Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).

Methods/Principal Findings

The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.

Conclusions/Significance

Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.     

Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation.

Methods for measuring rates of DNA synthesis, and thus cell proliferation, in humans had not been available until recently. We (D. C. Macallan, C. A. Fullerton, R. A. Neese, K. Haddock, S. S. Park, and M. K. Hellerstein, 1998, Proc. Natl. Acad. Sci. USA 95, 708-713) recently developed a stable isotope-mass spectrometric technique for measuring DNA synthesis by labeling the deoxyribose (dR) moiety of purine deoxyribonucleotides through the de novo nucleotide synthesis pathway. The original analytic approach had limitations, however. Here, we describe technical improvements that increase yield, stability, sensitivity, and reproducibility of the method. The purine deoxyribonucleoside, deoxyadenosine (dA), is directly isolated from hydrolysates of DNA by using an LC18 SPE column. Two derivatives were developed for analyzing the dR moiety of dA alone (without the base), an aldonitrile-triacetate derivative, and a reduced pentose-tetraacetate (PTA) derivative. The PTA derivative in particular exhibited greater stability (no degradation after several weeks), greater GC/MS signal, and much less abundance sensitivity of isotope ratios (i.e., less dependence of mass isotopomer abundances on the amount of material injected into the mass spectrometer source), compared to previous derivatives of dA. The need for complex, multidimensional abundance corrected standard curves was thereby avoided. Using the PTA derivative, dR enrichments from DNA of fully turned over cells of rodents with 2H2O enrichments in body water of 2.2-2.8% were 9.0-9.5%, and less than 1.0 microg DNA (ca. 2 x 10(5) cells) was required for reproducible analyses. In summary, these methodologic advances allow measurement of stable isotope incorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and experimental animals, with fewer cells, greater reproducibility, and less labor. Many applications of this approach can be envisioned.     

Mariculture and Natural Production of the Antitumoural (+)-Discodermolide by the Caribbean Marine Sponge Discodermia dissoluta

Biotechnological research on marine organisms, such as ex situ or in situ aquaculture and in vitro cell culture, is being conducted to produce bioactive metabolites for biomedical and industrial uses. The Caribbean marine sponge Discodermia dissoluta is the source of (+)-discodermolide, a potent antitumoural polyketide that has reached clinical trials. This sponge usually lives at depths greater than 30 m, but at Santa Marta (Colombia) there is a shallower population, which has made it logistically possible to investigate for the first time, on ways to supply discodermolide. We thus performed in situ, 6-month fragment culture trials to assess the performance of this sponge in terms of growth and additional discodermolide production and studied possible factors that influence the variability of discodermolide concentrations in the wild. Sponge fragments cultured in soft mesh bags suspended from horizontal lines showed high survivorship (93 %), moderate growth (28 % increase in volume) and an overall rise (33 %) in the discodermolide concentration, equivalent to average additional production of 8 μg of compound per millilitre of sponge. The concentration of discodermolide in wild sponges ranged from 8 to 40 μg mL^?1. Locality was the only factor related to discodermolide variation in the wild, and there were greater concentrations in peripheral vs. basal portions of the sponge, and in clean vs. fouled individuals. As natural growth and regeneration rates can be higher than culture growth rates, there is room for improving techniques to sustainably produce discodermolide.     

Defense Responses in Two Ecotypes of Lotus japonicus against Non-Pathogenic Pseudomonas syringae

Lotus japonicus is a model legume broadly used to study many important processes as nitrogen fixing nodule formation and adaptation to salt stress. However, no studies on the defense responses occurring in this species against invading microorganisms have been carried out at the present. Understanding how this model plant protects itself against pathogens will certainly help to develop more tolerant cultivars in economically important Lotus species as well as in other legumes. In order to uncover the most important defense mechanisms activated upon bacterial attack, we explored in this work the main responses occurring in the phenotypically contrasting ecotypes MG-20 and Gifu B-129 of L. japonicus after inoculation with Pseudomonas syringae DC3000 pv. tomato. Our analysis demonstrated that this bacterial strain is unable to cause disease in these accessions, even though the defense mechanisms triggered in these ecotypes might differ. Thus, disease tolerance in MG-20 was characterized by bacterial multiplication, chlorosis and desiccation at the infiltrated tissues. In turn, Gifu B-129 plants did not show any symptom at all and were completely successful in restricting bacterial growth. We performed a microarray based analysis of these responses and determined the regulation of several genes that could play important roles in plant defense. Interestingly, we were also able to identify a set of defense genes with a relative high expression in Gifu B-129 plants under non-stress conditions, what could explain its higher tolerance. The participation of these genes in plant defense is discussed. Our results position the L. japonicus-P. syringae interaction as a interesting model to study defense mechanisms in legume species.     

