Plasmodium vivax Malaria in Pregnant Women in the Brazilian Amazon and the Risk Factors Associated with Prematurity and Low Birth Weight: A Descriptive Study

Introduction

Plasmodium vivax is the most prevalent malaria species in the American region. Brazil accounts for the higher number of the malaria cases reported in pregnant women in the Americas. This study aims to describe the characteristics of pregnant women with malaria in an endemic area of the Brazilian Amazon and the risk factors associated with prematurity and low birth weight (LBW).

Methods/Principal Findings

Between December 2005 and March 2008, 503 pregnant women with malaria that attended a tertiary health centre were enrolled and followed up until delivery and reported a total of 1016 malaria episodes. More than half of study women (54%) were between 20–29 years old, and almost a third were adolescents. The prevalence of anaemia at enrolment was 59%. Most women (286/503) reported more than one malaria episode and most malaria episodes (84.5%, 846/1001) were due to P. vivax infection. Among women with only P. vivax malaria, the risk of preterm birth and low birth weight decreased in multigravidae (OR, 0.36 [95% CI, 0.16–0.82]; p = 0.015 and OR 0.24 [95% CI, 0.10–0.58]; p = 0.001, respectively). The risk of preterm birth decreased with higher maternal age (OR 0.43 [95% CI, 0.19–0.95]; p = 0.037) and among those women who reported higher antenatal care (ANC) attendance (OR, 0.32 [95% CI, 0.15–0.70]; p = 0.005).

Conclusion

This study shows that P. vivax is the prevailing species among pregnant women with malaria in the region and shows that vivax clinical malaria may represent harmful consequences for the health of the mother and their offsprings particularly on specific groups such as adolescents, primigravidae and those women with lower ANC attendance.     

Development and Validation of a Luminescence-based,Medium-Throughput Assay for Drug Screening in Schistosoma mansoni

Background

Schistosomiasis, one of the world’s greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs.

Methodology/Principal Findings

The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery.

Conclusions

The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.     

Generation of Luciferase-Expressing Leishmania infantum chagasi and Assessment of Miltefosine Efficacy in Infected Hamsters through Bioimaging

Background

The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters.

Methodology/Principal Findings

A transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival.

Conclusions/Significance

Luciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before drug treatment and therefore allowing distribution of animals in groups with equivalent mean parasite burden. Miltefosine was effective in vivo in an L. infantum chagasi experimental model of infection.     

A low-protein diet during pregnancy prevents modifications in intercellular communication proteins in rat islets

Background

Gap junctions between β-cells participate in the precise regulation of insulin secretion. Adherens junctions and their associated proteins are required for the formation, function and structural maintenance of gap junctions. Increases in the number of the gap junctions between β-cells and enhanced glucose-stimulated insulin secretion are observed during pregnancy. In contrast, protein restriction produces structural and functional alterations that result in poor insulin secretion in response to glucose. We investigated whether protein restriction during pregnancy affects the expression of mRNA and proteins involved in gap and adherens junctions in pancreatic islets. An isoenergetic low-protein diet (6% protein) was fed to non-pregnant or pregnant rats from day 1–15 of pregnancy, and rats fed an isocaloric normal-protein diet (17% protein) were used as controls.

Results

The low-protein diet reduced the levels of connexin 36 and β-catenin protein in pancreatic islets. In rats fed the control diet, pregnancy increased the levels of phospho-[Ser^279/282]-connexin 43, and it decreased the levels of connexin 36, β-catenin and beta-actin mRNA as well as the levels of connexin 36 and β-catenin protein in islets. The low-protein diet during pregnancy did not alter these mRNA and protein levels, but avoided the increase of levels of phospho-[Ser^279/282]-connexin 43 in islets. Insulin secretion in response to 8.3 mmol/L glucose was higher in pregnant rats than in non-pregnant rats, independently of the nutritional status.

