Trypanosoma cruzi: Insights into naphthoquinone effects on growth and proteinase activity

In this study we compared the effects of naphthoquinones (α-lapachone, β-lapachone, nor-β-lapachone and Epoxy-α-lap) on growth of Trypanosoma cruzi epimastigotes forms, and on viability of VERO cells. In addition we also experimentally analyzed the most active compounds inhibitory profile against T. cruzi serine- and cysteine-proteinases activity and theoretically evaluated them against cruzain, the major T. cruzi cysteine proteinase by using a molecular docking approach. Our results confirmed β-lapachone and Epoxy-α-lap with a high trypanocidal activity in contrast to α-lapachone and nor-β-lapachone whereas Epoxy-α-lap presented the safest toxicity profile against VERO cells. Interestingly the evaluation of the active compounds effects against T. cruzi cysteine- and serine-proteinases activities revealed different targets for these molecules. β-Lapachone is able to inhibit the cysteine-proteinase activity of T. cruzi proteic whole extract and of cruzain, similar to E-64, a classical cysteine-proteinase inhibitor. Differently, Epoxy-α-lap inhibited the T. cruzi serine-proteinase activity, similar to PMSF, a classical serine-proteinase inhibitor. In agreement to these biological profiles in the enzymatic assays, our theoretical analysis showed that E-64 and β-lapachone interact with the cruzain specific S2 pocket and active site whereas Epoxy-α-lap showed no important interactions. Overall, our results infer that β-lapachone and Epoxy-α-lap compounds may inhibit T. cruzi epimastigotes growth by affecting T. cruzi different proteinases. Thus the present data shows the potential of these compounds as prototype of protease inhibitors on drug design studies for developing new antichagasic compounds.     

Human salivary gland branching morphogenesis: morphological localization of claudins and its parallel relation with developmental stages revealed by expression of cytoskeleton and secretion markers

Development of salivary glands is a highly complex and dynamic process termed branching morphogenesis, where branched structures differentiate into mature glands. Tight junctions (TJ) are thought to play critical roles in physiological functions of tubular organs, contributing to cell polarity and preventing lateral movement of membrane proteins. Evidence demonstrated that claudins are directly involved in TJ formation and function. Using immunohistochemistry and immunofluorescence we have mapped the distribution of claudins-1, 2, 3, 4, 5, 7 and 11 and compared it with the expression of differentiation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. Expression of all claudins, except claudin-2 was detected in the various phases of human salivary gland development, up to fully mature salivary gland. The expression of all claudins increased according to the progression of salivary gland maturation evidenced by the classical markers-cytokeratin 14, cytokeratin low molecular weight, smooth muscle actin and human secretory component. Tight junction proteins-claudins appear to be important in the final shape and physiological functions of human salivary glands and are parallel related with markers of salivary gland differentiation.     

Prediction of Complex Human Traits Using the Genomic Best Linear Unbiased Predictor

Despite important advances from Genome Wide Association Studies (GWAS), for most complex human traits and diseases, a sizable proportion of genetic variance remains unexplained and prediction accuracy (PA) is usually low. Evidence suggests that PA can be improved using Whole-Genome Regression (WGR) models where phenotypes are regressed on hundreds of thousands of variants simultaneously. The Genomic Best Linear Unbiased Prediction (G-BLUP, a ridge-regression type method) is a commonly used WGR method and has shown good predictive performance when applied to plant and animal breeding populations. However, breeding and human populations differ greatly in a number of factors that can affect the predictive performance of G-BLUP. Using theory, simulations, and real data analysis, we study the performance of G-BLUP when applied to data from related and unrelated human subjects. Under perfect linkage disequilibrium (LD) between markers and QTL, the prediction R-squared (R²) of G-BLUP reaches trait-heritability, asymptotically. However, under imperfect LD between markers and QTL, prediction R² based on G-BLUP has a much lower upper bound. We show that the minimum decrease in prediction accuracy caused by imperfect LD between markers and QTL is given by (1−b)², where b is the regression of marker-derived genomic relationships on those realized at causal loci. For pairs of related individuals, due to within-family disequilibrium, the patterns of realized genomic similarity are similar across the genome; therefore b is close to one inducing small decrease in R². However, with distantly related individuals b reaches very low values imposing a very low upper bound on prediction R². Our simulations suggest that for the analysis of data from unrelated individuals, the asymptotic upper bound on R² may be of the order of 20% of the trait heritability. We show how PA can be enhanced with use of variable selection or differential shrinkage of estimates of marker effects.     

