Evaluating mortality rates and causalities in a critically endangered felid across its whole distribution range

The conservation of endangered species requires accurate data, and knowledge of cause-specific mortality rates is one of the most important issues. In recent years, conservation programs for the critically endangered Iberian lynx Lynx pardinus have been developed on the basis of mortality data derived 30 years ago from the small Doñana population. Thus, there is an urgent need for an update of mortality rates and causes in both populations (Sierra Morena and Doñana). Here we use radio-tracking information from the whole range of the Iberian lynx to quantify mortality rates and identify their causes. Between 2006 and 2011, we radio-tagged 78 Iberian lynxes from its two remaining populations (39 from Sierra Morena and 39 from Doñana). Mortality events were evaluated to identify causes, and cause-specific annual mortality rates (AMR) were obtained using the nonparametric cumulative incidence function estimator. Overall, AMR was estimated at 0.16?±?0.05 (0.19?±?0.09 in Sierra Morena and 0.12?±?0.07 in Doñana). Disease was the main cause of mortality both for the whole population and the Doñana population. Poaching was the main cause of mortality in Sierra Morena. Our results suggest that the best strategy for conserving this species is to focus action on decreasing the fatal effect of disease and poaching. Given the possible existence of an underlying inbreeding-mediated immunosuppression, genetic management aimed at increasing the genetic diversity of this population is also recommended.     

Analysis of trophic structure of two carnivore assemblages by means of guild identification

We evaluated the existence of trophic guild structure, considering seasonal and annual variation, in two terrestrial carnivore assemblages: one from Santa Cruz province (Argentinean Patagonia, composed by six carnivore species), and the other from Doñana National Park (SW Spain, composed by five carnivore species). To identify trophic guilds, we first studied seasonal and annual diets of predators, calculated trophic overlap among species pairs, and then constructed overlap matrices (similarity matrices). We determined guild membership objectively by entering the similarity matrices into the clustering technique unweighted pair-group method with arithmetic averaging. Carnivores from both assemblages were grouped, respectively, into four feeding guilds. Lagomorphs and rodents promoted the formation of two feeding guilds in both study sites, although the taxonomic composition of predator species that composed them was different. The ungulates-edentates feeding guild was only present at Santa Cruz, whereas the birds and reptiles feeding guild was only present at Doñana. Invertebrates and fruits were the base for the formation of a guild composed by species of the same taxonomic origin both in Santa Cruz and Doñana. Guild structure of Santa Cruz and Doñana assemblages did not exhibit seasonal or annual variation, although the specific guild composition changed over the two studied periods for both assemblages. This structure probably responded to discontinuities in resource spectra in Santa Cruz and fluctuations in rabbit abundance in Doñana. Our results support the hypothesis that establishes that guilds are originated by opportunistic convergence of species on abundant and energetically rewarding resources.     

Characterization of aerobic heterotrophic bacteria in cold and nutrient-poor freshwater ecosystems

Aerobic heterotrophic bacteria present in the surface water of three cold and nutrient-poor lakes in the Chilean Patagonia (Alto Reino, Las Dos Torres and Venus) were analysed for genetic similarity and metabolic diversity using 16S ribosomal DNA and the Biolog EcoPlate^TM system, respectively. Bacterial fingerprints of water samples in enriched and non-enriched nutrient broth demonstrated a >50% fingerprinting similarity between the lakes. Metabolic activity was also similar. However, the Biolog EcoPlate^TM system carbon substrates revealed functional diversity. Lake Las Dos Torres showed the most fingerprinting similarity between enriched and non-enriched cold water samples. The amounts of living and viable bacteria were also higher in this lake’s water sample, suggesting a predominance of facultative oligotrophic groups. DNA sequencing analysis demonstrated the presence of phylum Bacteroidetes in Lake Alto Reino; phyla Bacteroidetes and Gammaproteobacteria in Lake Las Dos Torres; and phyla Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria in Lake Venus. Although each lake had a unique bacterial community structure, the different bacterial groups may be performing similar metabolic functions, given the similarity in extreme environmental conditions.     

