Revealing the latent mobilization capability of the staphylococcal bacteriocinogenic plasmid pRJ9

Plasmid pRJ9 is a non-self-mobilizable bacteriocinogenic plasmid from Staphylococcus aureus. Despite this feature, DNA sequencing and RT-PCR experiments showed that it presents a Mob region with three genes (mobCAB), transcribed as an operon. In silico analysis of the Mob proteins encoded by pRJ9 showed that they present all the conserved functional features reported until present as being essential for plasmid mobilization. Moreover, they showed a high identity to Mob proteins encoded by mobilizable plasmids from Staphylococcus spp., especially to those encoded by plasmid pRJ6, which presents four mob genes (mobCDAB). A putative oriT region was also found upstream of the pRJ9 mob operon. pRJ9 could only be successfully mobilized by pGO1 when pRJ6 was present in the same strain. Further experiments showed that the pRJ9 oriT can be recognized by the pRJ6 Mob proteins, confirming its functionality. As pRJ9 does not possess a mobD gene while pRJ6 does, the absence of this gene was believed to be responsible for its lack of mobilization. However, conjugation experiments with a donor strain carrying also mobD cloned into an S. aureus vector showed that pRJ9 does not become mobilized even in the presence of the protein MobD encoded by pRJ6. Therefore, the reasons for pRJ9 failure to be mobilized are presently unknown.     

In situ forming chitosan hydrogels prepared via ionic/covalent co-cross-linking

In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.     

SIMPLOT: Simulating the impacts of fire severity on sustainability of eucalyptus forests in Portugal

SIMPLOT is a forest simulator for eucalyptus mainly driven by wood demand. It was developed to predict the evolution of the eucalyptus plantations in Portugal by combining forest inventory data with growth models taking into account the effect of different drivers such as wood demand, hazards occurrence and percentage of land use changes. The use of simulators for scenario analysis can be a powerful tool to explore policy options and to illustrate the consequences of different management alternatives. In the past years Portugal has been marked by extremely severe forest fires of great environmental impact. This paper shows simulation runs for two main scenario lines: the wood demand line and the wildfires line. In the first one, the simulator is used to identify a reasonable wood demand out of three different wood demands combined with a low/medium intensity fire scenario. The selected wood demand combined with three fire scenarios of increasing severity and a fourth one disregarding the existence of recent severe wildfires builds the second scenario line. The purpose of this study is to evaluate the impact of different magnitudes of forest fires occurrence on the sustainability of eucalyptus plantations starting with NFI data gathered in 1997 during a horizon of 28 years. The simulations reflect a constant level of afforestation and deforestation and assume that no changes took place between different management alternatives. These simulations provide some insight on the impact of different wood demand and different magnitudes/frequency of severe wildfires: it is not only the number and magnitude of severe wildfires that make a difference, but it is also the number and magnitude of medium wildfires that follow an extremely severe one. Furthermore, the inter-annual variability of wildfire occurrence affects carbon stock and carbon sequestration in a different way. The occurrence of severe wildfires has an immediate effect on carbon sequestration. The lower values are registered in the same year in which the most severe wildfires occur. On the other hand, the occurrence of severe wildfires has more permanent consequences on carbon stocks than on carbon sequestration. The more severe and numerous are the wildfires the more difficult and at long-term will be to recover the carbon stocks in the forest. Results have also shown that if a higher wood demand compatible with the expected increase of pulp industry capacity would have been considered this would have had drastic impacts on eucalyptus forest sustainability due to overharvesting in order to meet the desired wood demand.     

On the representability of complete genomes by multiple competing finite-context (Markov) models

A finite-context (Markov) model of order k yields the probability distribution of the next symbol in a sequence of symbols, given the recent past up to depth k. Markov modeling has long been applied to DNA sequences, for example to find gene-coding regions. With the first studies came the discovery that DNA sequences are non-stationary: distinct regions require distinct model orders. Since then, Markov and hidden Markov models have been extensively used to describe the gene structure of prokaryotes and eukaryotes. However, to our knowledge, a comprehensive study about the potential of Markov models to describe complete genomes is still lacking. We address this gap in this paper. Our approach relies on (i) multiple competing Markov models of different orders (ii) careful programming techniques that allow orders as large as sixteen (iii) adequate inverted repeat handling (iv) probability estimates suited to the wide range of context depths used. To measure how well a model fits the data at a particular position in the sequence we use the negative logarithm of the probability estimate at that position. The measure yields information profiles of the sequence, which are of independent interest. The average over the entire sequence, which amounts to the average number of bits per base needed to describe the sequence, is used as a global performance measure. Our main conclusion is that, from the probabilistic or information theoretic point of view and according to this performance measure, multiple competing Markov models explain entire genomes almost as well or even better than state-of-the-art DNA compression methods, such as XM, which rely on very different statistical models. This is surprising, because Markov models are local (short-range), contrasting with the statistical models underlying other methods, where the extensive data repetitions in DNA sequences is explored, and therefore have a non-local character.     