Conclusion

Short-term protein restriction during pregnancy prevented the Cx43 phosphorylation, but this event did not interfer in the insulin secretion.     

Soil Bacterial Community Response to Differences in Agricultural Management along with Seasonal Changes in a Mediterranean Region

Land-use change is considered likely to be one of main drivers of biodiversity changes in grassland ecosystems. To gain insight into the impact of land use on the underlying soil bacterial communities, we aimed at determining the effects of agricultural management, along with seasonal variations, on soil bacterial community in a Mediterranean ecosystem where different land-use and plant cover types led to the creation of a soil and vegetation gradient. A set of soils subjected to different anthropogenic impact in a typical Mediterranean landscape, dominated by Quercus suber L., was examined in spring and autumn: a natural cork-oak forest, a pasture, a managed meadow, and two vineyards (ploughed and grass covered). Land uses affected the chemical and structural composition of the most stabilised fractions of soil organic matter and reduced soil C stocks and labile organic matter at both sampling season. A significant effect of land uses on bacterial community structure as well as an interaction effect between land uses and season was revealed by the EP index. Cluster analysis of culture-dependent DGGE patterns showed a different seasonal distribution of soil bacterial populations with subgroups associated to different land uses, in agreement with culture-independent T-RFLP results. Soils subjected to low human inputs (cork-oak forest and pasture) showed a more stable bacterial community than those with high human input (vineyards and managed meadow). Phylogenetic analysis revealed the predominance of Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes phyla with differences in class composition across the site, suggesting that the microbial composition changes in response to land uses. Taken altogether, our data suggest that soil bacterial communities were seasonally distinct and exhibited compositional shifts that tracked with changes in land use and soil management. These findings may contribute to future searches for bacterial bio-indicators of soil health and sustainable productivity.     

Phosphoprotein levels,MAPK activities and NFκB expression are affected by fisetin

Flavonoids, polyphenolic phytochemicals, are ubiquitous in plants and are commonly present in the human diet. They may exert diverse beneficial effects, including antioxidant and anticarcinogenic activities. The present study was designed to evaluate three biomolecules that play important roles in the apoptotic process: mitogen-activated protein kinases, protein phosphatases and NFκB, using HL60 cells treated with fisetin as an experimental model. Our results demonstrated that cells treated with fisetin presented high expression of NFκB, activation of MAPK p38 and an increase of phosphoprotein levels; inhibition of enzymes involved in redox status maintenance were also observed. Our findings reinforce the hypothesis that fisetin is likely to exert beneficial and/or toxic actions on cells not through its potential as antioxidant but rather through its modulation of protein kinase and phosphatase signaling cascades. Additionally, our results also indicate that the cellular effects of fisetin will ultimately depend on the cell type and on the extent to which they associate with the cells, either by interactions at the membrane or by uptake into the cytosol.     

High connectivity and migration potentiate the invasion of Limnoperna fortunei (Mollusca: Mytilidae) in South America

Even after almost 30 years of Limnoperna fortunei introduction into South America, it is still unclear how the source and propagules are connected. Here, we present genetic evidence of population connectivity and gene flow of L. fortunei propagules from Asia into South America, proposing the main invasion routes into South America. To achieve that we expanded the sampling effort to cover all occurrence points of L. fortunei in South America. We sequenced the mtDNA COI gene and genotyped eight microsatellite loci (ML), and we evaluated the genetic source of the recently introduced population in Sobradinho hydroelectric power plant reservoir in Northeast Brazil. Our results revealed that China is the main genetic source of propagules for the Sobradinho population. We also found COI haplotypes and ML genotypes unique to South American populations, demonstrating a bridgehead effect likely caused by local mutation, adaptation, and admixture patterns that are maintained by high levels of gene flow among them. However, two genetic barriers were also detected. We concluded that L. fortunei is a well-established invader and is still rapidly expanding in Brazil, and the Amazon hydrographic basin is under an alarming threat of invasion.

