Transglycosylation abilities of ß-d-galactosidases from GH family 2.

The ability to predict the transglycosylation activity of glycosidases by in silico analysis was investigated. The transglycosylation abilities of 7 different ß-d-galactosidases from GH family 2 were tested experimentally using 7 different acceptors and p-nitrophenyl-ß-d-galactopyranoside as a donor of galactosyl moiety. Similar transglycosylation abilities were confirmed for all enzymes originating from bacteria belonging to Enterobacteriaceae, which were able to use all tested acceptor molecules. Higher acceptor selectivity was observed for all others used bacterial strains. Structure models of all enzymes were constructed using homology modeling. Ligand-docking method was used for enzymes-transglycosylation products models construction and evaluation. Results obtained by in silico analysis were compared with results arisen out of experimental testing. The experiments confirmed that significant differences in transglycosylation abilities are caused by small differences in active sites composition of analyzed enzymes. According to obtained result, it is possible to conclude that homology modeling may serve as a quick starting point for detection or exclusion of enzymes with defined transglycosylation abilities, which can be used for subsequent synthesis of e.g., pharmaceutically interesting glycosides. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02715-w.

Paraphyly of Conodiplostomum Dubois, 1937.

Adult trematodes of the genera Conodiplostomum Dubois, 1937 and Neodiplostomum Railliet, 1919 (Trematoda: Diplostomidae) parasitize the intestines of birds of prey, owls and, rarely, passeriform birds. Although the family is taxonomically unsettled, molecular phylogenetics have not been applied to analyze Conodiplostomum and Neodiplostomum and the reference DNA sequences from adult Diplostomidae are scarce and limit studies of their indistinct larval forms. We analyze the Conodiplostomum and Neodiplostomum spp. found during the examination of Czech birds performed from 1962 to 2017, and we provide comparative measurements and host spectra, including prevalence and intensity; we also provide and analyze the sequences of four DNA loci from eight diplostomid species. Molecular phylogenetic analysis suggested that Conodiplostomum spathula (Creplin, 1829), the type species of this genus, is nested in Neodiplostomum. Thus, we suggest the rejection of Conodiplostomum spathula (Creplin, 1829) and the resurrection of Neodiplostomum spathula (Creplin, 1829) La Rue, 1926 and reclassification of all species of Conodiplostomum with the neodiplostomulum type of metacercariae to Neodiplostomum as well. Conodiplostomum canaliculatum (Nicoll, 1914) is reclassified as Neodiplostomum spathulaeforme (Brandes, 1888). The molecular analysis suggested that Conodiplostomum perlatum (Ciurea, 1911), the species with the neascus type of metacercariae, belongs to Crassiphialinae Sudarikov, 1960. We erect the genus Ciureatrema gen. nov. Heneberg & Sitko and reclassify Conodiplostomum perlatum (Ciurea, 1911) as Ciureatrema perlatum (Ciurea, 1911) and establish it as a type species of Ciureatrema gen. nov. Further research should focus on the evolution of the neascus and neodiplostomulum types of metacercariae, as well as the evolution of the genital cone and pseudosuckers in Diplostomidae.

First evidence of changes in enzyme kinetics and stability of glucokinase affected by somatic cancer-associated variations.

Recent investigation of somatic variations of allosterically regulated proteins in cancer genomes suggested that variations in glucokinase (GCK) might play a role in tumorigenesis. We hypothesized that somatic cancer-associated GCK variations include in part those with activating and/or stabilizing effects. We analyzed the enzyme kinetics and thermostability of recombinant proteins possessing the likely activating variations and the variations present in the connecting loop I and provided the first experimental evidence of the effects of somatic cancer-associated GCK variations. Activating and/or stabilizing variations were common among the analyzed cancer-associated variations, which was in strong contrast to their low frequency among germinal variations. The activating and stabilizing variations displayed focal distribution with respect to the tertiary structure, and were present in the surroundings of the heterotropic allosteric activator site, including but not limited to the connecting loop I and in the active site region subject to extensive rearrangements upon glucose binding. Activating somatic cancer-associated variations induced a reduction of GCK's cooperativity and an increase in the affinity to glucose (a decline in the S0.5 values). The hotspot-associated variations, which decreased cooperativity, also increased the half-maximal inhibitory concentrations of the competitive GCK inhibitor, N-acetylglucosamine. Concluded, we have provided the first convincing biochemical evidence establishing GCK as a previously unrecognized enzyme that contributes to the reprogramming of energy metabolism in cancer cells. Activating GCK variations substantially increase affinity of GCK to glucose, disrupt the otherwise characteristic sigmoidal response to glucose and/or prolong the enzyme half-life. This, combined, facilitates glucose phosphorylation, thus supporting glycolysis and associated pathways.

Central European Strigeidae Railliet, 1919 (Trematoda: Strigeidida): Molecular and comparative morphological analysis suggests the reclassification of Parastrigea robusta Szidat, 1928 into Strigea Abildgaard, 1790.

