Malformations and mortality in the Asian Common Toad induced by exposure to pleurolophocercous cercariae (Trematoda: Cryptogonimidae)

Malformations and increased mortality due to infection by the digenetic trematode, Riberioa ondatrae have been reported for many species of amphibians. Severe malformations have also been reported in the Common Hourglass Tree Frog, Polypedates cruciger induced by pleurolophocercous cercariae in Sri Lanka in addition to the changes in the behaviour, development and survival of the host. We exposed pre-limb bud stage tadpoles (Gosner stages 25–26) of the Asian Common Toad, Duttaphrynus melanostictus to the same pleurolophocercous type cercariae under laboratory conditions. Molecular and morphological identification showed that these cercariae belonged Acanthostomum burminis infecting freshwater snakes as definitive hosts. These cercariae induced malformations (27.8%) and reduced survival to metamorphosis (53.8%). The magnitude of the effects increased with the dose of cercariae. Types of malformations were mainly axial, such as scoliosis and kyphosis. Severe limb malformations such as extra or missing limbs as reported for amphibians exposed to R. ondatrae were not observed in the D. melanostictus. Same authors reported a higher percentage of malformations previously when P. cruciger was exposed to the cercariae A. burminis compared to D. melanostictus. However, tadpoles of D. melanostictus, which are smaller compared to those of P. cruciger, experienced higher mortality than P. cruciger tadpoles. Trematode induced malformations and mortality in amphibians are highly variable and depend on multiple factors such as host species differences such as resistance to infection and tolerance, life-history characteristics such as size at metamorphosis and length of the metamorphosis period, and other factors such as size of the amphibian at the time of trematode exposure.     

Identification of triacylglycerol and steryl ester synthases of the methylotrophic yeast Pichia pastoris

In yeast like in many other eukaryotes, fatty acids are stored in the biologically inert form of triacylglycerols (TG) and steryl esters (SE) as energy reserve and/or as membrane building blocks. In the present study, we identified gene products catalyzing formation of TG and SE in the methylotrophic yeast Pichia pastoris. Based on sequence homologies to Saccharomyces cerevisiae, the two diacylglycerol acyltransferases Dga1p and Lro1p and one acyl CoA:sterol acyltransferase Are2p from P. pastoris were identified. Mutants bearing single and multiple deletions of the respective genes were analyzed for their growth phenotype, lipid composition and the ability to form lipid droplets. Our results indicate that the above mentioned gene products are most likely responsible for the entire TG and SE synthesis in P. pastoris. Lro1p which has low fatty acid substrate specificity in vivo is the major TG synthase in this yeast, whereas Dga1p contributes less to TG synthesis although with some preference to utilize polyunsaturated fatty acids as substrates. In contrast to S. cerevisiae, Are2p is the only SE synthase in P. pastoris. Also this enzyme exhibits some preference for certain fatty acids as judged from the fatty acid profile of SE compared to bulk lipids. Most interestingly, TG formation in P. pastoris is indispensable for lipid droplet biogenesis. The small amount of SE synthesized by Are2p in a dga1?lro1? double deletion mutant is insufficient to initiate the formation of the storage organelle. In summary, our data provide a first insight into the molecular machinery of non-polar lipid synthesis and storage in P. pastoris and demonstrate specific features of this machinery in comparison to other eukaryotic cells, especially S. cerevisiae.     

Common Membrane Trafficking Defects of Disease‐Associated Dynamin 2 Mutations

Dynamin (Dyn) is a multidomain and multifunctional GTPase best known for its essential role in clathrin‐mediated endocytosis (CME). Dyn2 mutations have been linked to two human diseases, centronuclear myopathy (CNM) and Charcot‐Marie‐Tooth (CMT) disease. Paradoxically, although Dyn2 is ubiquitously expressed and essential for embryonic development, the disease‐associated Dyn2 mutants are autosomal dominant, but result in slowly progressing and tissue‐specific diseases. Thus, although the cellular defects that cause disease remain unclear, they are expected to be mild. To gain new insight into potential pathogenic mechanisms, we utilized mouse Dyn2 conditional knockout cells combined with retroviral‐mediated reconstitution to mimic both heterozygous and homozygous states and characterized cellular phenotypes using quantitative assays for several membrane trafficking events. Surprisingly, none of the four mutants studied exhibited a defect in CME, but all were impaired in their ability to support p75/neurotrophin receptor export from the Golgi, the raft‐dependent endocytosis of cholera toxin and the clathrin‐independent endocytosis of epidermal growth factor receptor (EGFR). While it will be important to study these mutants in disease‐relevant muscle and neuronal cells, given the importance of neurotrophic factors and lipid rafts in muscle physiology, we speculate that these common cellular defects might contribute to the tissue‐specific diseases caused by a ubiquitously expressed protein.     