     

Leukocyte transepithelial migration in lung induced by DMSA functionalized magnetic nanoparticles

Magnetic nanoparticles surface-covered with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute a promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. However, they can also induce a transient transendothelial migration of leukocytes in the organ as a side effect after endovenous administration of MNPs-DMSA. We demonstrated that monocytes/macrophages constitute the main subpopulation of leukocytes involved in this process. Our recent research found that MNPs-DMSA upregulated the mRNA expression of E-, L- and P-selectin and macrophage-1 antigen and increased concentration of tumor necrosis factor α (TNFα) in lung, in a time dependent manner. The critical relevance of the β₂ integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our work characterizes mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung and identifies β₂ integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. In addition, MNPs-DMSA could be used as modulators of lung immune response.Key words: magnetic nanoparticles, DMSA, nanobiotechnology, transepithelial migration, cell adhesion molecules, integrins, monocytes, lungNanotechnology deals with structures of 100 nm or smaller in at least one dimension and has the potential to create many new materials and devices with a vast range of applications. Materials can be produced that are nanoscale in one dimension (for example, very thin surface coatings), in two dimensions (for example, nanowires and nanotubes) or in all three dimensions (for example, nanoparticles).Magnetic nanoparticles (MNPs) are a class of nanoparticles that can be manipulated using a magnetic field. MNPs are traditionally ferrite-based materials with the general formula MFe₂O₄, where M is a doubly charged metal-ion, such as iron, nickel or cobalt. Magnetic fluids (MFs) are colloidal mixtures composed of MNPs suspended in a carrier fluid, usually an organic or inorganic solvent. There is an increasing interest in developing biocompatible MFs for biomedical applications¹ for instance, for detection of circulating tumor cells,² contrast agents for magnetic resonance imaging³ and in an experimental cancer treatment called magnetic hyperthermia in which the fact that nanoparticles heat when they are placed in an alternative magnetic field is used.⁴ Another potential use includes attaching magnetic nanoparticles to drug/gene for targeting purposes.⁵ In order to be used for medical applications, magnetic nanoparticles are coated with a surfactant to prevent their agglomeration (due to van der Waals and magnetic forces) and allow the association of MNPs surface with different molecules.^6,7In previous studies, we have shown that MNPs surface-coated with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) (Fig. 1), with average diameter of about 9 nm, presented preferential distribution in the lung tissue, after intravenous administration in mice.^8–10 This target specificity of MNPs-DMSA offers a unique property that may be successfully exploited for the treatment of lung diseases.¹¹ In addition, we reported that the presence of MNPs-DMSA in the lung led to trafficking of leukocytes from blood vessels into pulmonary parenchyma and airspace and that interleukin-1 (IL-1) and interleukin-6 (IL-6) were overexpressed.¹² IL-1 acts as a trigger that activates a cascade of cytokine production and induces the production of a wide range of immunomodulatory cytokines.¹³ IL-6 is among the mediators regulated by IL-1 and is often increased in inflammatory processes in the lung.¹³ These differential expressions were particularly associated with blood vessels and cells of airway ducts suggesting that they could have some role during the recruitment process of inflammatory cells, as observed in histological analyses. In fact, these cytokines are commonly associated with the activation of cells concerning the expression of adhesion surface proteins.¹³ This is in agreement with several studies that described the requirement of IL-1α production in rat airways for full polymorphonuclear cell migration in models for immune-complex deposition or inhalation of cement dust, coal dust or diesel exhaust particles.^14–16Open in a separate windowFigure 1Schematic representation of DMSA-functionalized maghemite nanoparticles.Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. A mechanistic understanding of cellular interactions with synthetic surfaces, particularly in the context of inflammatory and healing responses, has been a major goal of biomaterial science.Leukocyte trafficking in the lung involves transendothelial migration, migration in tissue interstitium and transepithelial migration. In addition, leukocyte emigration involves regulatory mechanisms including complement activation, cytokine regulation, chemokine production, activation of adhesion molecules and their respective counter receptors. The process is presumably initiated and modulated by the production of early response cytokines such as IL-1 and tumor necrosis factor (TNF) from lung cells, especially from alveolar macrophages, setting the stage for leukocyte migration through endothelium.¹⁷ On the other hand, ensuing production of interleukin-10 (IL-10) brings into play powerful anti-inflammatory factors that strongly regulate inflammatory responses, functioning as intrinsic regulators of the lung inflammatory response.