Search for new tools to combat Gram-negative resistant bacteria among amine derivatives of 5-arylidenehydantoin

A series of amine-alkyl derivatives of 5-arylidenehydantoin 3–21 was evaluated for their ability to improve antibiotic effectiveness in two strains of Gram-negative Enterobacter aerogenes: the reference strain (ATCC-13048) and the chloramphenicol-resistant derivative over-producing the AcrAB-TolC efflux pump (CM-64). Three antibiotics, chloramphenicol, nalidixic acid and sparfloxacin were used as markers of efflux pump activity. New compounds (5–16) were obtained within 3–4 step synthesis using Knoevenagel condensation, Mitsunobu reaction and microwave aided N-alkylation. Molecular modeling based structure–activity relationship (SAR) studies were performed. The most active compounds: (Z)-5-(4-(diethylamino)benzylidene)-3-(2-hydroxy-3-(4-(2-hydroxyethyl)piperazin-1-yl)propyl)imidazolidine-2,4-dione (14) and (Z)-5-(2,4-dimethoxybenzylidene)-3-(2-hydroxy-3-(isopropylamino)propyl)imidazolidine-2,4-dione (15) induced fourfold decrease of minimal inhibition concentration (MIC) of all tested antibiotics in the strain CM-64 overexpressing the AcrAB-TolC pump.     

Population specific fitness response of Drosophila subobscura to lead pollution

Abstract Differences in heavy metal tolerance among separate populations of the same species have often been interpreted as local adaptation. Persistence of differences after removing the stressor indicates that mechanisms responsible for the increased tolerance were genetically determined. Drosophila subobscura Collin (Diptera: Drosophilidae) populations were sampled from two localities with different history of heavy metal pollution, and reared for eight generations in the laboratory on a standard medium and on media with different concentrations of lead (Pb). To determine whether flies from different natural populations exposed to the Pb‐contaminated media in the laboratory show population specific variability in fitness components over generations, experimental groups with different concentrations of lead were assayed in three generations (F₂, F₅, and F₈) for fecundity, developmental time, and egg‐to‐adult viability. On the contaminated medium, fecundity was reduced in later generations and viability was increased, irrespective of the environmental origin of populations. For both populations, developmental time showed a tendency of slowing down on media with lead. Faster development was observed in later generations. Preadaptation to contamination, meaning higher fecundity, higher viability, and faster egg to adult development in all studied generations, was found in D. subobscura originating from the locality with a higher level of heavy metal pollution.     

Changes in L-phenylalanine ammonia-lyase activity and isoflavone phytoalexins accumulation in soybean seedlings infected with Sclerotinia sclerotiorum

Soybean [Glycine max (L.) Merr.] cultivars (Meli, Alisa, Sava and 1511/99) were grown up to V1 phase (first trifoliate and one node above unifoliate) and then inoculated with Sclerotinia sclerotiorum (Lib.) de Bary under controlled conditions. Changes in L-phenylalanine ammonia-lyase (PAL) activity and isoflavone phytoalexins were recorded 12, 24, 48 and 72 h after the inoculation. Results showed an increase in PAL activity in all four examined soybean cultivars 48 h after the inoculation, being the highest in Alisa (2-fold higher). Different contents of total daidzein, genistein, glycitein and coumestrol were detected in all samples. Alisa and Sava increased their total isoflavone content (33.9% and 6.2% higher than control, respectively) as well as 1511/99, although 48 h after the inoculation its content decreased significantly. Meli exhibited the highest rate of coumestrol biosynthesis (72 h after the inoculation) and PAL activity (48 h after the inoculation). All investigated cultivars are invariably susceptible to this pathogen. Recorded changes could point to possible differences in mechanisms of tolerance among them.     