��-Amyloid Causes Depletion of Synaptic Vesicles Leading to Neurotransmission Failure

Alzheimer disease is a progressive neurodegenerative brain disorder that leads to major debilitating cognitive deficits. It is believed that the alterations capable of causing brain circuitry dysfunctions have a slow onset and that the full blown disease may take several years to develop. Therefore, it is important to understand the early, asymptomatic, and possible reversible states of the disease with the aim of proposing preventive and disease-modifying therapeutic strategies. It is largely unknown how amyloid β-peptide (Aβ), a principal agent in Alzheimer disease, affects synapses in brain neurons. In this study, we found that similar to other pore-forming neurotoxins, Aβ induced a rapid increase in intracellular calcium and miniature currents, indicating an enhancement in vesicular transmitter release. Significantly, blockade of these effects by low extracellular calcium and a peptide known to act as an inhibitor of the Aβ-induced pore prevented the delayed failure, indicating that Aβ blocks neurotransmission by causing vesicular depletion. This new mechanism for Aβ synaptic toxicity should provide an alternative pathway to search for small molecules that can antagonize these effects of Aβ.     

Molecular physiology of glutamine and glutamate biosynthesis in developing seedlings of conifers

Nitrogen is a limiting factor in tree growth and development. The incorporation of ammonium ions in carbon skeletons is catalyzed by the sequential action of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). Most studies on nitrogen‐assimilating enzymes have been reported for annual crop plants. Knowledge of these enzymes in woody plants is much more limited, particularly at the molecular level. Here, we review current available information on glutamine/glutamate biosynthesis and chloroplast development in conifers.     

The use of an unoccupied aerial vehicle to survey shark species over sand and rocky-reef habitats in a marine protected area

Cabo Pulmo National Park was established in 1995 and has since seen a large increase in fish biomass. An unoccupied aerial vehicle (UAV) was used to survey shallow coastal habitat in which lemon sharks (Negaprion brevirostris), bull sharks (Carcharhinus leucas) and Pacific nurse sharks (Ginglymostoma unami) were recorded. Sharks were more common in the afternoon, potentially using warmer shallow areas to behaviourally thermoregulate. This study highlights UAV surveying to be a viable tool for species identification, a limitation of previous terrestrial surveys conducted in the area.     

Use of RmInt1, a group IIB intron lacking the intron-encoded protein endonuclease domain, in gene targeting

The group IIA intron Ll.LtrB from Lactococcus lactis and the group IIB intron EcI5 from Escherichia coli have intron-encoded proteins (IEP) with a DNA-binding domain (D) and an endonuclease domain (En). Both have been successfully retargeted to invade target DNAs other than their wild-type target sites. RmInt1, a subclass IIB3/D intron with an IEP lacking D and En domains, is highly active in retrohoming in its host, Sinorhizobium meliloti. We found that RmInt1 was also mobile in E. coli and that retrohoming in this heterologous host depended on temperature, being more efficient at 28°C than at 37°C. Furthermore, we programmed RmInt1 to recognize target sites other than its wild-type site. These retargeted introns efficiently and specifically retrohome into a recipient plasmid target site or a target site present as a single copy in the chromosome, generating a mutation in the targeted gene. Our results extend the range of group II introns available for gene targeting.     

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.