Implication of an Asp residue in the ribonucleolytic activity of hirsutellin A reveals new electrostatic interactions at the active site of ribotoxins

Ribotoxins are fungal extracellular ribonucleases that specifically cleave ribosomes leading to cell-death via apoptosis. α-Sarcin is the ribotoxin studied in deepest detail, and therefore constitutes the referential protein for the whole family. It has been demonstrated that ribotoxin activity depends on a very precise structural microenvironment in which electrostatic interactions among residues in the active site are of the highest importance. Hirsutellin A (HtA) has been recently described as the smallest ribotoxin known to date, encompassing all the abilities of previously characterized members of this family into a shorter sequence. Comparison of HtA and α-sarcin three-dimensional structures suggested that residues presumably forming the catalytic triad of HtA would be His 42, Glu 66, and His 113. Within this same idea, the presence of an Asp residue (Asp 40) in a position equivalent to α-sarcin Tyr 48 is highlighted as a novelty in this field. In this work, substitution mutants H42Q, E66Q and H113Q, as well as double and triple mutants in all possible combinations, are studied regarding their ribonucleolytic activity and cytotoxicity. Implication of these three residues in the ribotoxin activity of HtA is confirmed, though none of them is strictly essential for ribosomal cleavage. Studies with mutants D40N and D40N/E66Q demonstrate an important role for Asp 40 in the activity of HtA and establish a new set of electrostatic interactions different from the one described for already known ribotoxins.     

Operation of an aerobic granular pilot scale SBR plant to treat swine slurry

A pilot-scale Sequencing Batch Reactor was operated during 307 days in order to treat swine slurry characterized by its high variable composition: organic and nitrogen applied loading rates and C/N ratio were 1.4–6.3 kg COD_s/(m³ d), 0.5–2.5 kg N/(m³ d) and 1.9–9.4 g COD_s/(g N), respectively. Aerobic granules successfully developed in the reactor and their physical properties remained rather stable despite the feeding composition variability. Organic and ammonia removal efficiency reached 61–73% and 56–77%, respectively, however ammonia was mainly oxidized to nitrite. The reactor had a good biomass retention capacity to select for granular biomass. However, its efficiency to retain the solids present in the feeding was low. Aerobic granulation in SBR systems appears as an interesting alternative to treat slurry in small livestock facilities where the implementation of anaerobic digestion systems is not a feasible option or the removal of nitrogenous compounds is required.     

Disturbances, elevation, topography and spatial proximity drive vegetation patterns along an altitudinal gradient of a top biodiversity hotspot

The correlation between vegetation patterns (species distribution and richness) and altitudinal variation has been widely reported for tropical forests, thereby providing theoretical basis for biodiversity conservation. However, this relationship may have been oversimplified, as many other factors may influence vegetation patterns, such as disturbances, topography and geographic distance. Considering these other factors, our primary question was: is there a vegetation pattern associated with substantial altitudinal variation (10–1,093 m a.s.l.) in the Atlantic Rainforest—a top hotspot for biodiversity conservation—and, if so, what are the main factors driving this pattern? We addressed this question by sampling 11 1-ha plots, applying multivariate methods, correlations and variance partitioning. The Restinga (forest on sandbanks along the coastal plains of Brazil) and a lowland area that was selectively logged 40 years ago were floristically isolated from the other plots. The maximum species richness (>200 spp. per hectare) occurred at approximately 350 m a.s.l. (submontane forest). Gaps, multiple stemmed trees, average elevation and the standard deviation of the slope significantly affected the vegetation pattern. Spatial proximity also influenced the vegetation pattern as a structuring environmental variable or via dispersal constraints. Our results clarify, for the first time, the key variables that drive species distribution and richness across a large altitudinal range within the Atlantic Rainforest.     

Phospholipase D1 downregulation by α-synuclein: Implications for neurodegeneration in Parkinson's disease

We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration.     

Relationships between cell surface protease and acid phosphatase activities ofLeishmania promastigote

A correlation between the ratio of the cell surface protease activity to phosphatase activity and the complexity of the pattern of cell surface exposed polypeptides ofLeishmania promastigotes was demonstrated for various strains grown under similar conditions. The ratio of the cell surface protease activity to acid phosphatase activity was high forL. major andL.b. panamensis and it correlates with the expression of a single polypeptide of 63 KDa on their cell surface. Intermediate and lower ratios of these enzymatic activites relate with more complex radio-iodinated patterns: two main bands inL.b. guyanensis (70 and 58 KDa) andL.b. braziliensis (72 and 60 KDa) and three main bands 65, 50, 27 KDa in allL.m. mexicana strains tested. Evidence is presented that the acid phosphatase located on theL.m. mexicana cell surface is not an artifact due to a secondary absorption of the secreted acid phosphatase from the culture medium. These results confirm theLeishmania antigen cell surface heterogeneity. The implications on the biology ofLeishmania and the clinical manifestation of leishmaniasis are discussed.     