The spatial and temporal patterns of falciparum and vivax malaria in Perú: 1994–2006

Background

Malaria is the direct cause of approximately one million deaths worldwide each year, though it is both preventable and curable. Increasing the understanding of the transmission dynamics of falciparum and vivax malaria and their relationship could suggest improvements for malaria control efforts. Here the weekly number of malaria cases due to Plasmodium falciparum (1994–2006) and Plasmodium vivax (1999–2006) in Perú at different spatial scales in conjunction with associated demographic, geographic and climatological data are analysed.

Methods

Malaria periodicity patterns were analysed through wavelet spectral analysis, studied patterns of persistence as a function of community size and assessed spatial heterogeneity via the Lorenz curve and the summary Gini index.

Results

Wavelet time series analyses identified annual cycles in the incidence of both malaria species as the dominant pattern. However, significant spatial heterogeneity was observed across jungle, mountain and coastal regions with slightly higher levels of spatial heterogeneity for P. vivax than P. falciparum. While the incidence of P. falciparum has been declining in recent years across geographic regions, P. vivax incidence has remained relatively steady in jungle and mountain regions with a slight decline in coastal regions. Factors that may be contributing to this decline are discussed. The time series of both malaria species were significantly synchronized in coastal (ρ = 0.9, P < 0.0001) and jungle regions (ρ = 0.76, P < 0.0001) but not in mountain regions. Community size was significantly associated with malaria persistence due to both species in jungle regions, but not in coastal and mountain regions.

Conclusion

Overall, findings highlight the importance of highly refined spatial and temporal data on malaria incidence together with demographic and geographic information in improving the understanding of malaria persistence patterns associated with multiple malaria species in human populations, impact of interventions, detection of heterogeneity and generation of hypotheses.     

Attenuating effect of melatonin on pyridoxal-stimulated release of adrenomedullary catecholamines in the rat

AimsTo investigate the ability of melatonin (MEL) to suppress adrenomedullary catecholamine (CAT) release in the rat, with pyridoxal (PL) being used as an adrenomedullary stimulus and liver and gastrocnemius muscle glycogenolysis acting as indices of CAT release.Main methodsMEL (1–4 mg/kg, i.p.) and PL (300 mg/kg, i.p.) were administered separately or together to male Sprague–Dawley rats (275–300 g), and blood samples for the assay of plasma glucose and CATs were periodically collected for up to 3 h after PL. Immediately thereafter, the liver and gastrocnemius muscle were surgically removed and used for the assay of glycogen. The role of adrenoceptors in PL-induced glycogenolysis was examined by parallel experiments in which idazoxan (IDX, 1 mg/kg), propranolol (PRO, 2 mg/kg) or metoprolol (MET, 2 mg/kg) were administered alongside MEL. In addition, MEL (4 mg/kg) was co-administered with taurine (TAU, 2.4 mmol/kg), a known adrenomedullary membrane stabilizer.Key findingsMEL attenuated the release of adrenomedullary CATs and accompanying liver and gastrocnemius muscle glycogenolysis due to PL in a dose-dependent manner. A co-treatment with MEL and an adrenoceptor blocker had a greater attenuating effect on PL-induced glycogenolysis and hyperglycemia than MEL but without impinging on the CAT levels seen with MEL alone. Evidence of maximal inhibitory action by MEL on PL-induced plasma CAT elevation was suggested by the about equal levels of plasma CATs after treatments with MEL and with MEL plus TAU.SignificanceThe present study demonstrates the modulatory effect of MEL of exogenous origin on adrenomedullary CAT secretion when present in supraphysiological concentrations.     