Comparison between the complete mtDNA sequences of the blue and the fin whale,two species that can hybridize in nature

The sequence of the mitochondrial DNA (mtDNA) molecule of the blue whale (Balaenoptera musculus) was determined. The molecule is 16,402 by long and its organization conforms with that of other eutherian mammals. The molecule was compared with the mtDNA of the congeneric fin whale (B. physalus). It was recently documented that the two species can hybridize and that male offspring are infertile whereas female offspring may be fertile. The present comparison made it possible to determine the degree of mtDNA difference that occurs between two species that are not completely separated by hybridization incompatibility. The difference between the complete mtDNA sequences was 7.4%. Lengths of peptide coding genes were the same in both species. Except for a small portion of the control region, disruption in alignment was usually limited to insertion/deletion of a single nucleotide. Nucleotide differences between peptide coding genes ranged from 7.1 to 10.5%, and difference at the inferred amino acid level was 0.0–7.9%. In the rRNA genes the mean transition difference was 3.8%. This figure is similar in degree to the difference (3.4%) between the 12S rRNA gene of humans and the chimpanzee. The mtDNA differences between the two whale species, involving both peptide coding and rRNA genes, suggest an evolutionary separation of 5 million years. Although hybridization between more distantly related mammalian species may not be excluded, it is probable that the blue and fin whales are nearly as different in their mtDNA sequences as hybridizing mammal species may be. Correspondence to: Ú. Árnason     

Intrathecal administration of autologous mesenchymal stromal cells for spinal cord injury: Safety and efficacy of the 100/3 guideline

Background aims

Cell therapy with autologous mesenchymal stromal cells (MSCs) in patients with spinal cord injury (SCI) is beginning, and the search for its better clinical application is an urgent need.

RAPD-PCR analysis of Staphylococcus aureus strains isolated from bovine and human hosts

Staphylococcus aureus is one of the most important pathogens in humans and animals. In this study eighty strains were analyzed by RAPD-PCR to assess the genetic relationship between S. aureus isolates from bovine and human hosts. Results were compared with those obtained by biotyping. Fifty-two percent of the S. aureus isolates belonged to a host specific biotype (human, bovine and poultry). Bovine and human ecovars were the most prevalent. Dendrogram obtained by RAPD results showed that all the isolates clustered into eleven groups (A-K) at a relative genetic similarity of less than 30% when analyzed with the three primers. Group A clustered 95% of the human host isolates and the remaining groups (B-K) clustered the bovine host isolates. Principal coordinate analysis also showed that the isolates could be arbitrarily divided into two groups, bovine and human, by the second coordinate. Only 9 isolates (11%) were not clustered into these groups. The genetic diversity among the S. aureus isolates from bovine hosts is relatively low compared to that of isolates from human hosts. There were no statistically significant differences among isolated from bovine and human hosts. This study shows that RAPD-PCR assayed with three primers can be successfully applied to assess the genetic relationship of S. aureus isolates from different hosts.     

New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase,Emerging from Nonconventional Codon Usage and Directed Mutation

