percentage of live cells, 5D-ALT was slightly better than SD-ALT: 40.6% (36.6%-60.0%) vs. 53.2% (50.9%-57.2%) (p = 0.12).In order to assess whether 5D-ALT was better than SD-ALT, we compared only the median between the percentage of live cells and the number of cfu/ml, as SD and 5D therapies were not comparable according to the total number of cells in both the ALT group and the SLT group. We found that 5D-ALT was significantly better than SD-ALT in terms of the median (IQR) cfu/ml recovered in the sonicate: 0.0 (0.0-0.0) vs. 2.15 × 10When we analyzed whether leakage of lock solutions occurred through the ETT, we did not recover any other solution in either the SD or the 5D lock therapies.P. aeruginosa biofilm (72 h) in ETTs significantly reduced cell viability.Our in vitro data demonstrated that 5D-ALT using SDD solution applied on mature VAP is considered a major nosocomial infection, with a frequency ranging from 9% to 46% , 30, 31.Biofilm formation on the ETT surface plays a key role in the pathogenesis of VAP, as it prevents the action of antibiotics and host defense cells . PersistP. aeruginosa biofilm was associated with a considerable reduction in the number of viable cells (both cfu/ml and percentage of live cells). As for duration of lock therapy, even though the median percentage of live cells in the sonicate of the ETT segments treated with SD-ALT was significantly lower than in the control group , 5D-ALT not only significantly reduced the median percentage of live cells by >50% , but it also reduced the median number of cfu/ml . Our findings were also corroborated by direct observation of the biofilm in the ETT segments using CLSM and SEM, as 5D-ALT had a thinner biomass and the bacillus was deformed. This major cell damage observed by SEM was caused by tobramycin and colimycin, since both drugs produced leakage of cellular components, with the result that the cells were deformed [Several preventive measures, such as subglottic aspiration and SDD, have proven effective in the reduction of VAP rates among patients under mechanical ventilation . In addideformed .We believe that the difference in the total number of cells observed in both periods of lock therapy is due to marked dislodgement of dead bacteria from the biofilm on the ETT surface in the 5D model . MoreoveP. aeruginosa biofilm, our data support using the ALT not only as a therapeutic measure once the biofilm is established, but also as a preventive measure in patients who are expected to be under mechanical ventilation for more than 48 h (from the beginning of intubation until extubation or until discharge from the ICU). Moreover, the combination of compounds in the SDD solution would cover most microorganisms causing VAP. However, despite in ICUs with low levels of antibiotic resistance there is no evidence that universal use of SDD increases antibiotic resistance, in ICUs with high endemic levels of antibiotic resistance, SDD may increase the selective pressure for antibiotic-resistant microorganisms [Escherichia coli and Staphylococcus aureus) should be tested.Finally, even though our in vitro model was based on a static pre-established rganisms –20. Routrganisms . BesidesP. aeruginosa biofilm in ETT can be significantly eradicated after applying ALT with SDD solution in the subglottic space. Future studies are needed to further evaluate the efficacy of this approach when combined with SDD as a prophylactic measure for VAP in patients under mechanical ventilation.This is the first in vitro study demonstrating by culture and microscopy that


Akodon montensis Thomas, 1913 were collected in the Serra dos Órgãos National Park (SONP) in November 2014 and July 2015. The fecal material was analyzed, and a prevalence of 7.5% was recorded for a new coccidian species of the genus Eimeria Schneider, 1875, with part of its endogenous development recorded in the small intestine. The oocysts of a new coccidian species of genus Eimeria are ellipsoidal to subspherical. The wall is bi-layered, c. 1.5 μm (1.3–1.6 μm) thick, outer layer rough. Oocyst (n = 126) mean length is 25.3 μm (21.0–28.0 μm), with a width of 20.2 μm (17.0–22.0 μm) and mean length/width (L:W) ratio of 1.3 (1.2–1.4). Polar granule is present, with the oocyst residuum as a large spherical to subspherical globule. Sporocyst shape (n = 126) is ellipsoidal, with a mean length of 11.8 μm (9.3–14.4 μm), width of 7.9 μm (6.7–9.3 μm), and mean L:W ratio of 1.5 (1.4–1.7). Sporocysts with nipple-like Stieda body and sub-Stieda body are absent. A sporocyst residuum formed by several globules, usually along the sporocyst wall. This is the first record of Eimeria in the montane grass mouse from Brazil.A total of 53 specimens of the montane grass mouse,  Eimeria spp. are protozoan parasites with worldwide distribution that might cause enteritis in many vertebrate species ], and Besnoitia akodoni Dubey, Sreekumar, Rosenthal, Lindsay, Grisard and Vitor, Akodon montensis.Akodon Meyen, 1833 has 44 species distributed throughout South America , Akodon serrensis , Akodon paranaensis Christoff et al., 2000, and Akodon montensis Thomas, 1913 , in the municipality of Petrópolis, Rio de Janeiro State, Brazil . This park is located near the center of the Serra do Mar Ecological Corridor  and Sherman® traps set on the ground, a total of 90 live traps set per day. Additionally, 20 pitfall traps, made of 100-l buckets, were buried in the ground, along four transects, a capture effort of 80 pitfalls traps set per day. Each trapping session lasted for ten consecutive days. All animals were captured alive and euthanized a posteriori for sample collection. Voucher specimens were deposited in the scientific collection of the National Museum  at the Universidade Federal do Rio de Janeiro (UFRJ). All the samples were collected during the Rede Bioma (Biome Network) project “Inventories: Patterns of Diversity, Biogeography, and Endemism of Mammals, Birds, Amphibians, Fruit Flies, and Parasites in the Atlantic Forest,” which is supported by a consortium of Brazilian research agencies, financed by CNPq PPBio Rede Bioma.Rodents were captured in November 2014 and