Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.     

The new kappa-KTx 2.5 from the scorpion Opisthacanthus cayaporum

The kappa-KTx family of peptides, which is the newest K⁺-channel blocker family from scorpion venom, is present in scorpions from the families Scorpionidae and Liochelidae. Differently from the other scorpion KTx families, the three-dimensional structure of the known kappa-KTxs toxins is formed by two parallel α-helices linked by two disulfide bridges. Here, the characterization of a new kappa-KTx peptide, designated kappa-KTx 2.5, derived from the Liochelidae scorpion Opisthacanthus cayaporum, is described. This peptide was purified by HPLC and found to be identical to OcyC8, a predicted mature sequence precursor (UniProtKB C5J89) previously described by our group. The peptide was chemically synthesized and the circular dichroism (CD) spectra of both, native and synthetic, conducted at different temperatures in water and water/trifluoroethanol (TFE), showed a predominance of α-helices. The kappa-KTx 2.5 is heat stable and was shown to be a blocker of K⁺-currents on hKv1.1, and hKv1.4, with higher affinity for Kv1.4 channels (IC₅₀ = 71 μM). Similarly to the other kappa-KTxs, the blockade of K⁺-channels occurred at micromolar concentrations, leading to uncertainness about their proper molecular target, and consequently their pharmacologic effect. In order to test other targets, kappa-KTx2.5 was tested on other K⁺-channels, on Na⁺-channels, on bacterial growth and on smooth muscle tissue, a known assay to identify possible bradykinin-potentiating peptides, due to the presence of two contiguous prolines at the C-terminal sequence. It has no effect on the targets used except on hKv1.1, and hKv1.4 expressed in Chinese hamster ovary cells. Since the only plausible function found for kappa-KTx2.5 seems to be the blockade of K⁺-channels, a discussion regarding the analysis of structure-function relationships is included in this communication, based on sequence alignments of members of the kappa-KTx toxin family, and on computational simulation of a structural model of the kappa-KTx2.5-Kv1.2 complex.     

Isolation of Helicobacter pylori from the intestinal mucosa of patients with Crohn's disease

BACKGROUND: Helicobacter species are associated with inflammatory bowel disease in rodents and in nonhuman primates. Therefore, we prospectively investigated the presence of Helicobacter species in the intestinal mucosa of patients with and without Crohn's disease by culture and polymerase chain reaction (PCR) assays. MATERIALS AND METHODS: Mucosal fragments were obtained from the ileum, different colon regions, and rectum of 43 patients with Crohn's disease and of 74 patients without inflammatory bowel disease. RESULTS: Helicobacter pylori strains, identified by 16S rRNA gene sequencing, were more frequently isolated and PCR-detected in the intestinal mucosa of patients with ulcerative colitis-like Crohn's disease than in intestinal mucosa of the control group. Otherwise, anti-H. pylori immunoglobulin G levels were significantly lower in fibrostenosing and fistulating Crohn's disease subgroups. No other Helicobacter species were found in the intestinal mucosa of the patients. CONCLUSIONS: Although our results suggest an association between the presence of H. pylori in the intestine and ulcerative colitis-like phenotype of Crohn's disease, H. pylori infection in the actual causality of Crohn's disease is still to be determined.     

When phylogeny and ecology meet: Modeling the occurrence of Trichoptera with environmental and phylogenetic data

Ecological studies are increasingly considering phylogenetic relationships among species. The phylogeny is used as a proxy or filter to improve statistical tests and retain evolutionary elements, such as niche conservation. We used the phylogenetic topology to improve the model for occurrence of Trichoptera genera in Cerrado (Brazilian Savanna) streams. We tested whether parameters generated by logistic models of occurrence, using phylogenetic signals, are better than models generated without phylogenetic information. We used a model with Bayesian updating to examine the influence of stream water pH and phylogenetic relationship among genera on the occurrence of Trichoptera genera. Then, we compared this model with the logistic model for each Trichoptera genus. The probability of occurrence of most genera increased with water pH, and the phylogeny‐based explicit logistic model improved the parameters estimated for observed genera. The inferred relationship between genera occurrence and stream pH improved, indicating that phylogeny adds relevant information when estimating ecological responses of organisms. Water with elevated acidity (low pH values) may be restrictive for the occurrence of Trichoptera larvae, especially if the regional streams exhibit neutral to alkaline water, as is observed in the Cerrado region. Using phylogeny‐based modeling to predict species occurrence is a prominent opportunity to extend our current statistical framework based on environmental conditions, as it enables a more precise estimation of ecological parameters.     