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k_cat/K_m) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes'' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.β-Fructofuranosidases (EC 3.2.1.26) are enzymes of biotechnological interest that catalyze the release of β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. In general, they exhibit a high degree of sequence homology, and based on their amino acid sequences, they fall into family 32 of the glycosyl-hydrolases (GH), along with invertases, inulinases, and fructosyltransferases (http://www.cazy.org). The GH32 family has been studied intensely, and some three-dimensional structures are now available, such as that of inulinase from Aspergillus awamorii (26), fructan-exohydrolase from Cichorium intybus (CiFEH) (34, 36), or invertase from Thermotoga maritima (2, 3) and Arabidopsis thaliana (35). These proteins contain a five-blade β-propeller N-terminal catalytic module and a C-terminal β-sandwich domain (19). Multiple-sequence alignment of GH32 proteins, which are included in the GH-J clan together with the GH68 proteins of the inulosucrase family, reveals the presence of three conserved motifs, each containing a key acidic residue (in boldface) implicated in substrate binding and hydrolysis: Asn-Asp-Pro-Asn-Gly (NDPNG), Arg-Asp-Pro (RDP), and Glu-Cys (EC) (28). These conserved residues are implicated in a double-displacement reaction in which a covalent glycosyl-enzyme intermediate is formed. Thus, the catalytic mechanism proposed for the Saccharomyces cerevisiae invertase implies that Asp23 (NDPNG) acts as a nucleophile and Glu204 (EC) acts as the acid/base catalyst (29), whereas Asp309 (RDP) of Acetobacter diazotropicus levansucrase influences the efficiency of sucrose hydrolysis (7) and Arg188 and Asp189 of the latter motif define the substrate binding and specificity of exoinulinase from A. awamorii toward fructopyranosyl residues (26).As well as hydrolyzing sucrose, β-fructofuranosidases may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose skeleton by different glycosidic bonds, depending on the source of the enzyme (12, 21, 31). FOS act as prebiotics, and they exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, and osteoporosis (16). Currently, FOS are mainly produced by Aspergillus fructosyltransferase in industry (10, 31), providing a mixture of FOS with an inulin-type structure that contains β-(2→1)-linked fructose oligomers (¹F-FOS: 1-kestose or nystose). Curiously, when the link between two fructose units (⁶F-FOS: 6-kestose) or between fructose and the glucosyl moiety (⁶G-FOS: neokestose) involves a β-(2→6) link, the prebiotic properties of the FOS may be enhanced beyond that of commercial FOS (23).The yeast Schwanniomyces occidentalis (also called Debaryomyces occidentalis) produces a number of extracellular enzymes that make it of interest in biotechnology. Several of its amylolytic enzymes have been characterized, including amylases and glucoamylase (1, 9), as well as an invertase (17). In addition, we also characterized an extracellular β-fructofuranosidase (Ffase) from this yeast that hydrolyzes sucrose, 1-kestose, and nystose (5). This enzyme exhibited a transfructosylating activity that efficiently produces the trisaccharides 6-kestose and 1-kestose in the ratio 3:1, generating the highest 6-kestose yield yet reported, as far as we know. The Ffase three-dimensional structure has recently been solved (6) and represented as a homodimer, each modular subunit arranged like other GH32 enzymes. The Asp50 (NDPNG) and Glu230 (EC) located at the center of the propeller are the catalytic residues implicated in substrate binding and hydrolysis, whereas Arg178 and Asp179 form the RDP motif (6).The genetic codes of some yeasts incorporate certain variations. For example, while CUG was believed to be a universal codon for leucine, in the cytoplasm of certain species of the genus Candida (15) it encodes a serine, as in Pichia farinosa (33). The reassignment of this codon is mediated by a novel serine-tRNA that acquired a leucine 5′-CAG-3′ anticodon (25).Here, we show that deviation from the standard use of the CUG leucine codon to encode serine was correlated with the transferase capacity and specificity of the Ffase enzyme. Indeed, the S196L substitution enhanced the transferase activity of the enzyme 3-fold. Several site-directed mutants were generated and characterized to study their transferase capacities. These results are considered on the basis of the enzymes'' three-dimensional structure, which enables a novel putative binding site of sucrose that serves as a water substitute donor in the hydrolytic reaction yielding the tranglycosylation product 6-kestose to be identified.     

Plants,microorganisms, and soil temperatures contribute to a decrease in methane fluxes on a drained Arctic floodplain

As surface temperatures are expected to rise in the future, ice‐rich permafrost may thaw, altering soil topography and hydrology and creating a mosaic of wet and dry soil surfaces in the Arctic. Arctic wetlands are large sources of CH₄, and investigating effects of soil hydrology on CH₄ fluxes is of great importance for predicting ecosystem feedback in response to climate change. In this study, we investigate how a decade‐long drying manipulation on an Arctic floodplain influences CH₄‐associated microorganisms, soil thermal regimes, and plant communities. Moreover, we examine how these drainage‐induced changes may then modify CH₄ fluxes in the growing and nongrowing seasons. This study shows that drainage substantially lowered the abundance of methanogens along with methanotrophic bacteria, which may have reduced CH₄ cycling. Soil temperatures of the drained areas were lower in deep, anoxic soil layers (below 30 cm), but higher in oxic topsoil layers (0–15 cm) compared to the control wet areas. This pattern of soil temperatures may have reduced the rates of methanogenesis while elevating those of CH₄ oxidation, thereby decreasing net CH₄ fluxes. The abundance of Eriophorum angustifolium, an aerenchymatous plant species, diminished significantly in the drained areas. Due to this decrease, a higher fraction of CH₄ was alternatively emitted to the atmosphere by diffusion, possibly increasing the potential for CH₄ oxidation and leading to a decrease in net CH₄ fluxes compared to a control site. Drainage lowered CH₄ fluxes by a factor of 20 during the growing season, with postdrainage changes in microbial communities, soil temperatures, and plant communities also contributing to this reduction. In contrast, we observed CH₄ emissions increased by 10% in the drained areas during the nongrowing season, although this difference was insignificant given the small magnitudes of fluxes. This study showed that long‐term drainage considerably reduced CH₄ fluxes through modified ecosystem properties.