July 2015 along ten transects in each expedition. The specimens were captured in Tomahawkw/v) solution of K2Cr2O7 and maintained at room temperature. On five consecutive days, the tube was opened and the solution was stirred vigorously to oxygenate it and promote the sporulation of any oocyst present in the faces. The samples were processed in a saturated sugar solution by the centrifugal flotation method (Sheather’s method), placed on slides examined under a microscope at a magnification of × 400  of the 53 Eimeria akodonensis n. sp.Type host: Akodon montensis Thomas, 1913 (Rodentia: Sigmodontinae), Symbiotype host .Type-material: The oocysts were preserved in 70% ethanol, based on Duszynski and Gardner (http://r1.ufrrj.br/lcc) at the Federal Rural University of Rio de Janeiro, in Seropédica, Rio de Janeiro, Brazil. Phototypes and line drawings were deposited together with the specimens. The catalog number is P-77/2017. Gardner . The samSporulation time: Unknown.Site of infection: Small intestine.Prevalence: 4 of 53 (7.5%).Etymology: The specific epithet is derived from the name of the host genus.\Exogenous stageDescription Figs. –3 and 4FSporulated oocystn = 126) is ellipsoidal to subspherical, wall bi-layered, 1.5 μm (1.3–1.6) thick, outer layer rough. Oocyst length is 25.3 μm (21.0–28.0), with a width of 20.2 μm (17.0–22.0) and length/width (L/W) ratio of 1.3 μm (1.2–1.4). Polar granule is present, with oocyst residuum as a large spherical to subspherical globule.Oocyst shape (Sporocystn = 126)is ellipsoidal with length of 11.8 μm (9.3–14.4), width of 7.9 μm (6.7–9.3), and (L/W) ratio of 1.5 μm (1.4–1.7). Sporocysts with nipple-like Stieda body and sub-Stieda body are absent. A sporocyst residuum formed by several globules, usually along the sporocyst wall.Sporocyst shape  in length and 11.8 μm 5.5–17.3) in width : s Carini , EimeriaEimeria akodonensis n. sp. were compared with data for coccidia of the family Eimeriidae described from other cricetid hosts in Brazil and Venezuela , and does not have a polar granule, its wall is smooth and not very thick, and the residua of the sporocysts are made up of fine granules. Eimeria ojastii  and both have a polar granule, although the sporocysts are slightly different in their morphology and size (14 μm × 7 μm vs. 11.8 μm × 7.9 μm). Arcay-de-Peraza  than E. akodoni (30.6 × 20.4 μm). In addition, macrogametocytes of E. akodonensis n. sp. were sub-spherical, 20.9 × 16.2 μm (L/W ratio: 1.2 μm), and those of E. akodoni were ellipsoidal, 19.7 × 11.7 μm (L/W ratio: 1.6 μm). The effects of infection by Eimeria akodonensis n. sp. on the intestinal tissue of Akodon montensis varied considerably, from a total absence to a light and diffuse infiltration distributed in the intestinal mucosa composed of neutrophils and mononuclear cells. However, this infiltration was not found in association with the parasites themselves, but rather in the epithelium of the intestinal villi. Overall, the set of morphological and physiological characteristics found in Eimeria akodonensis n. sp. supports conclusively its description as a new species.Morphologically, the most similar of the four species to e-Peraza  parasitie-Peraza  describee-Peraza  found E.e-Peraza , merogon


The synthesis, isolation and characterization of isobenzofuran are described in this publication. Isobenzofuran is of general interest in synthetic and physical organic chemistry because it is one of the most reactive dienes known. A number of synthetic pathways have been published which all suffer from disadvantages such as low yields and difficult purification. We present a synthetic pathway to prepare isobenzofuran in laboratory scale with high yields, from affordable, commercially available starting materials. Isobenzofurans have been described as the most reactive dienes for Diels–Alder reactions –5. Their1O2 in photodynamic therapy , [4 + 4], 8 + 8] and [4 + 6] additions –10. High therapy . The mos + 8 and  therapy . However therapy . 1,3-Dip1) in solution  is about 12 h. A half-life of 2 h in CDCl3 has been previously reported ..8) with 1), this solution was reacted with acetylenedicarboxylic acid dimethyl ester  and product 10 was obtained with a yield of 78% relative to the precursor DMIBF , the benzene solution was carefully evaporated and the residue was subjected to column chromatography over silica gel (cyclohexane/ethyl acetate). We were able to isolate IBF (1) as a colorless solid (mp 20 °C) with a yield of 66%. The solid compound can be stored for up to 8 months at −15 °C without decomposition (polymerization).To further purify isobenzofuran (1) is one of the most reactive dienes in Diels–Alder reactions and other cycloadditions. For practical applications it has been generated and reacted in situ, because it rapidly dimerizes or polymerizes in solvents of medium polarity such as chloroform (t1/2 = 2 h). We observed longer half-lives in low polarity solvents . We have been able to purify the compound by chromatography and to isolate it as a colorless solid (mp 20 °C). In crystalline form, it is stable for 8 months at −15 °C without decomposition. Upon oxidative methoxylation of commercially available phthalane (85% yield) + calcd. for C8H6O, 118.04173; found, 118.04186. : DiisoprDimethyl 1,4-epoxy-1,4-dihydronaphthalene-2,3-dicarboxylate (10): Dimethyl acetylenedicarboxylate  was dissolved in benzene (25 mL) under a nitrogen atmosphere. A freshly prepared solution of isobenzofuran (1), which was prepared from DMIBF , prior to purification by chromatography (see procedure above) was added dropwise and stirred for 16 h at 50 °C. The crude product was purified by column chromatography on silica gel . A colorless oil was obtained. Yield: 223 mg ; IR (ATR): 2953 (w), 1710 (s), 1637 (m), 1435 (m), 1291 (m), 1250 (s), 1211 (s), 1109 (s), 1064 (m), 976 (m), 910 (m), 854 (s), 755 (s), 734 (m), 655 (s) cm−1; 1H NMR  δ 7.43 , 7.07 , 5.96 , 3.80  ppm; 13C NMR  δ 162.8, 151.2, 146.2, 126.1, 121.4, 84.8, 52.4 ppm; HRMS (EI) m/z: [M]+ calcd. for C12H12O5, 260.06847; found, 260.06800.File 1Analytical equipment and methods, experimental procedures and NMR spectra.


