Benznidazole biotransformation and multiple targets in <i>Trypanosoma</i> cruzi revealed by metabolomics

&lt;b&gt;Background&lt;/b&gt;&lt;p&gt;&lt;/p&gt; The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Methodology/Principal findings&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Conclusions/significance&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi

Restriction Site Extension PCR: A Novel Method for High-Throughput Characterization of Tagged DNA Fragments and Genome Walking

BACKGROUND: Insertion mutant isolation and characterization are extremely valuable for linking genes to physiological function. Once an insertion mutant phenotype is identified, the challenge is to isolate the responsible gene. Multiple strategies have been employed to isolate unknown genomic DNA that flanks mutagenic insertions, however, all these methods suffer from limitations due to inefficient ligation steps, inclusion of restriction sites within the target DNA, and non-specific product generation. These limitations become close to insurmountable when the goal is to identify insertion sites in a high throughput manner. METHODOLOGY/PRINCIPAL FINDINGS: We designed a novel strategy called Restriction Site Extension PCR (RSE-PCR) to efficiently conduct large-scale isolation of unknown genomic DNA fragments linked to DNA insertions. The strategy is a modified adaptor-mediated PCR without ligation. An adapter, with complementarity to the 3' overhang of the endonuclease (KpnI, NsiI, PstI, or SacI) restricted DNA fragments, extends the 3' end of the DNA fragments in the first cycle of the primary RSE-PCR. During subsequent PCR cycles and a second semi-nested PCR (secondary RSE-PCR), touchdown and two-step PCR are combined to increase the amplification specificity of target fragments. The efficiency and specificity was demonstrated in our characterization of 37 tex mutants of Arabidopsis. All the steps of RSE-PCR can be executed in a 96 well PCR plate. Finally, RSE-PCR serves as a successful alternative to Genome Walker as demonstrated by gene isolation from maize, a plant with a more complex genome than Arabidopsis. CONCLUSIONS/SIGNIFICANCE: RSE-PCR has high potential application in identifying tagged (T-DNA or transposon) sequence or walking from known DNA toward unknown regions in large-genome plants, with likely application in other organisms as well

Prevalence and risk factors for Giardia duodenalis infection among children: A case study in Portugal

<p>Abstract</p> <p>Background</p> <p><it>Giardia duodenalis </it>is a widespread parasite of mammalian species, including humans. The prevalence of this parasite in children residing in Portugal is currently unknown. This study intended to estimate <it>G. duodenalis </it>infection prevalence and identify possible associated risk factors in a healthy paediatric population living in the District of the Portuguese capital, Lisbon.</p> <p>Methods</p> <p>Between February 2002 and October 2008, 844 children were randomly selected at healthcare centres while attending the national vaccination program. A stool sample and a questionnaire with socio-demographic data were collected from each child. <it>Giardia </it>infection was diagnosed by direct examination of stools and antigen detection by ELISA.</p> <p>Results</p> <p>The population studied revealed a gender distribution of 52.8% male and 47.2% female. Age distribution was 47.4% between 0-5 years and 52.6% between 6-15 years.</p> <p>The prevalence of <it>Giardia </it>infection was 1.9% (16/844) when estimated by direct examination and increased to 6.8% (57/844) when ELISA results were added. The prevalence was higher among children aged 0-5 years (7.8%), than among older children (5.8%), and was similar among genders (6.9% in boys and 6.5% in girls). The following population-variables were shown to be associated risk factors for <it>G. duodenalis </it>infection: mother's educational level (odds ratio (OR)= 4.49; confidence interval (CI): 1.20-16.84), father's educational level (OR = 12.26; CI: 4.08-36.82), presence of <it>Helicobacter pylori </it>infection (OR = 1.82; CI: 1.05-3.15), living in houses with own drainage system (OR = 0.10; CI: 0.02-0.64) and reported household pet contact, especially with dogs (OR = 0.53; CI: 0.31-0.93).</p> <p>Conclusion</p> <p>The prevalence of giardiasis in asymptomatic children residing in the region of Lisbon is high. Several risk factors were associated with <it>Giardia </it>prevalence and highlight the importance of parents' education and sanitation conditions in the children's well being. The association between <it>G. duodenalis </it>and <it>H. pylori </it>seems an important issue deserving further investigation in order to promote prevention or treatment strategies.</p

Could giardiasis be a risk factor for low zinc status in schoolchildren from northwestern Mexico? A cross-sectional study with longitudinal follow-up

<p>Abstract</p> <p>Background</p> <p>Both giardiasis and zinc deficiency are serious health problems worldwide. In Mexico, the prevalence of <it>G. intestinalis </it>was estimated at 32% in 1994. It remains a health problem in northwestern Mexico. Recent surveys (1987, 1995, and 1999) reported zinc deficiency in the Mexican population. The association of giardiasis and malabsorption of micronutrients has been well documented, although the association with zinc remains controversial. This study investigated the association between giardiasis and zinc deficiency in schoolchildren from northwestern Mexico.</p> <p>Methods</p> <p>We combined a cross-sectional design with a longitudinal follow-up six months after parasite treatment. The baseline sample consisted of 114 schoolchildren (mean age 8.8 yr) from seven suburban public schools, grouped as <it>Giardia</it>-free (<it>n </it>= 65, 57%) and <it>Giardia</it>-infected (<it>n </it>= 49, 43%). Three stool analyses per child were done using Faust's method. Children with giardiasis received secnidazole. Serum zinc was determined by atomic absorption spectrophotometry. Height and weight were measured. Socioeconomic information was obtained in an oral questionnaire, and daily zinc intake was assessed using 24 hour-recalls. Pearson's correlation and ANCOVA and paired t-test analyses were used to determine the association between giardiasis and zinc status.</p> <p>Results</p> <p>Longitudinal analysis demonstrated a significant increase of the mean serum zinc levels in the <it>Giardia</it>-infected group six months after treatment (13.78 vs. 19.24 μmol/L μmol/L; p = 0.001), although no difference was found between the <it>Giardia</it>-free and the <it>Giardia</it>-infected groups (p = 0.86) in the baseline analysis. Z scores for W/A and H/A were lower in the <it>Giardia</it>-infected than in the <it>Giardia</it>-free group (p < 0.05). No difference was observed in the socioeconomic characteristics and mean daily intakes of zinc between the groups (p > 0.05).</p> <p>Conclusions</p> <p>Giardiasis may be a risk factor for zinc deficiency in schoolchildren from northwestern Mexico.</p

Experimental study of the quantum driven pendulum and its classical analogue in atoms optics

We present experimental results for the dynamics of cold atoms in a far detuned amplitude-modulated optical standing wave. Phase-space resonances constitute distinct peaks in the atomic momentum distribution containing up to 65% of all atoms resulting from a mixed quantum chaotic phase space. We characterize the atomic behavior in classical and quantum regimes and we present the applicable quantum and classical theory, which we have developed and refined. We show experimental proof that the size and the position of the resonances in phase space can be controlled by varying several parameters, such as the modulation frequency, the scaled well depth, the modulation amplitude, and the scaled Planck's constant of the system. We have found a surprising stability against amplitude noise. We present methods to accurately control the momentum of an ensemble of atoms using these phase-space resonances which could be used for efficient phase-space state preparation