^18,19Tissue infiltration by circulating leukocytes is a three-step process involving rolling on the endothelium, attachment to the endothelium and transmigration across the endothelial cells lining blood vessel walls (Fig. 2). Leukocyte migration out of the blood is initiated by leukocyte rolling on the luminal side of the endothelium, as mediated by the low-affinity receptors selectins (E-, L- and P-selectin).^20–22 Binding of selectins on leukocytes stimulates “outside-in” signals in these cells, increasing the affinity of the integrin family of receptors (cell surface receptors consisting of an α- and a β-subunit, which are grouped in distinct subfamilies based on β-subunit utilization), which then bind to endothelial cell adhesion molecules such as intercellular adhesion molecule-1 [(ICAM-1)/CD54] and vascular cellular adhesion molecule-1 (VCAM-1). Function-blocking studies have identified the β₁ (CD29) and β₂ (CD18) integrins as the major players involved in leukocyte adhesion and migration.²³ Leukocyte integrin affinity is also rapidly increased by “inside-out” signals from leukocyte chemokine receptors triggered by chemokines displayed on the surface of endothelial cells.²⁴ With an increase in leukocyte integrin receptor affinity, leukocyte rolling is arrested.²⁴Open in a separate windowFigure 2Schematic representation of leukocyte endothelial migration into lung parenchyma.Using immunohistochemistry, we demonstrated that following injection of MF-DMSA, the distribution pattern of E-selectin and members of the β₂ integrin subfamily (macrophage-1 antigen, Mac-1; leukocyte function associated antigen-1, LFA-1) was changed in the lung vessels, but not of β₁ integrin.¹⁰ For L and P selectins no differences were observed between treated and control animals. However, for E-selectin, labeling was found in the endothelium of veins and venules 12 h after MF-DMSA administration, but not in the lung''s vascular compartments of the control and 4 h treatment groups.¹² Concerning integrins, in the control group, leukocytes labeled with Mac-1 and LFA-1 were found only in post-capillary sites. Four hours after MF-DMSA administration, leukocytes expressing these β₂ integrins were also found in capillaries.¹⁰ Our findings expand on other studies showing that the capillary network constitutes an important migration site in the lung.²⁵ Thus, the modulation of Mac-1 and LFA-1 expression in leukocytes located inside capillaries supports the importance of these integrins and capillaries for migratory activity in the lung, in this case after MF-DMSA administration. However, we cannot discard the participation of larger vessels in the migration induced by MNPs-DMSA. In fact, some images from our laboratory have showed that this is also a route used by the leukocytes after injection of these nanoparticles (Fig. 3).Open in a separate windowFigure 3Light microscopy image of leukocytes containing MNPs-DMSA inside a vein. Note that the cells (yellow arrows) are close or attached to the endothelium.It is worth noting that 12 h after MF-DMSA administration, leukocytes labeled with LFA-1 were observed only in post-capillary sites, similar to the control. We speculated that the absence of LFA-1 labeling in capillaries in the period of 12 h after MF-DMSA administration is due to the accentuated decrease of LFA-1 expression levels in the leukocyte over the course of time. In fact, as will be discussed below, we obtained a decrease in the LFA-1 mRNA 12 h after MNPs-DMSA administration. This point of view is in agreement with other studies that demonstrated the distinct contribution of LFA-1 and Mac-1 to transendothelial migration in the lung.²⁶ While both Mac-1 and LFA-1 participate in transendothelial migration at the beginning of the inflammatory process, over time Mac-1 becomes the predominant member of the β₂ integrin subfamily mediating migration of leukocytes.²⁶These results raised several questions related to MNPs-DMSA administration, such as: what is the time profile of leukocyte migration into the airspace? Which is the principal leukocyte subpopulation involved in this process? Is it a fact that the mechanism by which the presence of MNPs-DMSA induces transendothelial migration of leukocytes into the lung is based on their ability to somehow change the expression of cell adhesion molecules on leukocytes and lung vascular endothelial cells? Is β₂ or β₁ integrin, or both, the main receptor involved in MNPs-DMSA leukocyte-induced migration?Recently, we uncovered some of these answers including the main adhesion molecules that are involved in this migration. We first determined that the number of leukocytes in the bronchoalveolar lavage fluid reached its peak 12 h after MNPs-DMSA administration, decreasing to normal values in 48–72 h. Cytologic and FACS analysis demonstrated that the main subpopulation of leukocytes involved in this process was monocyte/macrophage.