Shewanella spp., newly isolated from the gut of the sea cucumber Apostichopus japonicus . The whole-genome sequences reported here will expand the repertoire of genomic information for the members of the genus Shewanella and will provide important insights into their roles within microbial communities.In this study, we sequenced the genomes of two  Shewanella is composed of a diverse group of facultative anaerobic bacteria that are widely distributed in aquatic environments .The genus ronments –3. Several value , 5. Moreathogens , 7. The  members –3. Here,Shewanella spp. were first identified by 16S rRNA gene sequence analysis using EzTaxon-e  prepared with the Accel-NGS 2S PCR-free library kit  according to the manufacturer’s instructions. A total of 7,865,394 and 7,492,116 raw sequence reads were generated from S. colwelliana and S. algae, resulting in 255× and 235× coverages of the genomes, respectively. The raw sequence reads were filtered using Trimmomatic (de novo assembled with three different assemblers, including Abyss version 1.9 (de novo assembly using QUAST version 4.1 (S. colwelliana CSB03KR consisted of 58 scaffolds, covering 4,655,471 bp, the longest of which was 594,967 bp, and had a 45.4% GC content and an N50 of 241,596 bp. The draft genome for S. algae CSB04KR also consisted of 64 scaffolds, covering 4,803,356 bp, the longest of which was 301,346 bp, and had a 53.0% GC content and an N50 of 149,689 bp.The two isolated pure cultures of zTaxon-e . Genomicsion 1.9 , SOAPdension 1.9 . Finallysion 1.9 , which ssion 4.1 , the assS. colwelliana CSB03KR genome contains 4,336 genes, including 4,244 protein-coding sequences classified in 514 subsystems, 3 5S rRNAs, 4 SSU rRNAs, 3 LSU rRNAs, and 82 tRNAs. The annotated S. algae CSB04KR genome contains 4,358 genes, including 4,256 protein-coding sequences classified in 526 subsystems, 8 5S rRNAs, 1 SSU rRNA, 2 LSU rRNAs, and 91 tRNAs. In summary, the complete genomes of S. colwelliana CSB03KR and S. algae CSB04KR sequenced in this study will provide comprehensive insights into the microbial ecology of the gut in sea cucumbers.Genome annotation was performed with the RAST version 2.0 automatic annotation server , using tS. colwelliana CSB03KR and S. algae CSB04KR have been deposited in DDBJ/ENA/GenBank under the accession numbers MCBT00000000 and MBFW00000000, respectively.The complete genomes for


Methanohalophilus portucalensis strain FDF-1T, a halophilic methylotrophic methanogen isolated from the sediment of a saltern in Figeria da Foz, Portugal. The average nucleotide identity and DNA-DNA hybridization analyses show that Methanohalophilus mahii, M. halophilus, and M. portucalensis are three different species within the Methanosarcinaceae family.We report here the complete genome sequence (2.08 Mb) of  Methanohalophilus portucalensis strain FDF-1T  was described as a new species by Boone et al. , a 100-bp paired-end library sequenced on an Illumina HiSeq platform , and a PacBio RS library . The 13,835,892 paired reads of 300 bp were quality trimmed (Q30) and de novo assembled into contigs using SPAdes version 3.6.1    between M. halophilus and M. mahii, and 92.59%  between M. mahii and M. portucalensis. An ANI score below 95% has been defined for the delineation of a new species (in silico DNA-DNA hybridization (DDH) values using the genome-to-genome distance calculator GGDC2.1   and M. h SLPT 2,02,424 bp  species . Thus, t GGDC2.1 , which iCP017881.This whole-genome sequence has been deposited at GenBank under the accession number


The satisfactory outcome in the surgical treatment of obesity must include,in addition to weight loss, a significant change in the pre-existingcomorbidities and in the quality of life of the patients.  To evaluate the quality of life in the late postoperative period in patientsthat underwent videolaparoscopic sleeve gastrectomy.  Was applied the questionnaire “Bariatric Analysis and Reporting OutcomeSystem” (BAROS) in patients that underwent videolaparoscopic sleevegastrectomy.  A total of 47 patients between 21-60 years old were evaluated. The totalmean of the BMI before surgery was 43.06±5.87 kg/m². The average percentageof the reduction of excess weight after surgery was 85.46±23.6%. The scoreobtained by patients in the questionnaire about the improvement in thequality of life showed excellent (36.17%), very good (40.43%), good (21.28%)and reasonable (2.13%) results. There was clinical improvement after surgeryin all comorbidities investigated.  BAROS showed excellent results in 36.17%, very good in 40.43%, good in21.28% and reasonable in 2.13%. The weight loss was critical to improve thequality of life and offered the resolution or clinical improvement in all ofthe investigated comorbidities. It is considered a chronic disease, multifactorial and associatedwith several comorbidities and severe impairment of quality of life,The surgical approach to severe obesity emerged around 1950. During the followingyears, several techniques and analysis were developed and recognized this procedureas effective in both weight loss and reduction of comorbidities, providing greatersurvival compared to clinical treatments,,,,,The gastric sleeve is a restrictive procedure that consists of the removal of thegreat gastric curvature, starting from 4-6 cm from the pylorus to theesophagogastric angle, leaving the new reservoir with tubular and elongatedshapeHowever, as many factors besides weight are modified after this procedure, it isfundamental to evaluate the quality of life of these patients. Thus, in order toreinforce the importance of continuity of care in the postoperative period, theBariatric Protocol and Reporting Outcome System (BAROS) was developed in 1998. Thisinstrument emerged as a simple, inexpensive and reliable alternative for assessingthe self-perception