Development of a chemically defined medium for the production of the antibiotic platensimycin by Streptomyces platensis

The actinomycete Streptomyces platensis produces two compounds that display antibacterial activity: platensimycin and platencin. These compounds were discovered by the Merck Research Laboratories, and a complex insoluble production medium was reported. We have used this medium as our starting point in our studies. In a previous study, we developed a semi-defined production medium, i.e., PM5. In the present studies, by varying the concentration of the components of PM5, we were able to develop a superior semi-defined medium, i.e., PM6, which contains a higher concentration of lactose. Versions of PM6, containing lower concentrations of all components, were also found to be superior to PM5. The new semi-defined production media contain dextrin, lactose, MOPS buffer, and ammonium sulfate in different concentrations. We determined antibiotic production capabilities using agar diffusion assays and chemical assays via thin-layer silica chromatography and high-performance liquid chromatography. We reduced crude nutrient carryover from the seed medium by washing the cells with distilled water. Using these semi-defined media, we determined that addition of the semi-defined component soluble starch stimulated antibiotic production and that it and dextrin could both be replaced with glucose, resulting in the chemically defined medium, PM7.     

Renifer aniarum (Digenea: Reniferidae), an introduced North American parasite in grass snakes Natrix natrix in Calabria, southern Italy

Over the past decades, as a result of various human activities involving intentional or unintentional movement of animals, many helminth species have been introduced to new regions with several ecological and epidemiological implications for the native species. A high prevalence of infection with an introduced digenean Renifer aniarum, previously known only from North America, was found in the grass snake Natrix natrix in the Calabria region, southern Italy. Morphological and molecular comparison with North American R. aniarum has confirmed the identity of the Italian specimens. A total of 41 grass snakes were studied for R. aniarum infection. Of 24 snakes sampled between 2009 and 2010, 22 were positive for this parasite. In contrast, all 17 snakes sampled from museum collections between 1983 and 1994 were negative. Our results support the hypothesis that R. aniarum was perhaps introduced into this area during the 1990s by the translocation of the American bullfrog Lithobates (Rana) catesbeianus, a normal second intermediate host of the digenean in its native range in North America. Although the life cycle of R. aniarum is complex and includes 3 host stages, this parasite has found suitable first and second intermediate hosts as well as definitive hosts in Italy. Renifer aniarum was second only to the very common grass snake tapeworm Ophiotaenia europaea in both prevalence and abundance among 9 species of helminths recovered in our study.     

Multiple origins of European populations of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae), a liver parasite of ruminants

The giant liver fluke, Fascioloides magna, a liver parasite of free-living and domestic ruminants of Europe and North America, was analysed in order to determine the origin of European populations and to reveal the biogeography of this originally North American parasite on the European continent. The variable fragments of the mitochondrial cytochrome c oxidase subunit I (cox1; 384bp) and nicotinamide dehydrogenase subunit I (nad1; 405bp) were used. Phylogenetic trees and haplotype networks were constructed and the level of genetic structuring was evaluated using population genetic tools. In F. magna individuals originating from all European foci of infection (Italy, Czech Republic and Danube floodplain forests involving the territories of Slovakia, Hungary and Croatia) and from four of five major North American enzootic areas, 16 cox1 and 18 nad1 haplotypes were determined. The concatenated sequence set produced 22 distinct haplotypes. The European fluke populations were less diverse than those from North America in that they contained proportionately fewer haplotypes (eight), while a more substantial level of genetic diversity and a greater number of haplotypes (15) were recorded in North America. Only one haplotype was shared between the European (Italy) and North American (USA/Oregon and Canada/Alberta) flukes, supporting a western North American origin of the Italian F. magna population. Haplotypes found in Italy were distinct from those determined in the remaining European localities which indicates that introduction of F. magna to the European continent occurred more than once. In the Czech focus of infection, a south-eastern USA origin was revealed. Identical haplotypes, common to parasites from the Czech Republic and from an expanding focus in Danube floodplain forests, implies that the introduction of F. magna to the Danube region came from an already established Czech focus of infection.     