Evaluating Purifying Selection in the Mitochondrial DNA of Various Mammalian Species

Mitochondrial DNA (mtDNA), the circular DNA molecule inside the mitochondria of all eukaryotic cells, has been shown to be under the effect of purifying selection in several species. Traditional testing of purifying selection has been based simply on ratios of nonsynonymous to synonymous mutations, without considering the relative age of each mutation, which can be determined by phylogenetic analysis of this non-recombining molecule. The incorporation of a mutation time-ordering from phylogeny and of predicted pathogenicity scores for nonsynonymous mutations allow a quantitative evaluation of the effects of purifying selection in human mtDNA. Here, by using this additional information, we show that purifying selection undoubtedly acts upon the mtDNA of other mammalian species/genera, namely Bos sp., Canis lupus, Mus musculus, Orcinus orca, Pan sp. and Sus scrofa. The effects of purifying selection were comparable in all species, leading to a significant major proportion of nonsynonymous variants with higher pathogenicity scores in the younger branches of the tree. We also derive recalibrated mutation rates for age estimates of ancestors of these various species and proposed a correction curve in order to take into account the effects of selection. Understanding this selection is fundamental to evolutionary studies and to the identification of deleterious mutations.     

The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana

The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes (TH and DDC), in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.     

Vegetation of the <Emphasis Type="Italic">Hydrochari-Lemnete</Emphasis> and <Emphasis Type="Italic">Potametea</Emphasis> classes in the Danube-Tisza-Danube hydrosystem (Serbia)

Aquatic vegetation of Hydrochari-Lemnetea and Potametea classes in the Danube-Tisza-Danube hydrosystem (Hs DTD) was studied in 2009–2012, by applying the standard Braun-Blanquet method. The canal network vegetation comprises 14 associations, with Trapetum natantis and Ceratophylletum demersi being the most widely distributed. Hs DTD is also a habitat for several important endangered species, which serve as edificators of the following phytocenoses: Nymphaeetum albae, Nymphaeetum albo-luteae, Nymphoidetum peltatae, Trapetum natantis, Lemno-Spirodeletum, Salvinio-Spirodeletum polyrrhizae, Lemno-Utricularietum vulgaris, Potametum nodosi, Myriophyllo-Potametum and Najadetum marinae. In the studied vegetation, we also found an invasive phytocenosis Elodeetum canadensis that did not have an expanding tendency, and Ceratophyllo demersi-Vallisnerietum spiralis that had this tendency, which made monitoring its stands necessary. Physico-chemical analyses of water, conducted at localities in which the studied phytocenoses thrive, revealed that the development and distribution of most phytocenoses is closely linked with specific habitat conditions. Among the studied parameters, the most significant for the phytocenoses differentiation were: pH, alkalinity, COD-MnO₄, BOD₅, NO ₃ ^? , NO ₂ ^? , PO ₄ ^3? and the concentration of total phosphorus.     

Pathologic Evaluation of Type 2 Porcine Reproductive and Respiratory Syndrome Virus Infection at the Maternal-Fetal Interface of Late Gestation Pregnant Gilts

The pathogenesis of fetal death caused by porcine reproductive and respiratory syndrome virus (PRRSV) remains unclear. The objective of this study was to improve our understanding of the pathogenesis by assessing potential relationships between specific histopathological lesions and PRRSV RNA concentration in the fetuses and the maternal-fetal interface. Pregnant gilts were inoculated with PRRSV (n = 114) or sham inoculated (n = 19) at 85±1 days of gestation. Dams and their litters were humanely euthanized and necropsied 21 days later. PRRSV RNA concentration was measured by qRT-PCR in the maternal-fetal interface and fetal thymus (n = 1391). Presence of fetal lesions was positively related to PRRSV RNA concentration in the maternal-fetal interface and fetal thymus (P<0.05 for both), but not to the distribution or severity of vasculitis, or the severity of endometrial inflammation. The presence of fetal and umbilical lesions was associated with greater odds of meconium staining (P<0.05 for both). The distribution and severity of vasculitis in endometrium were not significantly related to PRRSV RNA concentration in maternal-fetal interface or fetal thymus. Endometrial inflammation severity was positively related to distribution and severity of vasculitis in endometrium (P<0.001 for both). Conclusions from this study suggest that type 2 PRRSV infection in pregnant gilts induces significant histopathological lesions at maternal-fetal interface, but they are not associated with presence of PRRSV in the maternal-fetal interface at 21 days post infection. Conversely, fetal pathological lesions are associated with presence of PRRSV in the maternal-fetal interface and fetal thymus, and meconium staining is significantly associated with the presence of both fetal and umbilical lesions observed 21 days post infection.     

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) as a potential industrial biocatalyst

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) [poly(GMA‐co‐EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low‐cost, easy‐to‐prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA‐co‐EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA‐co‐EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:43–53, 2016