of quality of life in postoperative patientsThe BAROS protocol consists of three major areas of investigation , from which a maximum score ofthree points is obtained for each category, totaling a maximum of nine points. Thescore of the category “weight loss” is given by the percentage loss of excess weight(%PEP). In “medical conditions” the individual scores higher as there is clinicalimprovement or cure of one, several or all of the pre-existing comorbidities.Finally, the “quality of life questionnaire” includes questions about the practiceof physical activities, social activities, work performance, sexual interest andimprovement in the general conditionTherefore, the purpose of this study was to apply this protocol in patients whounderwent vertical gastrectomy by videolaparoscopy.This study is the result of a cross-sectional study conducted between the months ofApril and May of 2016. The sample was selected from a clinical database of patientsundergoing laparoscopic vertical gastrectomy surgery by the staff of “Clínica deCirurgia do Aparelho Digestivo VIDAR”  between the years 2012and 2015. The project was approved by the Ethics Committee of the RegionalUniversity of Blumenau,SC, on April 28, 2016 (number 1519860).Patients were contacted and invited to participate in person filling out the printedBAROS questionnaire. The same questionnaire adapted in “Google Forms” platform wassent by e-mail if impossibility of displacement to the clinic. Patients exclusioncriteria were: a) who had any cognitive limitation to compromise responses to datacollection instrument; b) who refused to answer the questionnaire; c) who did notanswer a question of the form. In possession of the information obtained, the datawere divided into three categories, according to the period elapsed after surgery:a) up to six months; b) seven to 12 months; c) more than 12 months. Was performed using Epi Info software, version 7. The data level of significancewas p<0.05.2. Theaverage overweight of the patients was equal to 51.01±18.48 kg, an equivalent to40.93±7.65% excess of average body weight.A total of 47 patients were evaluated, of whom 76.6% (n=36) were female and 23.4%(n=11) male. The average age was 37.3±10.75 years. The average weight and BMI beforesurgery were, respectively, 121.05±22.56 kg and 43.06±5.87 kg/mAll 47 patients completed the questionnaire BAROS, offering the following results:36.17% (n=17) were classified as “excellent”; 40.43% (n=19) as “great”; 21.28%(n=10) as “good” and 2.13% (n=1) as “reasonable” improvement of quality of life. Nopatient had a score in the “bad” category. .There was no statistically significant difference between the score of men and women(p>0.05). The time of postoperative period was also not significant ininfluencing the BAROS score (p>0.05). In patients with more than six months afterthe surgery, it was observed predominance of excellent (71.58%) and great (76.84%)results. It was also found that the highest scores were obtained for those patientswho had lost more than 75% of overweight (p<0.05).For the five domains assessed by questionnaire, the results were as follows: a)General condition: all patients responded “very good” (n=40 or 85.11%) or “good”(n=7 or 14.89%); b) Social or family activities: 21 of 47 patients (44.68%) claimedto have been no change in this area, and one patient (2.13%) reported worsening ofsocial relationships. The “improved a lot” got 29.79% (n=14) and the “improved”23.40% (n=11); c) Physical activities “increased a lot” 40.43% (n=19), “increased”34.04% (n=16), “unchanged” 23.40% (n=11) and “decreased” 2.13% (n=1); d) Sexualinterest “greatly increased” 14.89% (n=7), “increased” 29.79% (n=14), “unchanged”44.68% (n=21), “decreased” 6.38% (n=3) and “greatly diminished” 4.26% (n=2); e)Working performance “increased a lot” 51.06% (n=24), “increased” 38.30% (n=18),“unchanged” 4.26% (n=2) and “decreased” 6.38% n=3, .The gender was not a significant factor in EWL% (p>0.05). A total of 69.4% ofwomen lost more than 75% overweight vs. 45.4% for men. However, men lost more totalweight in kilograms (72.7% lost between 40 and 80 kg against 38.9% of women).Patients with grade 3 obesity  statistically(p<0.05) obtained better results in the loss of excess weight: 65.9% of patientslost more than 50% of the excess of weight.The time of postoperative period exerted a significant influence (p<0.01) inreducing the BMI of patients: limited reductions, of less than 10 kg/m², were foundin individuals living a postoperative period of less than six months; in contrast,52.63% of the patients with more than 12 months of postoperative period, lostbetween 15 and 30 kg/m².Regarding comorbidities among patients, 55.3% (n=26) had hypertension, 15% (n=7) type2 diabetes mellitus, 27.6% (n=13) dyslipidemia, 44.7% (n=21) obstructive sleepapnea, 49% (n=23) suffered from some form of joint problem and 49% (n=23) haddepressive disorder before performing the surgical procedure to reduce weight. Thesecomorbidities were not diagnosed or classified by the authors, but self-reported bypatients included in this study.It was observed in the postoperative period that 77% (n=20) of patients who had highblood pressure achieved complete resolution of this disease and 38.4% were able tomaintain control of hypertension with fewer antihypertensive medications. Amongpatients with type 2 diabetes mellitus, 71.4% (n=5) had complete resolution ofdisease and 28.5% (n=2) were able to reduce medication for glycemic control. Thosewho had dyslipidemia, 92.3% (n=12) showed no more such comorbidity and among thosesuffering from obstructive sleep apnea syndrome, 57.1% (n=12) did not reportsymptoms. Of individuals who had some kind of joint problem, 61% (n=14) extinguishedthis disease and did not use medications anymore; 34.7% (n=8) were able to reducethe number of painkillers