Interleukin‐21 administration to aged mice rejuvenates their peripheral T‐cell pool by triggering de novo thymopoiesis

The vaccination efficacy in the elderly is significantly reduced compared to younger populations due to thymic involution and age‐related intrinsic changes affecting their naïve T‐cell compartment. Interleukin (IL)‐21 was recently shown to display thymostimulatory properties. Therefore, we hypothesized that its administration to ageing hosts may improve T‐cell output and thus restore a competent peripheral T‐cell compartment. Indeed, an increase in the production of recent thymic emigrants (RTEs) attributable to intrathymic expansion of early thymic progenitors (ETPs), double‐negative (DN), and double‐positive (DP) thymocytes as well as thymic epithelial cell (TEC) was observed in recombinant (r)IL‐21‐treated aged mice. In sharp contrast, no alterations in the frequency of bone marrow (BM)‐derived progenitors were detected following rIL‐21 administration. Enhanced production of naïve T cells improved the T‐cell receptor (TCR) repertoire diversity and re‐established a pool of T cells exhibiting higher levels of miR‐181a and diminished amounts of the TCR‐inhibiting phosphatases SHP‐2 and DUSP5/6. As a result, stimulation of T cells derived from rIL‐21‐treated aged mice displayed enhanced activation of Lck, ZAP‐70, and ERK, which ultimately boosted their IL‐2 production, CD25 expression, and proliferation capabilities in comparison with T cells derived from control aged mice. Consequently, aged rIL‐21‐treated mice vaccinated using a tyrosinase‐related protein 2 (Trp2)‐derived peptide exhibited a substantial delay in B16 tumor growth and improved survival. The results of this study highlight the immunorestorative function of rIL‐21 paving its use as a strategy for the re‐establishment of effective immunity in the elderly.     

The ftsA gene product participates in formation of the Escherichia coli septum structure.

The patterns of septation in filaments of Escherichia coli, formed as a consequence of the lack of an active ftsA gene product and then returned to permissive conditions, were analyzed in isogenic strains containing three different mutated alleles of ftsA. Septation was blocked for at least one doubling time at the potential septation sites that presumably contained inactive FtsA protein but not at those sites containing either the active gene product or no gene product at all. These results suggested a possible structural role for the ftsA gene product in the construction of the E. coli septum.     

HPLC studio: a novel software utility to perform HPLC chromatogram comparison for screening purposes

A new tool, HPLC Studio, was developed for the comparison of high-performance liquid chromatography (HPLC) chromatograms from microbial extracts. The new utility makes it possible to create a virtual chromatogram by mixing up to 20 individual chromatograms. The virtual chromatogram is the first step in establishing a ranking of the microbial fermentation conditions based on either the area or diversity of HPLC peaks. The utility was used to maximize the diversity of secondary metabolites tested from a microorganism and therefore increase the chances of finding new lead compounds in a drug discovery program.     

Analysis of cytotoxic activity at short incubation times reveals profound differences among Annonaceus acetogenins, inhibitors of mitochondrial Complex I

Annonaceous acetogenins are potent cytotoxic agents against tumor cell lines as well as potent inhibitors of mitochondrial Complex I (Degli Esposti and Ghelli Biochim Biophys Acta 1187:116–120, 1994; Degli Esposti et al. Biochem J 301(Pt 1):161–167, 1994; Tormo et al. Arch Biochem Biophys 369:119–126, 1999). Eighteen different ACGs belonging to seven structural sub-families were tested against six tumor and two non tumor cell lines in a MTT cytotoxicity assay to evaluate the correlation between mitochondrial Complex I inhibition and cytotoxic activity potency and selectivity. The results showed a substantial heterogeneity in potency and selectivity among the different compounds tested, although no clear overall structure-activity relationships could be established. To further characterize the biological activity of these compounds, four ACGs were selected based on their inhibition binding sites to Complex I, to evaluate their cytotoxic activity over a 15-minute to 48-hour period using a more sensitive time-course LDH cytotoxicity assay. Our results indicate that, although all of the ACGs were highly cytotoxic in HepG2 cell lines at 24 h, each sub-class behaves rather differently at shorter times. Perhaps other aspects related to how these compounds reach or bind to their target sites, or differences in their ability to cross the cell and/or the mitochondrial membranes, could help explain their different activities. This different behavior between ACGs may provide new clues for a better understanding of their potential antitumor properties.     

