Industrial Scale Isolation,Structural and Spectroscopic Characterization of Epiisopiloturine from Pilocarpus microphyllus Stapf Leaves: A Promising Alkaloid against Schistosomiasis

This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)-Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), ¹H and ¹³C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution.     

Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.     

Comparative larval ontogeny of two fish species (Characiformes and Siluriformes) endemic to the São Francisco River in Brazil

The aim of the present study was to perform comparative histological analyses of the ontogenetic development of two fish species endemic to the São Francisco River in Brazil: Prochilodus argenteus and Lophiosilurus alexandri. Histological analyses were performed every 24 h from the moment of hatching until 14 days post-hatching (dph) for the observation of larval development and until 39 dph for the observation of gonadal development. Whole larvae were fixed in Bouin's solution and the histological slides were stained with haematoxylin–eosin. Lophiosilurus alexandri larvae had a larger body size compared with P. argenteus larvae since hatching. Lophiosilurus alexandri larvae had mouth opening and pigmentation of the eyes upon hatching, whereas these events were observed at 1 dph in P. argenteus larvae. The visualisation and the inflation of the swim bladder occurred at 1 and 3 dph, respectively, in the P. argenteus, whereas these events occurred at 2 and 8 dph, respectively, in L. alexandri. Yolk granules were absorbed at 4 dph in P. argenteus and the 10 dph in L. alexandri. At 7 dph, the digestive tube was more differentiated in L. alexandri than P. argenteus and at 14 dph, the digestive system of both species had features of their eating habits: broad stomach and short intestine in L. alexandri, typical of carnivorous habits; stomach with a mechanical function and long intestine in P. argenteus, typical of detritivorous habits. The epithelial lining tissue, formed by a single layer of cells in the newly hatched larvae (0 dph), differentiated throughout the study, exhibiting scales in P. argenteus and numerous club cells in the middle epithelial region of L. alexandri at 39 dph. Undifferentiated gonads with somatic cells and primordial germ cells were observed at 39 dph, with caudal-cranial migration since 1 dph in both species. The anatomic changes during the ontogeny of P. argenteus and L. alexandri larvae are directly associated with the evolutionary history of each species, which explains their feeding habits, behaviour and distribution in the environment: Prochilodus argenteus is detritivorous and actively swims in the water column, whereas L. alexandri is carnivorous and inhabits bottom regions. At 39 dph neither species exhibited sexual differentiation.     

A simple topographical model to predict Golden Eagle Aquila chrysaetos space use during dispersal

Many large raptors exploit or rely on anabatic and orographic winds which provide vertical lift, to supplement or provide the energy fuelling flight. Airspace is therefore a critical habitat for such large raptors and its use is subject to the underlying terrestrial topography, because particular topographical features are more likely to provide wind-energetic lift. Accordingly, ridges and/or ‘rugged topography’ are common preferred features in habitat use by large raptors. Our study aimed to provide a simple model of space use for a large raptor, the Golden Eagle Aquila chrysaetos, based on thousands of GPS telemetry records during juvenile dispersal of 92 birds tagged as nestlings between 2007 and 2016 across upland Scotland. Model development was based on the hypothesis that four topographical variables would be influential: slope, aspect, altitude and distance from ridge. The telemetry dataset was divided into training and two testing components. The first testing set was derived by a temporal split resulting in approximately equal sample size on records and some temporal overlap in individuals’ records with training data. The second testing set involved no individuals from the training set. Aspect was removed early in training model development because it was not influential. The model found that young Golden Eagles preferred, or used according to availability, space above slopes greater than 10°, at an altitude of ≥ 300 m, and within 300 m of a ridge. The test data were highly correlated with those from the training data in the model variables, and performance as regard to expected preferences from the model was improved in both test datasets, indicating the model was robust. Given the apparent universal nature of large raptor dependence on topography, that topography is relatively immutable according to time and use, and that topographical data are readily available, we commend our approach to other habitat preference studies of Golden Eagles and other large raptors elsewhere.     

Conformations Consistent with Charge Migration Observed in DNA and RNA X-ray Structures

Abstract

Electron holes are known to migrate along the DNA or RNA duplexes and to localize preferentially on successive guanines. The stationary point conformations of Gua pairs that can trap or let pass these holes have been characterized by quantum chemistry calculations. Here we show their recurrent occurrence in DNA and RNA X-ray structures, often in quadruplex conformations or in interaction with proteins, ligands or metal ions. These findings give support to the biological, possibly regulatory, roles of charge migration in cell functioning.     

Microbial activity of suspended biomass from a nitritation–anammox SBR in dependence of operational condition and size fraction

Single-stage nitritation–anammox combines the growth of aerobic ammonium-oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AnAOB) in one reactor. The necessary compromise of their milieu conditions often leads to the growth of nitrite-oxidizing bacteria (NOB). For this study, a sequencing batch reactor (SBR) for nitritation–anammox was operated for 180 days with sewage sludge reject water (removal capacity, 0.4 kg?N?m^?3?day^?1). The growth of NOB was favored by enhanced oxygen supply rather than extended aerobic phases. Suspended-type biomass from this SBR was taken regularly and sieved into three size fractions (all of them <1,000 μm). Batch experiments as well as fluorescence in situ hybridization were performed to study the distribution and activity of AnAOB, AOB, and NOB within those size fractions. Both the measured conversion rates and detected abundances decreased with increasing size fraction. The highest anammox conversion rates (15 g NH₄ ⁺–N per kilogram VSS per hour) and the highest abundances of Brocadia fulgida were found in the medium size fraction (100–315 μm). The batch experiments proved to be accurate tools for the monitoring of multiple processes in the reactor. The results were representative for reactor performance during the 6 months of reactor operation.