and other medicines. Finally, of patients who haddepression in preoperative period, 39.1% (n=9) reduced the number and/orconcentration of antidepressant medications and 48% (n=11) did not have symptomsanymore .2, 13.73±2.8 kg/m2 and 16.32±3.99kg/m2 and EWL% was 75.60±7.97% (n=3), 80.82±20.43% (n=25) and93.13±27.42% (n=19), respectively.In relation to the average weight loss, patients who had undergone surgery within sixmonths (n=3) lost 31.67±16.77 kg; between seven and 12 months (n=25), 38.36±8.94 kg;more than 12 months (n=19), 45.83±12.12 kg. The average reduction of BMI was10.53±3.83 kg/m2; overweight, 10.01±14.08 kg equivalent to 9.72±15.25%; the EWL%of all patients was 85.46±23.6% , dizziness (n=2),gastroesophageal reflux disease (n=1), hypotension (n=1), weakness (n=1), epigastricpain (n=1 ), tremor (n=1). No patient reported more than one complication and onlyone patient required reoperation, but did not justify the cause.,,,Several studies document the influence of weight loss in improving the quality oflifeThe average score of patients in the questionnaire of quality of life was 1.85±0.64,a maximum of three points. Similar data were obtained by Janik et al (2016) whichcompares the technical approach of sleeve and bypass with Roux-en-Y and found nosignificant differences.,The BAROS’ domains most highly evaluated by patients were “general condition”(average score of 0.92 up to 1.0), “working performance” (0.33/0.5) and “physicalactivity” (0.28/0.5). The areas “social activities” (0.2/0.5) and “sexual interest”(0.11/0.5) had the worst average. This happened because most patients have answeredthat there was no change in these two domains generating zero score. OtherstudiesLike other studies,Several studies show that the sleeve gastrectomy reduces mortality and thedevelopment of new comorbid conditions and worsening of already present diseases inobese individuals12 and up to 81%,The prevalence of diabetes among the obese patients in the preoperative period wassimilar to the study of Blume et al (2012) which showed a value of 14.7%. The fullresolution of this disease was similar to other studies that have obtained rates of47%,,In addition to the above comorbidities, the high frequency of psychological diseasesof obesity can lead to behavioral changes by the fact that these individuals arefrequent targets of prejudice and discriminationBAROS showed excellent results in 36.17%, optimum in 40.43%, good in 21.28% andreasonable in 2.13%. Weight loss was fundamental for the improvement in quality oflife and provided resolution or clinical improvement in all investigatedcomorbidities.


We aimed to explore whether protein kinase C (PKC) activation plays an important role in oxidative stress-induced afterdepolarizations. Methods: Action potentials and ion currents of isolated rabbit cardiomyocytes were recorded using the patch clamp technique. H2O2 (1 mM) was perfused to induce oxidative stress and the specific classical PKC inhibitor, Gö 6983 (1 μM), was applied to test the involvement of PKC. Results: H2O2 perfusion prolonged the action potential duration and induced afterdepolarizations. Pretreatment with Gö 6983 prevented the emergence of H2O2-induced afterdepolarizations. Additional application of Gö 6983 with H2O2 effectively suppressed H2O2-induced afterdepolarizations. H2O2 increased the late sodium current   and the L-type calcium current  , which were significantly reversed by Gö 6983 (p < 0.01). H2O2 also increased the transient outward potassium current (Ito) . However, Gö 6983 showed little effect on H2O2-induced enhancement of Ito. Conclusions: H2O2 induced afterdepolarizations via the activation of PKC and the enhancement of ICa,L and INa,L. These results provide evidence of a link between oxidative stress, PKC activation and afterdepolarizations.Background: Hydrogen peroxide (H Under pathological conditions, such as heart failure (HF)  and ische stress  and it i  ,5, trans,L) [to) ,7, late ) [Na,L) ,9, and f) [Na,L) ,8.Protein kinase C (PKC), a group of serine/threonine protein kinase enzymes, has been identified as an important regulator of a variety of cardiac responses including myocardial fibrosis, cardiomyocyte hypertrophy and inflammation, which are closely associated with heart failure and ischemia ,11,12. PCa,L and INa,L seems to be the underlying mechanism.Accumulating evidence suggests that increased oxidative stress could serve as an important arrhythmogemic factor in diseased hearts. ROS can persistently activate PKC via direct oxidative modification  and incr2O2.Action potentials (APs) were recorded from isolated rabbit ventricular myocytes using the whole-cell current clamp mode. To elicit EADs stably, the myocytes were paced at basic cycle lengths of 6 s . After t2O2 perfusion prolonged APD gradually and induced afterdepolarizations and TAs after approximately 7 min, while control experiments in parallel (without H2O2) showed that no electrical abnormalities occurred for up to 15 min (n = 9). APD 90 was prolonged from 276.7 ± 77.4 to 585.0 ± 65.0 ms  at 5 min of application of 1 mM H2O2. Examples of afterdepolarizations and TAs are shown in As shown in 2O2-induced afterdepolarizations by using the specific classical PKC inhibitor Gö 6983. Unlike those myocytes consistently presenting EADs approximately 7 min after exposure to 1 mM H2O2 . To further confirm the effect of PKC inhibition on H2O2-induced EADs, we applied another widely used selective PKC inhibitor, Bisindolylmaleimide (BIM). As expected, pretreatment with BIM (1 μM) prevented the emergence of H2O2-induced afterdepolarizations in six of six ventricular myocytes  are shown in 2O2 significantly enhanced ICa,L, which was effectively attenuated by Gö 6983. The peak amplitudes of ICa,L at +10 mV were measured for data analysis. As shown in Ca,L was elevated