1,3-Propanediol production in a two-step process fermentation from renewable feedstock

In this work, the production of 1,3-propanediol from glucose and molasses was studied in a two-step process using two recombinant microorganisms. The first step of the process is the conversion of glucose or other sugar into glycerol by the metabolic engineered Saccharomyces cerevisiae strain HC42 adapted to high (>200 g l⁻¹) glucose concentrations. The second step, carried out in the same bioreactor, was performed by the engineered strain Clostridium acetobutylicum DG1 (pSPD5) that converts glycerol to 1,3-propanediol. This two-step strategy led to a flexible process, resulting in a 1,3-propanediol production and yield that depended on the initial sugar concentration. Below 56.2 g l⁻¹ of sugar concentration, cultivation on molasses or glucose showed no significant differences. However, at higher molasses concentrations, glycerol initially produced by yeast could not be totally converted into 1,3-propanediol by C. acetobutylicum and a lower 1,3-propanediol overall yield was observed. In our hand, the best results were obtained with an initial glucose concentration of 103 g l⁻¹, leading to a final 1,3-propanediol concentration of 25.5 g l⁻¹, a productivity of 0.16 g l⁻¹ h⁻¹ and 1,3-propanediol yields of 0.56 g g⁻¹ glycerol and 0.24 g g⁻¹ sugar, which is the highest value reported for a two-step process. For an initial sugar concentration (from molasses) of 56.2 g l⁻¹, 27.4 g l⁻¹ of glycerol were produced, leading to 14.6 g l⁻¹ of 1.3-propanediol and similar values of productivity, 0.15 g l⁻¹ h⁻¹, and overall yield, 0.26 g g⁻¹ sugar.     

Particle capture into the lung made simple?

Understanding the impact distribution of particles entering the human respiratory system is of primary importance as it concerns not only atmospheric pollutants or dusts of various kinds but also the efficiency of aerosol therapy and drug delivery. To model this process, current approaches consist of increasingly complex computations of the aerodynamics and particle capture phenomena, performed in geometries trying to mimic lungs in a more and more realistic manner for as many airway generations as possible. Their capture results from the complex interplay between the details of the aerodynamic streamlines and the particle drag mechanics in the resulting flow. In contrast, the present work proposes a major simplification valid for most airway generations at quiet breathing. Within this context, focusing on particle escape rather than capture reveals a simpler structure in the entire process. When gravity can be neglected, we show by computing the escape rates in various model geometries that, although still complicated, the escape process can be depicted as a multiplicative escape cascade in which each elementary step is associated with a single bifurcation. As a net result, understanding of the particle capture may not require computing particle deposition in the entire lung structure but can be abbreviated in some regions using our simpler approach of successive computations in single realistic bifurcations. Introducing gravity back into our model, we show that this multiplicative model can still be successfully applied on up to nine generations, depending on particle type and breathing conditions.     

Identification of a Passiflora alata Curtis dimeric peptide showing identity with 2S albumins

Antifungal proteins and peptides, essential compounds for plant defense, have been isolated from several tissues of various plants. These proteins could be used as a natural alternative to control phytopathogenic fungi. In this report a heterodimeric antifungal protein named Pa-AFP1, showing higher identity with the 2S albumin family, was purified by using 70-100% ammonium sulfate saturation and further purification steps such as anionic exchange Q-Sepharose chromatography associated with HPLC reversed-phase C4 chromatography. Analysis by Tricine-SDS-PAGE revealed two peptidic molecular masses of approximately 4500 Da and 7000 Da, in the presence of β-mercaptoethanol, while by removing the reducing agent a single protein with molecular mass of about 11,500 Da was obtained. Moreover, dimer mass was confirmed by MALDI-TOF analyses (11,569.76 Da). The antifungal protein, named Pa-AFP1, efficiently inhibited the growth of filamentous fungi Colletotrichum gloeosporioides, and was added to a short list of 2S albumins with antimicrobial properties. Otherwise, this same peptide showed no activity toward bacteria and yeasts. In summary, this compound could be used in the future to develop biotechnological products for the control of phytopathogenic fungi.     