Two new species of Rhabdias (Nematoda: Rhabdiasidae) from the marine toad, Bufo marinus (L.) (Lissamphibia: Anura: Bufonidae), in Central America

Two new Rhabdias species are described from the lungs of the cane toad Bufo marinus (L.) from Costa Rica and Nicaragua. Rhabdias alabialis n. sp. differs from other known species of the genus by the remarkable morphology of its head end, i.e., the absence of lips or pseudolabia, the slitlike oral opening, and the triangular shape of the buccal capsule in apical view. Rhabdias pseudosphaerocephala n. sp. is identified as a form previously known in Central and South America as Rhabdias sphaerocephala Goodey, 1924, a species initially described from toads in Europe. The new species is differentiated from R. sphaerocephala based on head-end morphology and sequences of nuclear rDNA.     

Synthesis and structure-antituberculosis activity relationship of 1H-indole-2,3-dione derivatives

New series of 5-fluoro-1H-indole-2,3-dione-3-thiosemicarbazones 2a-k and 5-fluoro-1-morpholino/piperidinomethyl-1H-indole-2,3-dione-3-thiosemicarbazones 3a-r were synthesized. The structures of the synthesized compounds were confirmed by spectral data, elemental and single crystal X-ray diffraction analysis. The new 5-fluoro-1H-indole-2,3-dione derivatives, along with previously reported 5-nitro-1H-indole-2,3-dione-3-thiosemicarbazones 2l-v, 1-morpholino/piperidinomethyl-5-nitro-1H-indole-2,3-dione-3-thiosemicarbazones 4a-l, and 5-nitro-1H-indole-2,3-dione-3-[(4-oxo-1,3-thiazolidin-2-ylidene)hydrazones] 5a-s, were evaluated for in vitro antituberculosis activity against Mycobacterium tuberculosis H37Rv. Among the tested compounds, 5-nitro-1H-indole-2,3-dione-3-thiosemicarbazones (2p, 2r, and 2s) and its 1-morpholinomethyl derivatives (4a, 4e, 4g, and 4i) exhibited significant inhibitory activity in the primary screen. The antituberculosis activity of molecules with diverse skeletons was investigated by means of the Electronic-Topological Method (ETM). Ten pharmacophores and ten anti-pharmacophores that have been found by this form the basis of the system capable of predicting the structures of potentially active compounds. The forecasting ability of the system has been tested on structures that differ from those synthesized. The probability of correct identification for active compounds was found as equal to 93% in average. To obtain the algorithmic base for the activity prediction, Artificial Neural Networks were used after the ETM (the so-called combined ETM-ANN method). As the result, only 9 pharmacophores and anti-pharmacophores were chosen as the most important ones for the activity. By this, ANNs classified correctly 94.4%, or 67 compounds from 71.     

Description and phylogenetic relationships of Rodentolepis gnoskei n. sp. (Cyclophyllidea: Hymenolepididae) from a shrew Suncus varilla minor in Malawi

Rodentolepis gnoskei n. sp. is described based on specimens obtained from a lesser dwarf shrew Suncus varilla minor collected in the Nyika National Park, Malawi. The new species is one of the smallest hymenolepidids known from African shrews and is morphologically closest to two other miniature hymenolepidids from African shrews, Staphylocystis loossi and S. khalili. The new species differs from both of them by a much smaller strobila size and fewer proglottids. The rostellar hooks in the new species are more numerous and smaller in size than in S. loossi. The rostellar hooks in R. gnoskei n. sp. are almost three times shorter than hooks in S. khalili. The hook shape in both S. loossi and S. khalili is substantially different from that in the new species. Molecular phylogenetic analysis indicates that the new species is close to Rodentolepis fraterna, a parasite of rodents. The new species clearly differs from R. fraterna by the much shorter strobila, larger hooks, relatively longer cirrus sac, rate of proglottid development, the number of eggs per proglottid and parasitism in shrews. Although the new species fits the current diagnosis of Rodentolepis, its generic allocation is provisional and will likely be revised in the future because the type species of Rodentolepis, R. straminea, belongs to a different well supported clade. Thus, a new genus needs to be established for the lineage that includes R. fraterna and R. gnoskei n. sp. However, this systematic rearrangement is not recommended until Staphylocystis pistillum, the type species of Staphylocystis, is included in future phylogenetic analyses. Rodentolepis gnoskei n. sp. is the first tapeworm species reported from shrews in Malawi and the first species of cestode reported from S. varilla minor and any member of Suncus in Africa.