Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

Background  

Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica.     

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 °. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.     

Novel toxicity of native and HD Bacillus thuringiensis strains against to the sugarcane borer Diatraea saccharalis

Diatraea saccharalis (F.) (Lepidoptera: Pyralidae) is a pest that causes great economic losses to sugarcane producers in Mexico. In order to obtain alternatives for control of this pest, several Bacillus thuringiensis strains (native and from the Howard Dulmage collection) were tested. In bioassays, strains HD-133, HD-551, GM-7, GM-10, and GM-34 caused more than 50% mortality with a 50 g/ml spore-crystal complex concentration, and were selected as toxic strains. The lowest LC₅₀ value corresponded to GM-34 (33.21 g/ml). Cry1B and cry1C genes were detected by PCR analysis in the toxic strains. HD-133 and GM-10 habored cry1C gene, HD-551 and GM-7 strains harbored cry1B gene, while GM34 strain did not contain cry1B nor cry1C. An additional PCR analysis was performed to detect cry1A-type genes. All the toxic strains habor at least one cry1A-type gene. Immunoblotting revealed that all strains cross-reacted with an antiCry1A, and only the HD-551 gave a positive signal with antiCry1B polyclonal antisera. GM-7 crystal protein showed no cross-reaction with polyclonal Cry1B antiserum. The toxicity of these strains may be related to some member of the Cry1A toxin class.     

Peptide fragments as models to study the structure of a G-protein coupled receptor: the alpha-factor receptor of Saccharomyces cerevisiae

The alpha-factor tridecapeptide initiates mating in Saccharomyces cerevisiae upon interaction with Ste2p, its cognate G-protein coupled receptor (GPCR). This interaction is being used as a paradigm for understanding the structure and mechanism of activation of GPCRs by medium-sized peptides. In this article, the use of fragments of Ste2p to study its structure is reviewed. Methods of synthesis of peptides corresponding to both extramembranous and transmembrane domains of Ste2p are evaluated and problems that are encountered during synthesis and purification are described. The results from conformational analyses of the peptide fragments using fluorescence spectroscopy, CD, infrared spectroscopy, and NMR spectroscopy in organic-aqueous mixtures and in the presence of detergent micelles and lipid bilayers are critically reviewed. The data obtained to date provide biophysical evidence for the structure of different domains of Ste2p and indicate that peptides corresponding to these domains have unique biophysical tendencies. The studies carried out on Ste2p fragments indicate that valuable information concerning the structure of the intact receptor can be obtained by studying peptide fragments corresponding to domains of these polytopic integral membrane proteins.     

Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: a new method of evaluating sperm membrane integrity

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols. The present study evaluated the ability of an Annexin-V binding assay to detect early changes in sperm membrane integrity using flow cytometry (FC) in two different portions of the boar ejaculate, in cryopreserved semen. Using a split sample design, sperm motility was evaluated in fresh (controls) and frozen-thawed (FT) samples, both subjectively and by means of a computer-assisted motility assessment (CASA) system, while membrane integrity was assessed using Annexin-V (A) and propidium iodide (PI) staining in spermatozoa derived from the first sperm-rich fraction (Portion I) or the remaining ejaculate (Portion II). The A/PI technique revealed four sperm subpopulations, two PI negative (either A- (alive) or A+ (apoptotic)); and two PI positive (dead cells), either A+ (dead, late apoptotic or early necrotic cells) or A- (dead, late necrotic cells). Significant differences were found between the two portions of the ejaculate in the fresh (control) and FT samples. In the fresh controls, significantly more live, nonapoptotic spermatozoa (A-/PI-) were present in Portion I than in Portion II (P<0.001). Although apoptotic spermatozoa were detected in both semen portions, the frequency of live, early apoptotic (A+/PI-) cells was significantly lower in Portion I than in Portion II (P<0.001). Irrespective of the ejaculate portion considered, freezing and thawing significantly decreased the mean percentages of live spermatozoa (P<0.01), and dramatically increased the percentages of apoptotic or early necrotic cells (P<0.01), but not of early apoptotic cells (N.S.). The latter finding might suggest that apoptotic changes due to cryopreservation using the procedures applied in this trial are transient and lead to cell death. In conclusion, the Annexin-V binding assay was able to detect deleterious changes in the sperm plasma membrane at an earlier point than PI staining, thus representing a novel approach to investigating membrane integrity in this species. The finding that fewer spermatozoa in Portion I of the ejaculate showed early apoptosis post-freezing, suggests boar spermatozoa in this portion of the seminal plasma are less sensitive to the stress induced by cryopreservation.     

Is Leishmania (Viannia) peruviana a Distinct Species? A MLEE/RAPD Evolutionary Genetics Answer

A set of 38 Leishmania stocks from the Andean valleys of Peru was characterized by both Multilocus Enzyme Electrophoresis (MLEE) and Random Amplified Polymorphic DNA (RAPD). Data were analyzed in terms of taxonomy and evolutionary genetics. Synapomorphic MLEE and RAPD characters, clear-cut clustering, and strong agreement between the phylogenies inferred from either MLEE or RAPD supported the view that Leishmania (Viannia) peruviana and Leishmania (Viannia) braziliensis correspond to two closely related, but distinct monophyletic lines (clades) and can therefore be considered as "discrete typing units" (DTUs). The question whether the L. (V.) peruxviana DTU deserves species status is dependent upon the desirability of it, in terms of epidemiological and medical relevance. A previous Orthogonal Field Alternating Gel Electrophoresis (OFAGE) analysis of the same L. (V.) peruviana isolates was published by Dujardin et al. (1995b). The data from the different markers (i.e. MLEE, RAPD and OFAGE) were compared by population genetics analysis. RAPD and OFAGE provided divergent results, since RAPD showed a strong linkage disequilibrium whereas OFAGE revealed no apparent departure from panmictic expectation. MLEE showed no linkage disequilibrium. Nevertheless, contrary to OFAGE, this is most probably explainable by the limited variability revealed by this marker in L. (V.) peruviana (statistical type II error). RAPD data were consistent with the hypothesis that the present L. (V.) peruviana sample displays a basically clonal population structure with limited or no genetic exchange. Disagreement between RAPD and OFAGE can be explained either by accumulation of chromosomal rearrangements due to amplification/deletion of repeated sequences, or by pseudo-recombinational events.     

Genomic rearrangements in trypanosomatids: an alternative to the "one gene" evolutionary hypotheses?

Most molecular trees of trypanosomatids are based on point mutations within DNA sequences. In contrast, there are very few evolutionary studies considering DNA (re) arrangement as genetic characters. Waiting for the completion of the various parasite genome projects, first information may already be obtained from chromosome size-polymorphism, using the appropriate algorithms for data processing. Three illustrative models are presented here. First, the case of Leishmania (Viannia) braziliensis/L. (V.) peruviana is described. Thanks to a fast evolution rate (due essentially to amplification/deletion of tandemly repeated genes), molecular karyotyping seems particularly appropriate for studying recent evolutionary divergence, including eco-geographical diversification. Secondly, karyotype evolution is considered at the level of whole genus Leishmania. Despite the fast chromosome evolution rate, there is qualitative congruence with MLEE- and RAPD-based evolutionary hypotheses. Significant differences may be observed between major lineages, likely corresponding to major and less frequent rearrangements (fusion/fission, translocation). Thirdly, comparison is made with Trypanosoma cruzi. Again congruence is observed with other hypotheses and major lineages are delineated by significant chromosome rearrangements. The level of karyotype polymorphism within that "species" is similar to the one observed in "genus" Leishmania. The relativity of the species concept among these two groups of parasites is discussed.