Alterations in the dynamics of inflammation, proliferation and apoptosis in subcutaneous implants of lupus-prone mice

Wound repair is a complex process that involves inflammation, proliferation, extracellular matrix deposition/remodeling and apoptosis. Autoimmune diseases profoundly affect the healing process. We have used histological parameters to characterize the recruitment of mast cells and the proliferative activity and apoptosis in the fibrovascular tissue induced by subcutaneous polyether-polyurethane sponge implants in lupus-prone New Zealand White (NZW) and in control Balb/c mouse strains at days 10 and 21 post implantation. Fibrovascular tissue infiltration (hematoxylin and eosin staining), mast cell number (Dominici staining) and cellular proliferation (AgNOR staining) peaked early (day 10) but collagen deposition (picrosirius red staining) and apoptosis remained high in implants of NZW mice during the experimental period. In contrast, implants of Balb/c animals showed a progressive increase in mast cell recruitment and cellular proliferation but apoptosis fell from day 10 to 21 post-implantation. This divergent response early mast cells recruitment, excessive collagen deposition and disturbed removal of apoptotic cells from the site of injury in NZW mice implies that the genotype trait of NZW mice is a determining factor in abnormal healing response.     

Further characterization and mode of action of dactylomelin-P, an antibacterial protein isolated from the ink of the sea hare Aplysia dactylomela (Rang, 1828)

Sea hares are well known, nearly shell-less, marine opisthobranchs that use a complex repertoire of chemicals for defense and communication instead of a conventional gastropod shell. The most conspicuous characteristic of these invertebrates is the secretion of ink, which is rich in bioactive proteins. Many of these proteins belong to a family of L-amino acid oxidases (L-AAOs). In the current study, we aimed to determine whether dactylomelin-P, an antibacterial protein isolated from the ink of Aplysia dactylomela, could act as an L-AAO. We also investigated its biochemical properties and antibacterial mechanism of action. We found that dactylomelin-P is an acidic protein (pI = 5.0), rich in glutamic acid/glutamine, aspartic acid/asparagine, tyrosine, serine, and proline. It was stable under a broad pH range (3.0-12.0), after heating to 55 °C for 30 min, and after treating with protease. Its N-terminal amino acid sequence was DGVCSNRRQCNKEVCGSSYDVAIVGA and showed high similarity to other sea hare proteins previously identified as L-AAOs. The L-AAO activity was confirmed in an enzymatic assay, which showed that dactylomelin-P could oxidize L-lysine and L-arginine. We also demonstrated that the bacteriostatic activity of dactylomelin-P was mediated by hydrogen peroxide generated in the enzymatic reaction, but it acted as a bactericide in the presence of L-lysine and L-arginine. Transmission electron microscopy analyses showed that dactylomelin-P bound to growth-phase bacteria without causing morphological alterations to the cells. The bactericidal effect seems to involve H₂O₂ and other reactive components since it was not counteracted by H₂O₂ scavengers. Our findings showed biochemical, functional, and phylogenetic similarities among L-AAOs isolated from sea hares; this offers new insight into the evolution of these proteins and their roles in chemical defense.     

Non-linear models based on simple topological indices to identify RNase III protein members

Alignment-free classifiers are especially useful in the functional classification of protein classes with variable homology and different domain structures. Thus, the Topological Indices to BioPolymers (TI2BioP) methodology (Agüero-Chapin et al., 2010) inspired in both the TOPS-MODE and the MARCH-INSIDE methodologies allows the calculation of simple topological indices (TIs) as alignment-free classifiers. These indices were derived from the clustering of the amino acids into four classes of hydrophobicity and polarity revealing higher sequence-order information beyond the amino acid composition level. The predictability power of such TIs was evaluated for the first time on the RNase III family, due to the high diversity of its members (primary sequence and domain organization). Three non-linear models were developed for RNase III class prediction: Decision Tree Model (DTM), Artificial Neural Networks (ANN)-model and Hidden Markov Model (HMM). The first two are alignment-free approaches, using TIs as input predictors. Their performances were compared with a non-classical HMM, modified according to our amino acid clustering strategy. The alignment-free models showed similar performances on the training and the test sets reaching values above 90% in the overall classification. The non-classical HMM showed the highest rate in the classification with values above 95% in training and 100% in test. Although the higher accuracy of the HMM, the DTM showed simplicity for the RNase III classification with low computational cost. Such simplicity was evaluated in respect to HMM and ANN models for the functional annotation of a new bacterial RNase III class member, isolated and annotated by our group.     