Strigeidae Railliet, 1919 are digenean parasites of birds and mammals that are characteristic by their cup-shaped forebody and bilobed holdfast organ. Despite that the family is taxonomically unsettled, particularly due to a very limited number of visible autapomorphic identification features, molecular phylogenetics have never been applied to analyze the relationships among European members of Strigeidae except for the genus Ichthyocotylurus. Here, we analyze the Strigeidae found during the examination of Czech birds performed from 1962 to 2017, and we provide comparative measurements and host spectra, including prevalence and intensity; we also provide and analyze sequences of four DNA loci of 12 of the Strigeidae species. We suggest the reclassification of Parastrigea robusta Szidat, 1928 as Strigea robusta (Szidat, 1928) Heneberg and Sitko, 2018 comb. n. The genera Strigea Abildgaard, 1790 and Parastrigea Szidat, 1928 appear paraphyletic, and morphological diagnostic features of genera within Strigeini Dubois, 1936 are invalid. The mute swan Cygnus olor hosts two Cotylurus spp., Cotylurus syrius Dubois, 1934 and a second species with molecular identification features shared in part with Cotylurus cornutus (Rudolphi, 1808) and Cotylurus gallinulae Lutz, 1928. New host records are provided for seven species. Analyses of non-European genera of the Strigeidae are needed to provide an updated key to Strigeini.

An outbreak of philophthalmosis in Larus michahellis and Larus fuscus gulls in Iberian Peninsula.

Trematodes of the genus Philophthalmus Loos, 1899 are the eye parasites of birds and mammals, which use freshwater snails as their first intermediate hosts. Here we examined the presence of philophthalmids in a total of 1515 gulls (589 Larus fuscus and 926 Larus michahellis) admitted between January 2010 and October 2016 for rehabilitation at Olhão (Portugal), by the use of combined morphological and molecular analysis. We recorded the first infected L. fuscus and L. michahellis in July and November 2015, respectively. The philophthalmids were located in the conjunctival sac or under the nictitating membrane. Gulls infected with Philophthalmus lucipetus Rudolphi, 1819 presented no clinical signs, while those infected with Philophthalmus lacrymosus Braun, 1902 presented serious eye damage in the same host species. The prevalence of P. lucipetus reached 3.6% in L. fuscus and 0.8% in L. michahellis; the prevalence of P. lacrymosus reached 0.3% and 0.0%, respectively. The outbreak of P. lucipetus likely started in a narrowly defined area, since the first six cases, found between July and October 2015, originated from a single municipality, and only later more cases started to be retrieved from other municipalities of Portugal. These findings represent the first records of both philophthalmids in the Iberian Peninsula, their first records in L. michahellis and the first record of P. lacrymosus in L. fuscus. Further follow-up of the outbreak and the identification of intermediate hosts are needed.

Evidence-based tailoring of bioinformatics approaches to optimize methods that predict the effects of nonsynonymous amino acid substitutions in glucokinase.

Computational methods that allow predicting the effects of nonsynonymous substitutions are an integral part of exome studies. Here, we validated and improved their specificity by performing a comprehensive bioinformatics analysis combined with experimental and clinical data on a model of glucokinase (GCK): 8835 putative variations, including 515 disease-associated variations from 1596 families with diagnoses of monogenic diabetes (GCK-MODY) or persistent hyperinsulinemic hypoglycemia of infancy (PHHI), and 126 variations with available or newly reported (19 variations) data on enzyme kinetics. We also proved that high frequency of disease-associated variations found in patients is closely related to their evolutionary conservation. The default set prediction methods predicted correctly the effects of only a part of the GCK-MODY-associated variations and completely failed to predict the normoglycemic or PHHI-associated variations. Therefore, we calculated evidence-based thresholds that improved significantly the specificity of predictions (≤75%). The combined prediction analysis even allowed to distinguish activating from inactivating variations and identified a group of putatively highly pathogenic variations (EVmutation score <-7.5 and SNAP2 score >70), which were surprisingly underrepresented among MODY patients and thus under negative selection during molecular evolution. We suggested and validated the first robust evidence-based thresholds, which allow improved, highly specific predictions of disease-associated GCK variations.

Morphological and Molecular Assessment of Pentastomes from Gulls in Portugal.

During 2016, 153 gulls, including 64 Larus fuscus and 89 Larus michahellis, were found crippled in south Portugal. They died in Wildlife Rehabilitation and Investigation Center-RIAS and were necropsied. Reighardia sternae infected 2 (3%, n = 64) L. fuscus and 4 (4%, n = 89) L. michahellis. Molecular analysis confirmed the morphological identification on the basis of total body length, maximum body width, length of anterior and posterior hooks, total length of oral apparatus, and other features of oral apparatus of R. sternae. Both sequenced individuals in this study displayed 100% sequence identity to R. sternae individuals obtained previously from Larus ridibundus in Spain and to Reighardia sp. from Larus belcheri in Peru. Reighardia sternae is reported here for the first time in L. michahellis and for the first time from Portugal.