Microsatellite variation and evolution of human lactase persistence

The levels of haplotype diversity within the lineages defined by two single-nucleotide polymorphisms (SNPs) (–13910 C/T and –22018 G/A) associated with human lactase persistence were assessed with four fast-evolving microsatellite loci in 794 chromosomes from Portugal, Italy, Fulbe from Cameroon, São Tomé and Mozambique. Age estimates based on the intraallelic microsatellite variation indicate that the –13910*T allele, which is more tightly associated with lactase persistence, originated in Eurasia before the Neolithic and after the emergence of modern humans outside Africa. We detected significant departures from neutrality for the –13910*T variant in geographically and evolutionary distant populations from southern Europe (Portuguese and Italians) and Africa (Fulbe) by using a neutrality test based on the congruence between the frequency of the allele and the levels of intraallelic variability measured by the number of mutations in adjacent microsatellites. This result supports the role of selection in the evolution of lactase persistence, ruling out possible confounding effects from recombination suppression and population history. Reevaluation of the available evidence on variation of the –13910 and –22018 loci indicates that lactase persistence probably originated from different mutations in Europe and most of Africa, even if 13910*T is not the causal allele, suggesting that selective pressure could have promoted the convergent evolution of the trait. Our study shows that a limited number of microsatellite loci may provide sufficient resolution to reconstruct key aspects of the evolutionary history of lactase persistence, providing an alternative to approaches based on large numbers of SNPs.Electronic supplementary material Supplementary material is available for this article at     

The 29.9 kDa subunit of mitochondrial complex I is involved in the enzyme active/de-active transitions

Mitochondrial respiratory chain complex I undergoes transitions from active to de-activated forms. We have investigated the phenomenon in sub-mitochondrial particles from Neurospora crassa wild-type and a null-mutant lacking the 29.9 kDa nuclear-coded subunit of complex I. Based on enzymatic activities, genetic crosses and analysis of mitochondrial proteins in sucrose gradients, we found that about one-fifth of complex I with catalytic properties similar to the wild-type enzyme is assembled in the mutant. Mutant complex I still displays active/de-active transitions, indicating that other proteins are involved in the phenomenon. However, the kinetic characteristics of complex I active/de-active transitions in nuo29.9 differ from wild-type. The spontaneous de-activation of the mutant enzyme is much slower, implicating the 29.9 kDa polypeptide in this event. We suggest that the fungal 29.9 kDa protein and its homologues in other organisms may modulate the active/de-active transitions of complex I.     

Towards a structural understanding of the fibrillization pathway in Machado-Joseph's disease: trapping early oligomers of non-expanded ataxin-3

Machado-Joseph's disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract in the protein ataxin-3. Except for the polyglutamine region, proteins associated with polyglutamine diseases are unrelated, and for all of these diseases aggregates containing these proteins are the major components of the nuclear proteinaceous deposits found in the brain. Aggregates of the expanded proteins display amyloid-like morphological and biophysical properties. Human ataxin-3 containing a non-pathological number of glutamine residues (14Q), as well as its Caenorhabditis elegans (1Q) orthologue, showed a high tendency towards self-interaction and aggregation, under near-physiological conditions. In order to understand the discrete steps in the assembly process leading to ataxin-3 oligomerization, we have separated chromatographically high molecular mass oligomers as well as medium mass multimers of non-expanded ataxin-3. We show that: (a) oligomerization occurs independently of the poly(Q)-repeat and it is accompanied by an increase in beta-structure; and (b) the first intermediate in the oligomerization pathway is a Josephin domain-mediated dimer of ataxin-3. Furthermore, non-expanded ataxin-3 oligomers are recognized by a specific antibody that targets a conformational epitope present in soluble cytotoxic species found in the fibrillization pathway of expanded polyglutamine proteins and other amyloid-forming proteins. Imaging of the oligomeric forms of the non-pathological protein using electron microscopy reveals globular particles, as well as short chains of such particles that likely mimic the initial stages in the fibrillogenesis pathway occurring in the polyglutamine-expanded protein. Thus, they constitute potential targets for therapeutic approaches in Machado-Joseph's disease, as well as valuable diagnostic markers in disease settings.     

Genomic analysis of Grapevine Retrotransposon 1 (Gret1) in Vitis vinifera

The complete sequence of the first retrotransposon isolated in Vitis vinifera, Gret1, was used to design primers that permitted its analysis in the genome of grapevine cultivars. This retroelement was found to be dispersed throughout the genome with sites of repeated insertions. Fluorescent in situ hybridization indicated multiple Gret1 loci distributed throughout euchromatic portions of chromosomes. REMAP and IRAP proved to be useful as molecular markers in grapevine. Both of these techniques showed polymorphisms between cultivars but not between clones of the same cultivar, indicating differences in Gret1 distribution between cultivars. The combined cytological and molecular results suggest that Gret1 may have a role in gene regulation and in explaining the enormous phenotypic variability that exists between cultivars.     

Diet of Southern Muriquis in Continuous Brazilian Atlantic Forest

We systematically collected data on feeding behavior for one group of 33–39 southern muriquis (Brachyteles arachnoides) in Parque Estadual Carlos Botelho (PECB), São Paulo State, Brazil (37,432.45 ha of continuous Atlantic Forest), between January and December 1995. We determined food item consumption from instantaneous scans of behavior. Fruits were the most eaten food items in all 12 mo (40–80% of scan in every mo, average = 71.3%). Muriquis ate young leaves more than mature leaves or flowers. Our results are consistent with previous findings at the same and neighboring forest sites that southern muriquis have a consistently frugivorous diet when inhabiting less disturbed habitats, but contrast with previous observations on oppportunistic frugivory in muriqui populations inhabiting fragmented forests. Sustained high levels of frugivory probably result from year-round availability of fruit within large continuous forests.