Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.     

Association of Pro-Inflammatory Cytokines and Iron Regulatory Protein 2 (IRP2) with Leishmania Burden in Canine Visceral Leishmaniasis

Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2) were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.     

Sustained Reduction of the Dengue Vector Population Resulting from an Integrated Control Strategy Applied in Two Brazilian Cities

Aedes aegypti has developed evolution-driven adaptations for surviving in the domestic human habitat. Several trap models have been designed considering these strategies and tested for monitoring this efficient vector of Dengue. Here, we report a real-scale evaluation of a system for monitoring and controlling mosquito populations based on egg sampling coupled with geographic information systems technology. The SMCP-Aedes, a system based on open technology and open data standards, was set up from March/2008 to October/2011 as a pilot trial in two sites of Pernambuco -Brazil: Ipojuca (10,000 residents) and Santa Cruz (83,000), in a joint effort of health authorities and staff, and a network of scientists providing scientific support. A widespread infestation by Aedes was found in both sites in 2008–2009, with 96.8%–100% trap positivity. Egg densities were markedly higher in SCC than in Ipojuca. A 90% decrease in egg density was recorded in SCC after two years of sustained control pressure imposed by suppression of >7,500,000 eggs and >3,200 adults, plus larval control by adding fishes to cisterns. In Ipojuca, 1.1 million mosquito eggs were suppressed and a 77% reduction in egg density was achieved. This study aimed at assessing the applicability of a system using GIS and spatial statistic analysis tools for quantitative assessment of mosquito populations. It also provided useful information on the requirements for reducing well-established mosquito populations. Results from two cities led us to conclude that the success in markedly reducing an Aedes population required the appropriate choice of control measures for sustained mass elimination guided by a user-friendly mosquito surveillance system. The system was able to support interventional decisions and to assess the program’s success. Additionally, it created a stimulating environment for health staff and residents, which had a positive impact on their commitment to the dengue control program.     

Phylogeographic Pattern and Extensive Mitochondrial DNA Divergence Disclose a Species Complex within the Chagas Disease Vector Triatoma dimidiata

Background

Triatoma dimidiata is among the main vectors of Chagas disease in Latin America. However, and despite important advances, there is no consensus about the taxonomic status of phenotypically divergent T. dimidiata populations, which in most recent papers are regarded as subspecies.

Methodology and Findings

A total of 126 cyt b sequences (621 bp long) were produced for specimens from across the species range. Forty-seven selected specimens representing the main cyt b clades observed (after a preliminary phylogenetic analysis) were also sequenced for an ND4 fragment (554 bp long) and concatenated with their respective cyt b sequences to produce a combined data set totalling 1175 bp/individual. Bayesian and Maximum-Likelihood phylogenetic analyses of both data sets (cyt b, and cyt b+ND4) disclosed four strongly divergent (all pairwise Kimura 2-parameter distances >0.08), monophyletic groups: Group I occurs from Southern Mexico through Central America into Colombia, with Ecuadorian specimens resembling Nicaraguan material; Group II includes samples from Western-Southwestern Mexico; Group III comprises specimens from the Yucatán peninsula; and Group IV consists of sylvatic samples from Belize. The closely-related, yet formally recognized species T. hegneri from the island of Cozumel falls within the divergence range of the T. dimidiata populations studied.

Conclusions

We propose that Groups I–IV, as well as T. hegneri, should be regarded as separate species. In the Petén of Guatemala, representatives of Groups I, II, and III occur in sympatry; the absence of haplotypes with intermediate genetic distances, as shown by multimodal mismatch distribution plots, clearly indicates that reproductive barriers actively promote within-group cohesion. Some sylvatic specimens from Belize belong to a different species – likely the basal lineage of the T. dimidiata complex, originated ∼8.25 Mya. The evidence presented here strongly supports the proposition that T. dimidiata is a complex of five cryptic species (Groups I–IV plus T. hegneri) that play different roles as vectors of Chagas disease in the region.     