from −6.11 ± 0.47 to −8.82 ± 0.61 pA/pF after H2O2 perfusion , and decreased to −6.21 ± 0.45 pA/pF after the addition of Gö 6983 . In the control experiments, the run-down of ICa,L was less than 5% [The effects of H than 5% .Na,L plays a key role in H2O2-induced afterdepolarizations [Na,L curves averaged by five replicated depolarizing voltage steps from −120 to −30 mV of one representative cell are shown in Na,L were measured at 200 ms and are summarized in Na,L was elevated from −0.09 ± 0.01 to −0.23 ± 0.02 pA/pF  and attenuated by 1 μM Gö 6983 to −0.11 ± 0.02 pA/pF .Iizations ,20,21. I2O2 and the PKC inhibitor on Ito were also analyzed. The representative current-voltage traces of Ito are displayed in n = 6) are shown in to at +50 mV were measured and are summarized in to was significantly elevated from 3.07 ± 0.62 to 3.88 ± 0.50 pA/pF after H2O2 perfusion , and decreased to 3.58 ± 0.46 pA/pF after the addition of Gö 6983 .The effects of H2O2-induced afterdepolarizations effectively, indicating PKC activation mediates oxidative stress-induced afterdepolarizations; (2) PKC inhibition by Gö 6983 attenuated H2O2-induced enhancement of ICa,L and INa,L, but not Ito.The present study provided the following novel findings: (1) the specific classical PKC inhibitor Gö 6983 both prevented and reversed the emergence of HPKC, a classical second messenger, is acknowledged to be involved in several pathological progressions including hypertrophy, fibrosis, inflammation, ischemic preconditioning and atherosclerosis. Thus, PKC serves as a potential therapeutic target for heart failure, hypertension and coronary heart diseases ,22. Prev2+ and diacylglycerol [2+ and diacylglycerol, mild oxidative stress can activate PKC by selectively oxidizing the regulatory domain [2O2-induced afterdepolarizations.Oxidative stress is involved in a variety of cardiovascular diseases, including heart failure and ischemic heart disease. Cardiac arrhythmias are prevalent in patients with these conditions. Afterdepolarization-mediated TAs play an important role in ROS-induced arrhythmias , but theglycerol . When inglycerol . It is ny domain . These f2O2 may affect ICa,L, INa,L and Ito via direct oxidative modification, intracellular signaling pathways or transcription and translation regulations [2O2 is capable of enhancing these major membrane currents significantly. In our study, adding the PKC inhibitor along with H2O2 for several minutes effectively reversed the H2O2-induced alterations on ICa,L and INa,L, suggesting the mechanistic role of PKC on the H2O2-induced intracellular signaling cascade. However, acute application of the PKC inhibitor failed to reverse the enhancement of Ito, indicating a minor role of Ito on H2O2-induced effects.Hulations . Our res2+ channel [+ channel [Ca,L facilitation and INa,L augmentation. Previous studies have demonstrated that the elevation of not only INa,L but also ICa,L was involved in the ROS-induced afterdepolarization formation [Na,L can reduce the repolarization reserve, while activation of ICa,L can generate EAD upstroke. It is known that augmentation of ICa,L can increase Ca2+ influx and enhance cellular Ca2+ loading. In addition, APD prolongation can also enhance cellular Ca2+ loading, and further promotes PKC activation. In our study, the inhibition of PKC activation significantly reversed H2O2-induced enhancement of both ICa,L and INa,L, these might result in reduced cellular Ca2+ loading by attenuating Ca2+ influx via ICa,L and shortened APD.When activated, PKC can phosphorylate downstream proteins including the L-type Ca channel  and volt channel , resultiormation ; thus, e2O2-induced afterdepolarizations [2+ level, they may share similar ionic mechanisms in oxidative stress-induced afterdepolarizations. Another study demonstrated similar inhibitory effects on intracellular Ca2+-induced INa,L by both inhibitors of PKC and CaMKII [The results collectively suggested that PKC signaling plays a central role in oxidative stress-induced afterdepolarizations, although activation of other kinases such as protein kinase A and Ca/calmodulin-dependent protein kinase II (CaMKII) cannot be ruled out, and it is beyond the present study protocol. Previous studies have demonstrated that CaMKII activation played a role in Hizations . As PKC d CaMKII . This evIt has already been demonstrated that ROS levels are elevated in a number of cardiovascular diseases and predispose the heart to arrhythmias. Free radical scavengers were supposed to be a potential therapy strategy for alleviating arrhythmias , but thi2PO4, 1.0 MgCl2, 10 glucose, 10 HEPES, 1.8 CaCl2 with pH adjusted to 7.4 with NaOH. The KB solution contained (in mM) 85 KOH, 50 K-glutamate, 30 KCl, 1.0 MgCl2, 20 taurine, 0.5 Ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), 10 glucose, 10 HEPES with pH adjusted to 7.4 with KOH. Chemicals and reagents were purchased from Sigma Aldrich  unless otherwise noted. BIM was purchased from Selleckchem . Nifedipine, Gö 6983 and BIM were dissolved in Dimethyl Sulfoxide (DMSO) as stock solution according to the manufacturer’s instructions before diluting into the superfusate solution at the final concentration. The final concentration of DMSO was less than 0.1%.The standard Tyrode solution and Kraft-Bruhe (KB) solution were made with Milli-Q grade water. The standard Tyrode solution contained (in mM) 135 NaCl, 5.4 KCl, 0.33 NaH2+-free Tyrode’s solution containing 1.4 mg/mL collagenase  and 0.1 mg/mL protease  for 15 to 30 min. After washing out the enzyme solution, the hearts were removed from the apparatus and swirled in a culture dish in KB solution. The Ca2+ concentration was gradually increased to 1.8 mM, and the cells were then kept at 4 °C until use.The use and care of animals conformed to the Guide for the Care and Use of Laboratory Animals