The adsorption of multimeric enzymes on very lowly activated supports involves more enzyme subunits: Stabilization of a glutamate dehydrogenase from Thermus thermophilus by immobilization on heterofunctional supports

Glutamate dehydrogenase (GDH) from Thermus thermophilus is a homotrimeric enzyme that tends to dissociate at acidic pH values. GDH is readily adsorbed on highly activated anionic exchangers (HAAE), but hardly adsorbed on lowly activated supports (LAAE) or on highly activated epoxy supports. When using amino-epoxy supports, GDH immobilized on HAAE-epoxy and more slowly on LAAE-epoxy supports. Both immobilized biocatalysts were incubated at pH 10 for different times to increase the multipoint covalent attachment. LAAE-epoxy-GDH was stable at pH 4 and 25 °C, the enzyme stability did not depend on the enzyme concentration and did not release any subunit to the supernatant, in opposition to the results obtained using HAAE-epoxy supports. The general application of this strategy to stabilize multimeric enzymes was verified by immobilizing a crude protein extract. It seems that proteins adsorb on LAAE by the larger region of their surface (that is the one that involves the highest number of enzyme subunits), since it is the only area large enough to permit a multipoint ionic exchange on this LAAE. On the contrary, using HAAE, some proteins may become adsorbed by clusters that were rich in anionic groups and located in a corner of the multimer, involving only some of the subunits in the enzyme immobilization. That way, a careful design of the design of the support permits to take full advantage of the immobilization on heterofunctional supports.     

Digestive capacities allow the Mexican long-nosed bat (Leptonycteris nivalis) to live in cold environments

Digestive capabilities of nectar-feeding vertebrates to assimilate sugars affect their ability to acquire and store energy and could determine the minimal temperatures at which these animals can survive. Here, we described the sugar digestive capability of Leptonycteris nivalis and related it with its capacity to live in cold environments. We measured the enzymatic activity, food intake rate and changes in body mass of bats feeding at four different sucrose concentrations (from 5 to 35% wt./vol.). Additionally, we used a mathematical model to predict food intake and compared it with the food intake of bats. L. nivalis was able to obtain ~ 111.3 kJ of energy regardless of the sugar concentration of their food. Also, bats gained ~ 2.57 g of mass during the experimental trials and this gain was independent of sugar concentration. The affinity (1 / K_m) of sucrase (EC 3.2.1.48) was one order of magnitude higher relative to that reported for its sister species Leptonycteris yerbabuenae (0.250 and 0.0189 mmol^? 1 L, respectively), allowing this species to have a higher energy intake rate. We propose that the high ability to acquire energy conferred L. nivalis the faculty to invade cold environments, avoiding in this way the ecological competition with its sympatric species L. yerbabuenae.     

Subterranean, herbivore-induced plant volatile increases biological control activity of multiple beneficial nematode species in distinct habitats

While the role of herbivore-induced volatiles in plant-herbivore-natural enemy interactions is well documented aboveground, new evidence suggests that belowground volatile emissions can protect plants by attracting entomopathogenic nematodes (EPNs). However, due to methodological limitations, no study has previously detected belowground herbivore-induced volatiles in the field or quantified their impact on attraction of diverse EPN species. Here we show how a belowground herbivore-induced volatile can enhance mortality of agriculturally significant root pests. First, in real time, we identified pregeijerene (1,5-dimethylcyclodeca-1,5,7-triene) from citrus roots 9-12 hours after initiation of larval Diaprepes abbreviatus feeding. This compound was also detected in the root zone of mature citrus trees in the field. Application of collected volatiles from weevil-damaged citrus roots attracted native EPNs and increased mortality of beetle larvae (D. abbreviatus) compared to controls in a citrus orchard. In addition, field applications of isolated pregeijerene caused similar results. Quantitative real-time PCR revealed that pregeijerene increased pest mortality by attracting four species of naturally occurring EPNs in the field. Finally, we tested the generality of this root-zone signal by application of pregeijerene in blueberry fields; mortality of larvae (Galleria mellonella and Anomala orientalis) again increased by attracting naturally occurring populations of an EPN. Thus, this specific belowground signal attracts natural enemies of widespread root pests in distinct agricultural systems and may have broad potential in biological control of root pests.