Rat heart GDNF: effect of chemical sympathectomy

Developmental studies indicate a role for GDNF in survival of motor, autonomic, and sensory neurons. However, no study attempted to demonstrate its participation in autonomic nerve regeneration. In this work, chemical sympathectomy by 6-hydroxydopamine provided the model for assessing heart GDNF expression during denervation and axonal regrowth. A glyoxylic acid-based histochemical technique evaluated the noradrenergic innervation. ELISA determined GDNF levels after concentrating heart homogenates. Light and ultrastructural in situ hybridization and immunocytochemistry were used for identifying cells expressing GDNF mRNA and protein. In control rats, the GDNF cardiac levels were significantly higher in 37-day-old animals in comparison with those aging 60 days. In sympathectomized rats, GDNF cardiac levels were significantly higher 7 days after sympathectomy and dropped to control levels at day 30. GDNF mRNA was expressed in atrial and ventricular myocytes from normal and sympathectomized rats. GDNF immunoreactivity occurred on atrial granules and quantitative analysis in electron micrographs confirmed ELISA-obtained data. In ventricular myocytes gold particles occurred sparsely. These findings constitute the first evidence for GDNF synthesis by cardiomyocytes and postulate a role for this factor soon after cardiac sympathetic denervation, probably in nerve regeneration. In atrial myocytes, GDNF is probably secreted by regulated pathway.     

Synchronized activation of sympathetic vasomotor, cardiac, and respiratory outputs by neurons in the midbrain colliculi

The superior and inferior colliculi are believed to generate immediate and highly coordinated defensive behavioral responses to threatening visual and auditory stimuli. Activation of neurons in the superior and inferior colliculi have been shown to evoke increases in cardiovascular and respiratory activity, which may be components of more generalized stereotyped behavioral responses. In this study, we examined the possibility that there are "command neurons" within the colliculi that can simultaneously drive sympathetic and respiratory outputs. In anesthetized rats, microinjections of bicuculline (a GABA(A) receptor antagonist) into sites within a circumscribed region in the deep layers of the superior colliculus and in the central and external nuclei of the inferior colliculus evoked a response characterized by intense and highly synchronized bursts of renal sympathetic nerve activity (RSNA) and phrenic nerve activity (PNA). Each burst of RSNA had a duration of ～300-400 ms and occurred slightly later (peak to peak latency of 41 ± 8 ms) than the corresponding burst of PNA. The bursts of RSNA and PNA were also accompanied by transient increases in arterial pressure and, in most cases, heart rate. Synchronized bursts of RSNA and PNA were also evoked after neuromuscular blockade, artificial ventilation, and vagotomy and so were not dependent on afferent feedback from the lungs. We propose that the synchronized sympathetic-respiratory responses are driven by a common population of neurons, which may normally be activated by an acute threatening stimulus.     