of Shanghai, China and was approved by the Institutional Animal Care and Use Committee of Xinhua Hospital . Hearts were removed from 23 adult New Zealand White rabbits (2 to 3 kg) anesthetized with intravenous pentobarbital, and were then perfused retrogradely in Langendorff apparatus at 37 °C with Ca2O2 (1 mM) and Gö 6983 (1 μM) were added to the bath superfusate. For AP recordings, patch pipettes (resistance 2 to 3 MΩ) were filled with pipette solution containing (in mM) 110 K-aspartate, 30 KCl, 5 NaCl, 0.1 EGTA,10 HEPES, 5 MgATP, 5 creatine phosphate, 0.05 cAMP, pH 7.2 with KOH. APs were elicited with 2 ms, 2–4 nA square pulses at basic cycle lengths of 6 s.All experiments were performed at 35 °C to 37 °C. Myocytes were perfused in standard Tyrode solution for 5 min and then patch-clamped using the whole-cell configuration. HCa,L recording, the cells were superfused with a modified Tyrode’s solution in which KCl was replaced by CsCl, and the patch pipettes (resistance 1 to 2 MΩ) were filled with an internal solution containing (in mM): 110 Cs-Aspartate, 30 CsCl, 5 NaCl, 10 HEPES, 5 MgATP, 5 creatine phosphate, 0.1 EGTA, 0.05 cAMP, pH 7.2 with CsOH. ICa,L was recorded using a series of 200 ms steps from −40 to +50 mV with increments of 10 mV from a holding potential of −80 mV prior to a 100 ms prepulse to −40 mV.For INa,L, the pipette solution contained (in mM) 110 Cs-aspartate, 30 CsCl, 10 HEPES, 0.5 EGTA, 0.2 Na3-GTP, 5 Na2-phosphocreatine, 5 MgATP, pH 7.2 with CsOH. The bath solution was the same as that used for ICa,L. Nifedipine (30 μM) was added to the bath solution to block calcium currents. INa,L was elicited by 300 ms pulses from −120 to −30 mV, and the amplitude was measured at 200 ms.To isolate Ito) recording, the pipette and bath solutions were the same as those for AP recording. TTX (10 μM) and Cd3+ (0.3 mM) were added into the Tyrode’s solution to inhibit INa,L and ICa,L. Ito was elicited at basic cycle lengths of 10 s by a series of 300 ms steps from −40 to +70 mV with increments of 10 mV from a holding potential of −70 mV prior to a prepulse to −40 mV.For transient outward potassium current  controlled by a personal computer using a Digidata 1440A acquisition board driven by pCLAMP 10 software . Data are presented as means ± standard error of means unless indicated. Statistical significance was assessed using unpaired Student’s Ca,L and INa,L, and PKC might be a potential antiarrhythmic therapeutic target.PKC activation mediates oxidative stress-induced afterdepolarizations via enhancement of I


The effect of substituents on the optical properties of donor-π-acceptor compounds having dicyanovinyl as the acceptor are studied using density functional theory and time-dependent density functional theory (DFT/TD-DFT). The optical transitions are thoroughly examined to reveal the impact of subtituents on both absorption and fluorescence, mainly through the modification of the structure in the excited state. The theoretical results have shown that TD-DFT calculations, with a hybrid exchange–correlation and the long-range corrected density functional PBEPBE with a 6–311++G** basis set, was reasonably capable of predicting the excitation energies, the absorption and the emission spectra of these molecules.A set of different donor-π-acceptor compounds having dicyanovinyl as the acceptor and aryl moieties as donors were synthesized by Knoevenagel condensation. The UV–visible absorption and fluorescence spectra were investigated in different solvents. The optical band gab energy (Eg) was linearly correlated with the Hammett resonance effect of the donor to reveal that the higher the value of Hammett resonance effect of a donor, the lower the Eg of the molecule. The photophysical data revealed that compounds M4–M6 are typical molecular rotors with fluorescence due to twisted intramolecular charge transfer. Compound M5 revealed the largest Stokes shift (11,089 cm Donor-π-conjugate-electron acceptor (D-π-A) compounds are characterized by having intramolecular charge transfer (ICT) character. These compounds are of great interest owing to their high molar absorptivity , amenabiIn this context, we have designed and prepared as series of different benzenoid compounds containing different numbers of methoxy groups, methyl group and dimethylamino group as electron donors compared with the unsubstantiated benzene ring and using dicyanovinyl as the electron acceptor group. It was hypothesized that having acceptor in one side of a conjugated system and connected with different donors on the other side would help understanding the ICT character of such compounds and its impact in their photophysical properties.In recent years, calculations of electronic structures in the excited states have been a focus of interest because of the development of computations based on Gaussian and the time dependent density functional theory (TDDFT) –23. AlsoTherefore, computational chemistry is thus necessary to get insight into the molecular structure, although according to our best knowledge no evidence of similar study for the dicyanovinyl effect on the ICT character of the model compounds selected in this study. In this work, interest resides in correlating the theoretically predicted electronic parameters with the accurate experimental results so as to provide possible explanations for the experimentally observed data.The compounds  were obtained by Knoevenagel condensation in a basic medium as shown in Scheme 3, CH3OH and CH3CN and the photophysical properties of these compounds are shown in Figs. *. The effect of the donor ability of the substituent groups is nicely correlated with the optical data. Substituting hydrogen atom in compound 1 with different donors shown in Scheme Absorption and fluorescence spectra of molecules M1–6) recorded in CHCl–6 recordg) was