Letter to the Editor—the Use of Micronucleus Assay on Buccal Mucosa Cells for Risk Assessment: Relevance of Cigarette Smoke and Cytogenotoxicity

Biological Trace Element Research -     

QSAR studies on the human sirtuin 2 inhibition by non-covalent 7,5,2-anilinobenzamide derivatives

Abstract

Sirtuin 2 is a key enzyme in gene expression regulation that is often associated with tumor proliferation control and therefore is a relevant anticancer drug target. Anilinobenzamide derivatives have been discussed as selective sirtuin 2 inhibitors and can be developed further. In the present study, hologram and three-dimensional quantitative structure–activity relationship (HQSAR and 3D-QSAR) analyses were employed for determining structural contributions of a compound series containing human sirtuin-2-selective inhibitors that were then correlated with structural data from the literature. The final QSAR models were robust and predictive according to statistical validation (q² and r²_pred values higher than 0.85 and 0.75, respectively) and could be employed further to generate fragment contribution and contour maps. 3D-QSAR models together with information about the chemical properties of sirtuin 2 inhibitors can be useful for designing novel bioactive ligands.

Communicated by Ramaswamy H. Sarma     

Expression of the Arcelin 1 gene from Phaseolus vulgaris L. in cowpea seeds (Vigna unguiculata L.) confers bruchid resistance

Persistent decrease in the productivity of cowpea (Vigna unguiculata L.) has been partly due to attack by bruchids including Zabrotes subfasciatus and Callosobruchus maculatus. Resistance to these insects in Phaseolus vulgaris L. has been shown to be associated with arcelins, a family of seed proteins encoded by a multigenic family of lectins on the APA locus. In this work, we report the construction of an expression vector containing Arc1 gene isolated from P. vulgaris and introduced into cowpea as a strategy to confer resistance to insect attack. Following transformation and selection, feeding experiments in which C. maculatus and Z. subfasciatus were fed with transgenic (L3 and L5) and non-transgenic (control) grains showed that introduced gene protected the transgenic line. Significant differences (p < .05 and p < .01) were found in the number of eggs laid, the number of emerging insects and the loss of grain mass in L3, compared with control, for both insects. Similar observations were made in L5 with the exception of the number of laid eggs. The strategy here described may form the basis for the development of a cowpea variety tolerant to bruchids in a crop cultivated by farmers throughout Latin America and Africa.     

Activity of sodium trimetaphosphate,associated or not with fluoride,on dual-species biofilms

Abstract

A response surface methodology was used to build a model to predict reductions in uropathogenic Escherichia coli biofilms in response to three compounds: cranberry extract [CB] at 3.0–9.0%, and caprylic acid [CAR] and thymol [TM] at 0.01%–0.05%. The predictive model for microbial reduction had a high regression coefficient (R²?=?0.9988), and the accuracy of the model was verified (R²?=?0.9527). Values of CAR, TM, and the quadratic term CAR² were the most significant (P?10 reduction) determined by ridge analysis were 8.3% CB +0.04% CAR +0.04% TM at 37?°C for 1?min. The model could be used to predict the most cost-efficient amounts of antimicrobial agents for anti-urinary tract infection products such as catheter lock solution and antimicrobial coatings for catheters.     

Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata

The aim of this study was to evaluate the effect of silver nanoparticles (SN) against Candida albicans and Candida glabrata adhered cells and biofilms. SN (average diameter 5 nm) were synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. Minimal inhibitory concentration (MIC) tests were performed for C. albicans (n = 2) and C. glabrata (n = 2) grown in suspension following the Clinical Laboratory Standards Institute microbroth dilution method. SN were applied to adhered cells (2 h) or biofilms (48 h) and after 24 h of contact their effect was assessed by enumeration of colony forming units (CFUs) and quantification of total biomass (by crystal violet staining). The MIC results showed that SN were fungicidal against all strains tested at very low concentrations (0.4–3.3 μg ml^?1). Furthermore, SN were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata ATCC, which in both cases showed a reduction ～90%. Regarding cell viability, SN were highly effective on adhered C. glabrata and respective biofilms. On C. albicans the effect was not so evident but there was also a reduction in the number of viable biofilm cells. In summary, SN may have the potential to be an effective alternative to conventional antifungal agents for future therapies in Candida-associated denture stomatitis.