Phylogenetic and Genome-Wide Deep-Sequencing Analyses of Canine Parvovirus Reveal Co-Infection with Field Variants and Emergence of a Recent Recombinant Strain

Canine parvovirus (CPV), a fast-evolving single-stranded DNA virus, comprises three antigenic variants (2a, 2b, and 2c) with different frequencies and genetic variability among countries. The contribution of co-infection and recombination to the genetic variability of CPV is far from being fully elucidated. Here we took advantage of a natural CPV population, recently formed by the convergence of divergent CPV-2c and CPV-2a strains, to study co-infection and recombination. Complete sequences of the viral coding region of CPV-2a and CPV-2c strains from 40 samples were generated and analyzed using phylogenetic tools. Two samples showed co-infection and were further analyzed by deep sequencing. The sequence profile of one of the samples revealed the presence of CPV-2c and CPV-2a strains that differed at 29 nucleotides. The other sample included a minor CPV-2a strain (13.3% of the viral population) and a major recombinant strain (86.7%). The recombinant strain arose from inter-genotypic recombination between CPV-2c and CPV-2a strains within the VP1/VP2 gene boundary. Our findings highlight the importance of deep-sequencing analysis to provide a better understanding of CPV molecular diversity.     

The Truncated Isoform of Somatostatin Receptor5 (sst5TMD4) Is Associated with Poorly Differentiated Thyroid Cancer

Somatostatin receptors (ssts) are expressed in thyroid cancer cells, but their biological significance is not well understood. The aim of this study was to assess ssts in well differentiated (WDTC) and poorly differentiated thyroid cancer (PDTC) by means of imaging and molecular tools and its relationship with the efficacy of somatostatin analog treatment. Thirty-nine cases of thyroid carcinoma were evaluated (20 PDTC and 19 WDTC). Depreotide scintigraphy and mRNA levels of sst-subtypes, including the truncated variant sst5TMD4, were carried out. Depreotide scans were positive in the recurrent tumor in the neck in 6 of 11 (54%) PDTC, and in those with lung metastases in 5/11 cases (45.4%); sst5TMD4 was present in 18/20 (90%) of PDTC, being the most densely expressed sst-subtype, with a 20-fold increase in relation to sst2. In WDTC, sst2 was the most represented, while sst5TMD4 was not found; sst2 was significantly increased in PDTC in comparison to WDTC. Five depreotide positive PDTC received octreotide for 3–6 months in a pilot study with no changes in the size of the lesions in 3 of them, and a significant increase in the pulmonary and cervical lesions in the other 2. All PDTC patients treated with octreotide showed high expression of sst5TMD4. ROC curve analysis demonstrated that only sst5TMD4 discriminates between PDTC and WDTC. We conclude that sst5TMD4 is overexpressed in PDTC and may be involved in the lack of response to somatostatin analogue treatment.     

Methylation of histone H3 at lysine 37 by Set1 and Set2 prevents spurious DNA replication

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Investigations into the phylogenetic position of Micrognathozoa using four molecular loci

Micrognathozoa is the most recently discovered higher metazoan lineage. The sole known species of the group, Limnognathia maerski, was originally reported from running freshwater in Disko Island (Greenland), and has recently been recorded from the subantarctic region. Because of the presence of a particular type of jaws formed of special cuticularized rods, similar to those of gnathostomulids and rotifers, the three metazoan lineages were considered closely related, and assigned to the clade Gnathifera. A phylogenetic comparison of four molecular loci for Limnognathia maerski and other newly generated sequences of mainly acoelomate animals showed that Micrognathozoa may constitute an independent lineage from those of Gnathostomulida and Rotifera. However, the exact position of Micrognathozoa could not be determined due to the lack of support for any given relationships and due to the lack of stability in the position of Limnognathia maerski under analysis of different loci and of different parameter sets for sequence comparison. Nuclear loci tend to place Micrognathozoa with the syndermatan/cycliophoran taxa, but the addition of the mitochondrial gene cytochrome c oxidase subunit I favors a relationship of Micrognathozoa to Entoprocta.     

Biocatalytic properties of Baeyer–Villiger monooxygenases in aqueous–organic media

The biocatalytic properties of three Baeyer–Villiger monooxygenases (phenylacetone monooxygenase, 4-hydroxyacetophenone monooxygenase and ethionamide monooxygenase) in a variety of aqueous–organic media were studied using organic sulfides as substrates. The influence of the nature and the concentration of the solvents, as well as of the substrates, on the activity and enantioselectivity of the enzymes was investigated in detail. Solvents were found to decrease, to a different extent, enzyme activity. High increases of enantioselectivity and also reversal of enantiopreference were observed depending on the enzyme and on the nature of the solvent and the substrate employed.     

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras

α₁-Protease inhibitor Portland (α₁PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α₁PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α₁PDX with those of serpin B8 demonstrated that both the P4–P1 RXXR recognition sequence as well as the P1′–P5′ sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298–300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α₁PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298–300 loop whose substitution in α₁PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298–300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α₁PDX inhibitors of PCs.     

Microgeographic differentiation among closely related species of Biomphalaria (Gastropoda: Planorbidae) from the Andean Altiplano

Direct development and water dependence entail limited vagility in freshwater fauna. In these organisms, the population structure is probably linked to restrictions imposed by the habitat. In this study we investigate the relative contribution of processes stimulating the divergence of populations of Biomphalaria costata (Biese, 1951) and Biomphalaria crequii (Courty, 1907), two freshwater snails occurring in two contiguous and fragmented closed basins from the Andean Altiplano using mitochondrial DNA (cytochrome c oxidase subunit I) sequences, shell morphometric and radular morphology. In order to clarify the species boundaries, a third allopatric species was included: Biomphalaria aymara Valdovinos & Stuardo, 1991. Molecular analyses recovered two distinct clades: one composed of B. aymara from the Isluga swamps and B. costata from Spring 1 in Salar de Carcote, the single spring occupied by this species, and another integrated by snails from 12 springs spread across the Salar de Carcote and the Salar de Ascotán assigned to B. crequii, originally described from the Salar de Ascotán. Unlike shell morphometrics, radular morphology was informative for distinguishing these species. The division of the lineages occurred in the Late Pleistocene. A subclade that includes snails from the southernmost springs in Salar de Ascotán suggests fragmentation of the distribution of B. crequii associated with landscape discontinuities. In addition to microvicariance signals, the private haplotypes scattered around both salt spans show that close‐range dispersal is a common biogeographic process in this species. As evolutionary units, the single isolated and restricted population of B. costata has a high priority for conservation. © 2013 The Linnean Society of London