Historical trends in the epidemiology of candidaemia: analysis of an 11-year period in a tertiary care hospital in Brazil

Candida species are an important cause of bloodstream infections (BSI). To evaluate the epidemiological, clinical and microbiological aspects of two cohorts {1994-1999 [period 1 (P1) ]; 2000-2004 [period 2 (P2) ]} of candidaemic patients, we performed a retrospective analysis from a laboratory-based survey. A total of 388 candidaemias were identified, with an incidence of 0.20/1,000 patient-days and a significant increase in P2 vs. P1 (0.25 vs. 0.15, p = 0.04). Cancer and prior antibiotic use were frequent and Candida albicans was the most prevalent species found (42.4%). Resistance to fluconazole was found in 2.47% of the strains. No differences were observed in the species distribution of Candida during the study periods. In the P2 cohort, there were higher prevalence of elderly individuals, cardiac, pulmonary and liver diseases, renal failure, central venous catheters and antibiotic therapy. In P1, there were higher prevalence of neurological diseases and chemotherapy. The crude mortality was 55.4%. In conclusion, our incidence rates remained high. Furthermore, the distribution pattern of Candida species and the fluconazole resistance profile remained unchanged. Moreover, we found a clear trend of higher prevalence of candidaemia among the elderly and among patients with comorbidities. Finally, it is necessary to discuss strategies for the prevention and control of Candida BSI in Brazil.     

From endosperm to triploid plants: a stepwise characterization of the de novo shoot organogenesis and morpho-agronomic aspects of an ornamental passion fruit (Passiflora foetida L.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Somaclonal variation during in vitro culture is often an undesirable phenomenon but may also be a source of genetic variation useful for breeders. The...     

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

The success of Mycobacterium tuberculosis (Mtb) as a human pathogen relies on its ability to resist eradication by the immune system. The identification of mechanisms that enable Mtb to persist is key for finding ways to limit latent tuberculosis, which affects one-third of the world's population. Here we show that conditional gene silencing can be used to determine whether an Mtb gene required for optimal growth in vitro is also important for virulence and, if so, during which phase of an infection it is required. Application of this approach to the prcBA genes, which encode the core of the mycobacterial proteasome, revealed an unpredicted requirement of the core proteasome for the persistence of Mtb during the chronic phase of infection in mice. Proteasome depletion also attenuated Mtb in interferon-gamma-deficient mice, pointing to a function of the proteasome beyond defense against the adaptive immune response. Genes that are essential for growth in vitro, in vivo or both account for approximately 20% of Mtb's genome. Conditional gene silencing could therefore facilitate the validation of up to 800 potential Mtb drug targets and improve our understanding of host-pathogen dynamics.     

High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris

Dipetarudin is a potent direct thrombin inhibitor that was genetically engineered as a chimera between dipetalogastin II and hirudin. Dipetarudin was initially cloned and purified from Escherichia coli, but with a very low yield of about 0.3 mg/l of culture medium. In this study, we report the production of dipetarudin in the methylotrophic yeast Pichia pastoris using pPIC9 vector. The His+ transformants were screened for the best expression performances by prolongation of the ecarin clotting time. An optimal dipetarudin's expression was reached by addition of methanol in culture medium to a final concentration of 0.5%, every 8h during 4 days. Secreted dipetarudin was purified essentially using a two-step purification scheme: anion exchange chromatography in a Resource Q column, followed by C18-reversed phase HPLC. About 150 mg purified dipetarudin was obtained from 1l culture supernatant. This yield is 500-fold higher than the yield obtained with the E. coli system. The molecular mass of dipetarudin calculated by MALDI-TOF (7450 Da) was in agreement with the mass calculated by the amino acid composition (7454 Da), indicating correct processing of the signal sequence. The Ki value of dipetarudin was 399+/-83 fM, which is in agreement with that calculated for the inhibitor isolated from E. coli. This efficient and cost-effective expression system facilitates large-scale production and purification of dipetarudin for further structural, functional and pharmacological investigations.