²⁷It is well known that the reticuloendothelial system, in particular macrophage cells, actively neutralizes and eliminates foreign matter from the body, including nonbiological particles. These and other particulated materials in the lung may lead to lung damage. In fact, transmission electron microscopy analysis clearly demonstrated an uptake of MNPs-DMSA by monocyte/macrophage cells,²⁷ indicating that this may be a mechanism of nanoparticle clearance used by the lung in order to avoid further damage. It is worth noting that an increase in the relative percentage of lymphocytes after MNPs-DMSA administration was also observed. The importance of this finding was not addressed in the paper, but we speculate that it could be important for the control of the inflammatory process initiated by the MNPs-DMSA injection. Failure in control of the inflammatory processes could potentially lead to chronic inflammatory diseases and pulmonary fibrosis. In spite of the fact that we did not determine which was the main source of the production of two different cytokines, one considered pro-inflammatory (TNFα) and the other anti-inflammatory (IL-10), we found an increase in the ratio of IL-10/TNFα cytokine release 12 h after MNPs-DMSA administration. This is clearly a signal that the inflammatory process was being controlled, in agreement with previous reports showing that IL-10 is able to limit the induction of cell adhesion molecules in the lung.²⁸ We presume that lymphocytes are taking part in this process. Further studies are necessary to clarify this point.The nature of the cells present in the pulmonary tissue parenchyma was not determined in this study. However, these cells were not able to cause tissue damage in the lung. We observed no histological or ultrastructural damage in the lung of animals treated with MNPs-DMSA, indicating that the nanoparticle-induced inflammation is not enough to cause chronic disease, such as pulmonary fibrosis.We then determined the effect of MNPs-DMSA on mRNA expression of selectins, integrin β₁ and integrin β₂.²⁷ We found that MNPs-DMSA upregulated the mRNA expression of E-, L- and P-selectin, as well as Mac-1. Further, using knockout mice (deficient in the β₂-subunit common to all β₂ integrins), we observed that, compared to wild-type mice, the recruitment of leukocytes to the airspace following administration of MNPs-DMSA was completely blocked in the former.²⁷ The fact that transmigration of β₂ integrin-deficient monocytes was affected when compared with wild-type monocytes strongly argues in favor of a major contribution by β₂ integrins to monocyte trans-epithelial migration in our system, which is additionally supported by the increase of mRNA of β₂ integrins, as cited above.We should remember, however, that the absence of change in LFA-1 and very late antigen-4 (VLA-4) mRNA does not exclude a role for them in leukocyte migration induced by MNPs-DMSA. Integrins are cell adhesion molecules constitutively expressed on the cell surface and also stored within intracellular vesicles.^29,30 In addition, transendothelial migration of leukocytes depends not only on the number of integrins on the cell surface but also on the change in conformation of these molecules reflecting their activation.³² Therefore, our results did not exclude the possibility that MNPs-DMSA induce the activation of LFA-1 and VLA-4 constitutively located on the surface of leukocytes or the translocation of these integrins from intracellular vesicles to the plasma membrane. On the other hand, the absence of a significant change in the mRNA expression of VCAM-1, which is the major endothelial cell ligand for VLA-4, can be regarded as an indirect indicator that VLA-4 is not involved in this process.The fact that an increase in the mRNA of Mac-1 occurred and there is no change in the mRNA levels of VLA-4 (and LFA-1) corroborates the hypothesis that migration of leukocytes induced by MNPs-DMSA is mainly dependent on β₂ integrins and not β₁ integrins pathway. In addition, we can presume that MAC-1 is the main β₂ integrin molecule involved in the process of leukocyte trafficking.The increased use of nanoparticles in medicine has raised concerns on their ability to gain access to privileged sites in the body. In fact, a study has shown that, in some cases, they can potentially cause damage to tissues located behind cellular barriers. Therefore, it is fundamental to understand the mechanisms underlying interactions between nanoparticles and the body, for their safe and effective use. In the case of MNPs-DMSA, we can use this knowledge for treatment of lung diseases when associated with drugs, as well as for downregulation or upregulation of the local immune system.One important question still unanswered about the use of magnetic nanoparticles in lung disease treatments is what could be expected if more than one dose is necessary in a short period of time. Recent research of Mejias et al.³¹ was close to answer this question. In their study the authors injected repeated doses (nine in total) of magnetic nanoparticles stabilized with DMSA, but unfortunately, they did not analyze the lungs, assuming that the particles would be stocked in the liver, spleen and kidney. For these organs, however, the authors did not refer to any observed damage. We believe that the answer to this question is related with several factors such as physical-chemical features of the nanoparticles (size, hydrodynamic radius, etc.) interval between the injections, amount of iron injected, among others. These features are also important for a second open question: what happens if the organ has a preexistent disease? Further studies are necessary to clarify this point. It is important to minimize, in all cases, the amount of injected iron, increasing, when possible, the amount of drug attached to the nanoparticles. The use of magnetic nanoparticles is already a reality as a contrast agent. It is possible that in the future they also can be used as drug delivery carriers.In resume our work characterizes mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung and identifies β₂ integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. In addition, MNPs-DMSA could be used as modulators of lung immune response.     

