CLAME: a new alignment-based binning algorithm allows the genomic description of a novel Xanthomonadaceae from the Colombian Andes

Background

Hot spring bacteria have unique biological adaptations to survive the extreme conditions of these environments; these bacteria produce thermostable enzymes that can be used in biotechnological and industrial applications. However, sequencing these bacteria is complex, since it is not possible to culture them. As an alternative, genome shotgun sequencing of whole microbial communities can be used. The problem is that the classification of sequences within a metagenomic dataset is very challenging particularly when they include unknown microorganisms since they lack genomic reference. We failed to recover a bacterium genome from a hot spring metagenome using the available software tools, so we develop a new tool that allowed us to recover most of this genome.

Results

We present a proteobacteria draft genome reconstructed from a Colombian’s Andes hot spring metagenome. The genome seems to be from a new lineage within the family Rhodanobacteraceae of the class Gammaproteobacteria, closely related to the genus Dokdonella. We were able to generate this genome thanks to CLAME. CLAME, from Spanish “CLAsificador MEtagenomico”, is a tool to group reads in bins. We show that most reads from each bin belong to a single chromosome. CLAME is very effective recovering most of the reads belonging to the predominant species within a metagenome.

Conclusions

We developed a tool that can be used to extract genomes (or parts of them) from a complex metagenome.

     

Validation of molecular markers linked to apospory in tetraploid races of bahiagrass, Paspalum notatum Flüggé

Tetraploid (2n = 4x = 40) races of Paspalum notatum Flüggé are important natural forage grasses for the tropical and subtropical areas of the Americas. Almost all natural accessions reproduce by obligate aposporous apomixis. Previous work on the species allowed the identification of several molecular markers completely linked to apospory, one component of apomictic reproduction. Moreover, after a fingerprinting characterization of a germplasm collection, 11 amplified fragment length polymorphism (AFLP) markers exclusive to apomictic accessions were detected. The objectives of this work were (1) to validate the presence of molecular markers linked to apospory in tetraploid races of different geographic origins, (2) to determine if markers specific to apomictic accessions were associated with the mode of reproduction, and (3) to develop single-locus markers of apospory that can be used for marker-assisted selection. Thirteen natural apomictic accessions were analyzed. Moreover, the parental plants Q4188 (non-aposporous) and Q4117 (aposporous) and 44 F1 progenies (36 non-aposporous, 8 aposporous) derived from them were used as a validation population. Nine markers [two random amplification of polymorphic DNA (RAPD) and seven AFLP] 100% linked to apospory in Q4117 were tested. Amplification reactions with the corresponding primers showed that all markers were present in the 13 aposporous (apomictic) accessions, but were absent in the non-aposporous controls. On the other hand, linkage analysis of the 11 AFLP markers specific to the apomictic accessions showed that all of them were linked in coupling to apospory (r = 0.00, LOD 13.245). Based on one AFLP (E36M37c), two sequence characterized amplification region (SCAR) markers (SPNA1 and SPNA2) co-segregating with the trait and present in the 13 apomictic accessions were developed. The presence of markers associated with apospory was conserved among tetraploid accessions of different geographic origins. Moreover, the single-locus markers SPNA1 and SPNA2 could be used for routine marker-assisted selection in hybrid populations segregating for apospory and to facilitate the isolation of apospory-related genes.     

Modulation of Ocular Surface Glycocalyx Barrier Function by a Galectin-3 N-terminal Deletion Mutant and Membrane-Anchored Synthetic Glycopolymers

Background

Interaction of transmembrane mucins with the multivalent carbohydrate-binding protein galectin-3 is critical to maintaining the integrity of the ocular surface epithelial glycocalyx. This study aimed to determine whether disruption of galectin-3 multimerization and insertion of synthetic glycopolymers in the plasma membrane could be used to modulate glycocalyx barrier function in corneal epithelial cells.

Methodology/Principal Findings

Abrogation of galectin-3 biosynthesis in multilayered cultures of human corneal epithelial cells using siRNA, and in galectin-3 null mice, resulted in significant loss of corneal barrier function, as indicated by increased permeability to the rose bengal diagnostic dye. Addition of β-lactose, a competitive carbohydrate inhibitor of galectin-3 binding activity, to the cell culture system, transiently disrupted barrier function. In these experiments, treatment with a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, but not full-length galectin-3, prevented the recovery of barrier function to basal levels. As determined by fluorescence microscopy, both cellobiose- and lactose-containing glycopolymers incorporated into apical membranes of corneal epithelial cells, independently of the chain length distribution of the densely glycosylated, polymeric backbones. Membrane incorporation of cellobiose glycopolymers impaired barrier function in corneal epithelial cells, contrary to their lactose-containing counterparts, which bound to galectin-3 in pull-down assays.

Conclusions/Significance

These results indicate that galectin-3 multimerization and surface recognition of lactosyl residues is required to maintain glycocalyx barrier function at the ocular surface. Transient modification of galectin-3 binding could be therapeutically used to enhance the efficiency of topical drug delivery.     

Iron-activated iron uptake: A positive feedback loop mediated by iron regulatory protein 1

The love-hate relationship between iron and living matter has generated mechanisms to maintain iron concentration in a narrow range, above and below which deleterious effects occur. At the cellular level, iron homeostasis is accomplished by the activity of the IRP proteins, which, under conditions of iron depletion, up-regulate the expression of the iron acquisition proteins TfR and DMT1. It has been shown that hydrogen peroxide activates IRP1 and that this activation mediates a potentially harmful increase in cell iron uptake. Here we show that IRP1 activity is also induced by iron-mediated oxidative stress. When cells were incubated with up to 20 M of iron, a typical decrease in IRP1 and IRP2 activity was observed. Interestingly, when iron was further increased to 40 or 80 M, IRP1 was reactivated in three of the four different cell lines tested, i.e., Caco-2 cells, N2A cells and HepG2 cells. In the fourth cell line (K562) IRP1 activity did not increase, but neither did it decrease. This response to iron was largely abrogated when the antioxidant N-acetyl cysteine was added along with iron to the culture medium. Thus, the effect of iron was mediated by oxidative stress. Increases in IRP1 activity were accompanied by increases in cell iron uptake, an indication that the activated IRP1 was functional in the activation of iron uptake. Hence, this iron-induced iron uptake feedback loop results in the increase of intracellular iron and increased oxidative stress.     

Internal physiology of live krill revealed using new aquaria techniques and mixed optical microscopy and optical coherence tomography (OCT) imaging techniques

The Guadalupe fur seal (Arctocephalus townsendi) is a specialist predator feeding on prey present in one trophic level. Data related to the diet of the Guadalupe fur seal are few. It is still unknown where most of the individuals forage or the composition of their diet. On Isla Guadalupe, the San Benito Archipelago and the Farallon Islands, fur seals primarily feed on pelagic and coastal squids. However, differences between colonies were found probably caused by differences in diversity and abundance of prey species over the continental shelf and the pelagic environment, and maybe due to the plasticity of the species in their foraging behavior. Diet composition of the Guadalupe fur seal might reflect adaptations to local and temporal environmental conditions. The aim of this work was to consider historical information, add new information, identify main prey species, and determine where in the marine regions the Guadalupe fur seals feed.