Terpenes Increase the Lipid Dynamics in the Leishmania Plasma Membrane at Concentrations Similar to Their IC50 Values

Although many terpenes have shown antitumor, antibacterial, antifungal, and antiparasitic activity, the mechanism of action is not well established. Electron paramagnetic resonance (EPR) spectroscopy of the spin-labeled 5-doxyl stearic acid revealed remarkable fluidity increases in the plasma membrane of terpene-treated Leishmania amazonensis promastigotes. For an antiproliferative activity assay using 5×10⁶ parasites/mL, the sesquiterpene nerolidol and the monoterpenes (+)-limonene, α-terpineol and 1,8-cineole inhibited the growth of the parasites with IC₅₀ values of 0.008, 0.549, 0.678 and 4.697 mM, respectively. The IC₅₀ values of these terpenes increased as the parasite concentration used in the cytotoxicity assay increased, and this behavior was examined using a theoretical treatment of the experimental data. Cytotoxicity tests with the same parasite concentration as in the EPR experiments revealed a correlation between the IC₅₀ values of the terpenes and the concentrations at which they altered the membrane fluidity. In addition, the terpenes induced small amounts of cell lysis (4–9%) at their respective IC₅₀ values. For assays with high cell concentrations (2×10⁹ parasites/mL), the incorporation of terpene into the cell membrane was very fast, and the IC₅₀ values observed for 24 h and 5 min-incubation periods were not significantly different. Taken together, these results suggest that terpene cytotoxicity is associated with the attack on the plasma membrane of the parasite. The in vitro cytotoxicity of nerolidol was similar to that of miltefosine, and nerolidol has high hydrophobicity; thus, nerolidol might be used in drug delivery systems, such as lipid nanoparticles to treat leishmaniasis.     

Digoxin reduces the mutagenic effects of Mitomycin C in human and rodent cell lines

Digoxin is a drug widely used to treat heart failure and studies have demonstrated its potential as anticancer agent. In addition, digoxin presents the potential to interact with a series of other compounds used in medicine. The aim of the present study was to evaluate in vitro the cytotoxicity, genotoxicity and mutagenicity of digoxin and its potential to interact with the mutagen Mitomycin C (MMC). The cytotoxicity of digoxin was assessed by employing the MTT method and the comet assay was performed to assess the genotoxicity of this medicine in CHO-K1 and HeLa cell lines. Besides, the cytokinesis-block micronucleus assay was performed to assess the mutagenicity and the antimutagenicity of this drug. The Ames assay was also performed with TA98 and TA100 strains of S. typhimurium. Results showed that digoxin was cytotoxic, genotoxic and mutagenic for HeLa and CHO-K1 cell lines at concentrations many times higher than those observed in human therapeutic conditions. Nevertheless, an antimutagenic effect against the mutagen MMC was observed on both cell lines in concentrations near those used therapeutically in humans. This chemoprotective effect observed is an interesting finding that should be better explored regarding its impact in anticancer chemotherapy.     

Iron Status and Helicobacter pylori Infection in Symptomatic Children: An International Multi-Centered Study

Objective

Iron deficiency (ID) and iron deficiency anaemia (IDA) are global major public health problems, particularly in developing countries. Whilst an association between H. pylori infection and ID/IDA has been proposed in the literature, currently there is no consensus. We studied the effects of H. pylori infection on ID/IDA in a cohort of children undergoing upper gastrointestinal endoscopy for upper abdominal pain in two developing and one developed country.

Methods

In total 311 children (mean age 10.7±3.2 years) from Latin America - Belo Horizonte/Brazil (n = 125), Santiago/Chile (n = 105) - and London/UK (n = 81), were studied. Gastric and duodenal biopsies were obtained for evaluation of histology and H. pylori status and blood samples for parameters of ID/IDA.

Results

The prevalence of H. pylori infection was 27.7% being significantly higher (p<0.001) in Latin America (35%) than in UK (7%). Multiple linear regression models revealed H. pylori infection as a significant predictor of low ferritin and haemoglobin concentrations in children from Latin-America. A negative correlation was observed between MCV (r = −0.26; p = 0.01) and MCH (r = −0.27; p = 0.01) values and the degree of antral chronic inflammation, and between MCH and the degree of corpus chronic (r = −0.29, p = 0.008) and active (r = −0.27, p = 0.002) inflammation.

Conclusions

This study demonstrates that H. pylori infection in children influences the serum ferritin and haemoglobin concentrations, markers of early depletion of iron stores and anaemia respectively.