Bacterial diversity from benthic mats of Antarctic lakes as a source of new bioactive metabolites

During the MICROMAT project, the bacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics. In all, 723 Antarctic heterotrophic bacteria belonging to novel and/or endemic taxa in the α-, β- and γ-subclasses of the Proteobacteria, the Bacteroidetes branch, and of the high and low percentage G+C Gram-positives, were isolated, cultivated in different media and at different temperatures, and then screened for the production of antimicrobial activities. A total of 6348 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 122 bacteria showed antibacterial activity against the Gram-positives Staphylococcus aureus and to a lower extent Enterococcus faecium, and versus the Gram-negative Escherichia coli. Few of these strains showed also some antifungal activity against Cryptococcus neoformans, Aspergillus fumigatus and to a lower extent Candida albicans. LC–MS fractionation of extracts from a subset of strains (hits) that exhibited relatively potent antibacterial activities evidenced a chemical novelty that was further investigated. Two strains of Arthrobacter agilis produced potent antibacterial compounds with activity against Gram-positives and possibly related to novel cyclic thiazolyl peptides. To our knowledge, this is the first report of new antibiotics produced by bacteria from benthic microbial mats from Antarctic lakes. With no doubts these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as antibiotic compounds.     

Native Species for Roadslope Revegetation: Selection,Validation, and Cost Effectiveness

Native species arise as an alternative to improve the poor success of traditional hydroseeding with commercial species in degraded areas. The objectives of this study were to (1) establish a procedure to select suitable native species for roadslope revegetation, (2) validate the procedure by means of field sowing experiments, and (3) assess cost effectiveness of the use of native species in hydroseeding as regards commercial ones. Vegetation surveys were performed in semiarid roadslopes of East Spain and species success evaluated according to abundance and frequency. A list of potentially suitable species for revegetation was produced and a subset of species selected and hydroseeded to check their suitability in hydroseeding. Cost effectiveness of the use of native species was estimated. Results support the suitability of the procedure for the selection of native species. Vegetation cover produced by the hydroseeded mixture of native species was high (between 43 and 70% throughout the 4 years) and was 4–20 times higher than the cover produced by a standard commercial seed mixture. The price of the selected seed mixture was 30 times that of the commercial one, but the cost of relevant ecological advantages provided by the use of native species was only twice that of the commercial species on the basis of cost effectiveness assessment. Practical consequences for restorers, policymakers, and practitioners are discussed, and the use of native species is encouraged to improve revegetation success of roadslopes and similar slope types especially in semiarid and arid environments.     

Polymorphism in the yclC-rpoS region of enterobacteria

The nucleotide sequence downstream from the rpoS gene in Enterobacter cloacae and Kluyvera cryocrescens contains the slyA-pad1-yclC genes. The DNA sequence of Enterobacter cloacae CETC960 shows a 2.6-kb insertion of unknown origin between rpoS and slyA. This 2.6-kb sequence has also been detected in species of Salmonella and in Pseudomonas aeruginosa, but not in the same location. This insertion has been detected in all the Enterobacter cloacae clinical strains studied although the size of the rpoS-yclC region was highly variable, possibly owing to the presence of insertions and/or deletions. The study of the rpoS-mutS region in other enterobacteria also showed variability in size. Our results support the idea of a variational hot spot in the rpoS-mutS region that could be related to pathogenesis and horizontal transfer.     

Sip,an integrase protein with excision,circularization and integration activities,defines a new family of mobile Staphylococcus aureus pathogenicity islands

We report the complete sequence of Staphylococcal pathogenicity island bovine 2 (SaPIbov2), encoding the biofilm-associated protein Bap. SaPIbov2 contains 24 open reading frames, including sip, which encodes a functional staphylococcal integrase protein. SaPIbov2 is bordered by 18 bp direct repeats. The integration site into the chromosome lies at the 3' end of a gene encoding GMP synthase. SaPIbov2 has extensive similarity to previously described pathogenicity islands of Staphylococcus aureus. The principal difference is that toxin genes present in the other pathogenicity islands are exchanged for a transposon-like element that carries the bap gene and genes encoding an ABC transporter and a transposase. Also, SaPIbov2 can be excised to form a circular element and can integrate site-specifically and RecA-independently at a chromosomal att site in a Sip-dependent manner. This was demonstrated both in S. aureus and with plasmid substrates ectopically in Escherichia coli. Thus, SaPIbov2 encodes a functional recombinase of the integrase family that promotes element excision and insertion/integration. In addition, we demonstrated that the presence of SaPIbov2 facilitated the persistence of S. aureus in an intramammary gland infection model. Finally, different bovine isolates of S. aureus were found to carry islands related to SaPIbov2, suggesting the existence of a family of related pathogenicity islands.