estimated from the onset wavelength of absorption using the equation of Eg = 1240/λab, onset. Figure g and Hammett resonance effect of donors. As shown in this figure, the higher the value of Hammett resonance effect of a donor, the lower the Eg of the molecule indicating the involvement of an intramolecular charge transfer (ICT) between donor and acceptor.As the donor groups are in conjunction with acceptor via π-system, thus it was reasonable to correlate the calculated band gap energy of all compounds with Hammett resonance effect . The optAnother interesting feature observed in Table The fluorescent intensity is a function of the free rotation of the molecular rotor and thus a higher fluorescence would be observed dependent on the nature of TICT and/or the fluorophore microenvironment. Since the solvents used are non-viscous solvents thus the huge fluorescence observed in compound M5 compared with other compounds is reflecting its twisted geometry that hampers the free rotation. It is worth noting Table  that com0), especially in M3 and M5 molecules. These results indicate that the connection between acceptor group and donor group for highly enhanced ICT character, which is important for the absorption spectra red-shift.The optimized geometries obtained by B3LYP/6-311++G** level of theory for the ground and excited states studied molecules are displayed in Figs. max (nm), frontier molecular orbitals contributions and oscillator strength (f) of the studied compounds (M) collected in Table abs of the studied molecules decreases in the following order M6 > M3 > M5 > M4 > M2 > M1 which is the same order of the band gap except with M3. This bathochromic effect from M1 (304.27 nm) to M3 (397.62) is obviously due to increased π delocalization. With the increasing of conjugation, the λabs arising from S0 → S1 electronic transition increase. The first excited states for all studied molecules are π → π∗ transitions which differ in the dominant configuration. The natural transition orbitals (NTO) displayed in Fig. The vertical excited first three singlet states, transitions energies, and oscillator strength using TD-DFT (PBEPBE) method started from the optimized structures have been calculated. The corresponding simulated UV–visible absorption spectra of all molecules in the gas phase using PBEPBE/6-311++G** level of theory displays in Fig. 3 solutions on a Bruker Avance 600 MHz spectrometer. Infrared spectra were performed on a PerkinElmer spectrum 100 FTIR spectrometer. Mass spectra were measured on a GCMS-QP1000 EX spectrometer at 70 eV. UV–visible absorption spectra were recorded with a Jasco V560 spectrophotometer . Fluorescence spectra were conducted on a Perkin-Elmer LS-55 Luminescence Spectrometer and uncorrected. Melting points were determined in open capillary tubes in a Stuart Scientific melting point apparatus SMP3 and are uncorrected.All solvents and reagents were purchased from Sigma-Aldrich Company and used as received. 2-(4-Methoxybenzylidene)malononitrile (M3) is commercially available at Life Chemicals, Canada and was used as received. 1H and 13C NMR spectra were recorded in CDClA mixture of aldehyde derivative (10 mmol), malononitrile (10 mmol), sodium acetate anhydrous (12 mmol) and ethanol absolute (30 ml) were stirred at room temperature for 24 h. Then, water was added to the reaction mixture to precipitate the product. The precipitate was filtered, washed water and then dried. Further purification by silica gel column chromatography afforded the corresponding product in good yield.1H NMR : ∂ 7.54 , 7.63 , 7.78 2), 7.90 . 13C NMR : ∂ 82.88, 112.57, 113.73, 129.67, 130.77, 130.93, 134.69, 159.9; ATR-IR: 3043, 2222, 1589, 1567, 1449; MS (m/z) for C10H6N2 (M−H)+: Calcd: 153.05, Found: 153.Solid, m.p:84 °C 1H NMR : ∂ 2.45 , 7.32 , 7.71 2), 7.8 . 13C NMR : ∂ 22.05, 81.27, 112.88, 114.04, 128.50, 130.41, 130.95, 146.41, 159.79; ATR-IR: 3035, 2221, 1605, 1584, 1553, 1509; MS (m/z) for C11H8N2 (M−H)+: Calcd: 167.07, Found: 167.Solid, m.p:135 °C 1H NMR : ∂ 3.14 2), 6.68 , 7.46 2), 7.81 . 13C NMR : ∂ 40.15, 71.95, 111.60, 114.95, 116.03, 119.31, 133.83, 154.22, 158.16; ATR-IR: 2920, 2207, 1607, 1560, 1515, 1385, 1357; MS (m/z) for C12H11N3 (M−H)+: Calcd: 196.1, Found: 196.Solid, m.p:180 °C 1HNMR : ∂ 3.83 , 6.69 , 7.03 , 7.68 2).13C NMR : ∂ 55.71, 83.12, 107.22, 108.24, 112.66, 113.68, 132.35, 160.14, 161.25; ATR-IR: 2966, 2229, 1603, 1577, 1458, 1426, 1310; MS (m/z) for C12H10N2O2 (M−H)+: Calcd: 213.07, Found: 213.Solid, m.p:89 °C 1H NMR : ∂ 3.90 , 3.97 , 7.18 , 7.65 2).13C NMR : ∂ 56.37, 61.30, 80.60, 108.26, 113.23, 114.02, 125.96, 143.97, 153.37, 159.45; ATR-IR: 2942, 2839, 2221, 1568, 1499, 1455, 1247, 1126.92; MS (m/z) for C13H12N2O3 (M−H)+: Caled: 243.1, Found: 243.Solid, m.p:145 °C max) and oscillator strengths (f) for the three states by using TD-B3LYP/6–311++G** level of theory. Moreover, three density functional, namely, PBEPBE [All calculations are performed using Gaussian 09 W  program , PBEPBE  with samIn this paper, different donor-π-acceptor compounds having dicyanovinyl as the acceptor and aryl moieties as donors were synthesized. Compared with all molecules investigated, molecule 5 showed the highest Stokes shift as well as the highest fluorescent intensity indicating a typical molecular rotor. Also, the energy Eg values were nicely correlated with the donor ability of the substituent as presented by Hammett resonance effect. UV–visible absorption maxima of the compounds were examined experimentally as well as computationally and the results obtained have shown that TD-DFT calculations, with a hybrid exchange–correlation and the long-range corrected density functional PBEPBE with a 6–311++G** basis set, was reasonably capable of predicting the excitation energies, the absorption and the emission spectra of these molecules.