Molecular and phylogenetic characterization of potentially toxic cyanobacteria in Tunisian freshwaters

This study presents a genetic characterization of 27 potentially toxic cyanobacterial strains isolated from seven reservoirs located in the north and centre of Tunisia. These strains belonged mainly to Microcystis aeruginosa, Cylindrospermopsis raciborskii and Planktothrix agardhii species. Their toxicological potential was evaluated by molecular biology tools, which showed that none of the isolated strains carried segments of the gene cluster responsible for the production of cylindrospermopsin and saxitoxin. The majority of Microcystis isolates were able to synthesize microcystin, since they presented the six characteristic segments of the microcystin synthetase mcy cluster (mcyA, -B, -C, -D, -E and -G). This was further confirmed by MALDI-TOF analysis that showed the presence of eight microcystin variants, including microcystin-LR. The taxonomic identification of the strains was assessed based on the variability of the 16S rRNA gene sequences. Furthermore, the 16S-23S rRNA ITS sequences of Microcystis isolates and rpoC1 sequences of Cylindrospermopsis strains were also used in the phylogenetic analysis.     

The role of roadsides in conserving Cerrado plant diversity

The structure of Cerrado vegetation (sensu stricto) along roadsides was compared with that of protected areas (reserves). The superior stratum of reserves had 2.7 times more individuals and 1.4 times more species than on roadsides, while in the intermediate stratum there were 1.7 times more individuals and 25 % more species in reserves. Additionally, roadsides had a lower prevalence of tree species with thin bark and from forest physiognomies. In the intermediate stratum of roadsides there was a lower proportion of forest species, and also fewer species dispersed by animals. These differences occur, possibly because the vegetation of their edges is cleared during the construction of the roads and part of the soil is taken as landfill. Later, they suffer from a higher fire frequency and high coverage of exotic grasses (80 % average). Moreover, it is likely that the absence of some dispersers on roadsides may affect the recruitment of young plants. Despite these differences, the roadsides are able to maintain at least 70 % of tree species and 72 % of shrub species found in reserves. These results show that roadside vegetation can contribute to the conservation of the flora of Cerrado. This is particularly important considering that over 60 % of the original area of this ecosystem has been destroyed and that less than 4 % is protected. With 32,012 km of roads in the Cerrado alone, the roadside vegetation may represent a cumulative area of 96,000 of hectares, an area larger than many of the Cerrado’s national parks.     

African Origin and Europe-Mediated Global Dispersal of The Cyanobacterium Microcystis aeruginosa

Microcystis aeruginosa is a bloom-forming cyanobacteria, which currently has a cosmopolitan distribution. Since M. aeruginosa can produce toxic compounds across all continents that it inhabits, it is of major public health relevance to assess its origin and dispersal. Thus, we conducted a worldwide study using 29 isolates representative of all the main continents, and used a concatenated genetic system for phylogenetic analyses consisting of four genetic markers (spanning ca. 3,485 bp). Our results support an early origin of M. aeruginosa in the African continent, with a subsequent dispersal to establish a second genetic pool in the European continent, from where M. aeruginosa then colonized the remaining continental regions. Our findings indicate that the European population has a cosmopolitan distribution, and is genetically closer to populations from Africa and North America. Our study also highlights the utility of using a concatenated dataset for phylogenetic inferences in cyanobacteria.