Use of Bacterially Expressed dsRNA to Downregulate Entamoeba histolytica Gene Expression

Background

Modern RNA interference (RNAi) methodologies using small interfering RNA (siRNA) oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules.

Principal Findings

Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA) targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica β-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite.

Conclusions

Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.     

Minimal CK2 activity required for yeast growth

Protein kinase CK2 is essential for the growth of Saccharomyces cerevisiae. Yeast cells that lack the functional genes coding for both the catalytic subunits of protein kinase CK2 can grow only if they are complemented by exogenous cDNAs coding for this subunit. A series of deletion mutants of CK2α from Xenopus laevis was constructed. These mutants that have carboxyl end deletions yield a CK2α product that varies over four orders of magnitude in its capacity to phosphorylate casein in vitro. Complementation of yeast RPG41-1a, a mutant defective in CKA1 and CKA2 genes, with wild-type X. laevis CK2α and with cDNAs coding for truncated CK2α having amino acids 1–328 and 1–327 resulted in cells that grew in gal-minimal media at 30 ^∘C as well as the cells harboring the yeast CKA2 gene. However, the growth was significantly diminished when cells were complemented with X. laevis CK2α containing 1–326 amido acids. This mutant has 0.6% of the catalytic activity of the wild-type enzyme. Yeast cells that expressed CK2α 1–324 and 1–323 which have 10-and 100-fold less activity, respectively, were not able to grow. The growth of cells containing the CK2α 1–326 mutant was very sensitive to temperature, and minimal growth was observed at 37 ^∘C. This mutant was also more sensitive to UV radiation but was not significantly affected by 0.4 M NaCl.Both authors contributed equally to this work     

Effect of lectin-binding to fibrinogen D and E domains on coagulation and fibrinolysis

The effect on fibrinogen coagulation and fibrinolysis of the mannose-specific lectins concanavalin A, its acetyl derivative and Lens culinaris agglutinin was studied. Concanavalin A and acetyl-concanavalin A, which bind to the four carbohydrate chains of fibrinogen, and L. culinaris agglutinin, which only binds to the carbohydrate present in fibrinogen D domains, has the same effect on the coagulation rate: an inhibition at low lectin concentrations and an increase at high concentrations. On the other hand, L. culinaris agglutinin does not alter fibrin crosslinking while acetyl-concanavalin A produces a slight inhibition of both gamma-gamma and alpha-polymer formation. However, this effect is very small when compared with the clear inhibitory effect produced by concanavalin A. Concanavalin A and acetyl-concanavalin A have an inhibitory effect on the rate of fibrin clot lysis proportional to the lectin concentration. Nearly 100% inhibition was obtained when two lectin-binding sites were occupied by either concanavalin A or acetyl-concanavalin A. However, L. culinaris agglutinin has a clearly weaker effect and more than 50% inhibition was not observed. The comparative study of the effect of the three lectins on fibrinolysis as well as on the formation of fibrinogen aggregates suggests that the inhibitory effect of concanavalin A and acetyl-concanavalin A is primarily due to their binding to the carbohydrate chains of fibrinogen E domain.     

Cryopreserved CD90+ cells obtained from mobilized peripheral blood in sheep: a new source of mesenchymal stem cells for preclinical applications

Mobilized peripheral blood (MPB) bone marrow cells possess the potential to differentiate into a variety of mesenchymal tissue types and offer a source of easy access for obtaining stem cells for the development of experimental models with applications in tissue engineering. In the present work, we aimed to isolate by magnetic activated cell sorting CD90+ cells from MPB by means of the administration of Granulocyte-Colony Stimulating Factor and to evaluate cell proliferation capacity, after thawing of the in vitro culture of this population of mesenchymal stem cells (MSCs) in sheep. We obtained a median of 8.2 ± 0.6 million of CD90+ cells from the 20-mL MPB sample. After thawing, at day 15 under in vitro culture, the mean CD90+ cells determined by flow cytometry was 92.92 ± 1.29 % and cell duplication time determined by crystal violet staining was 47.59 h. This study describes for the first time the isolation, characterization, and post-in vitro culture thawing of CD90+ MSCs from mobilized peripheral blood in sheep. This population can be considered as a source of MSCs for experimental models in tissue engineering research.     

EGb761 Blocks MPP+-Induced Lipid Peroxidation in Mouse Corpus Striatum

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generation of free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the damage to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP⁺). We investigated the effects of EGb761 pretreatment on MPP⁺ neurotoxicity. C-57 black mice were pretreated with EGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administration of MPP⁺, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 h and 24 h after MPP⁺ administration. Striatal dopamine content was analyzed by HPLC at the highest EGb761 dose at 2 h and 24 h after MPP⁺ administration. MPP⁺-induced LP was blocked (100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) the dopamine-depleting effect of MPP⁺ at 24 h. These results suggest that supplements of EGb761 may be effective at preventing MPP⁺-induced oxidative stress.     

RAPD and ISSR markers indicate diminished gene flow due to recent fragmentation of Polylepis australis woodlands in central Argentina

Two Polylepis australis BITT. populations differing in size were examined with the aim of determining future sampling strategies; assessing levels of genetic diversity and checking whether trees of different ages might vary in their genetic structure due to the effects of fragmentation. RADP and ISSR gave similar values of diversity. A re-sampling technique showed that for P. australis, 10 trees and 20 markers were enough to produce an unbiased estimator of heterozygosity. AMOVA suggested differences in allele frequencies between young and old trees in the small population (p = 0.052), but not in a large population (p = 0.864); suggesting that gene flow between the areas diminished in relatively recent times. This assumption is supported by the fact that allele frequencies among both woodlands were significantly different between the young (p < 0.0001), but not the older trees (p = 0.87).     

Mate Finding Via a Trail Sex Pheromone by <Emphasis Type="Italic">Aphytis melinus</Emphasis> DeBach (Hymenoptera: Aphelinidae) Males

Finding mates is frequently problematic for parasitoid wasps. In some parasitoid species, males rely on volatile, airborne sex pheromones for locating mates, while in others they rely on contact, trail sex pheromones. This study sought to shed light on the mate finding mechanism of males of Aphytis melinus. Specifically, the goal was to determine whether A. melinus males use airborne or contact pheromones, or both, for locating mates. The study showed that A. melinus males rely on a contact, trail sex pheromone for locating mates: A. melinus males responded to substrate-borne cues left by virgin females, while they did not respond to airborne cues from virgin females. Specifically, males more frequently encountered virgin females when the females walked across an arena to a fixed encounter point compared to when they were manually placed at the encounter point, and spent greater than expected time on surfaces previously visited by virgin females compared to control surfaces not visited by females. In contrast, males did not respond to airborne cues from virgin females in an airflow olfactometer nor to traps baited with virgin females in the field, and spent similar lengths of time on surfaces visited by newly-mated or 24-h mated females versus control surfaces not visited by females. The main effect of the trail sex pheromone on the behavior of A. melinus males was to direct their search and, so, increase the likelihood of encountering mates. This effect apparently is not preceded by longer-range attraction of males via an airborne female sex pheromone. Overall, the results of this study support a hypothesis in which A. melinus males searching on substrates on which females may be present rely exclusively on a trail sex pheromone to locate mates.