Non Ionising Radiation as a Non Chemical Strategy in Regenerative Medicine: Ca2+-ICR “In Vitro” Effect on Neuronal Differentiation and Tumorigenicity Modulation in NT2 Cells

In regenerative medicine finding a new method for cell differentiation without pharmacological treatment or gene modification and minimal cell manipulation is a challenging goal. In this work we reported a neuronal induced differentiation and consequent reduction of tumorigenicity in NT2 human pluripotent embryonal carcinoma cells exposed to an extremely low frequency electromagnetic field (ELF-EMF), matching the cyclotron frequency corresponding to the charge/mass ratio of calcium ion (Ca²⁺-ICR). These cells, capable of differentiating into post-mitotic neurons following treatment with Retinoic Acid (RA), were placed in a solenoid and exposed for 5 weeks to Ca²⁺-ICR. The solenoid was installed in a μ-metal shielded room to avoid the effect of the geomagnetic field and obtained totally controlled and reproducible conditions. Contrast microscopy analysis reveled, in the NT2 exposed cells, an important change in shape and morphology with the outgrowth of neuritic-like structures together with a lower proliferation rate and metabolic activity alike those found in the RA treated cells. A significant up-regulation of early and late neuronal differentiation markers and a significant down-regulation of the transforming growth factor-α (TGF-α) and the fibroblast growth factor-4 (FGF-4) were also observed in the exposed cells. The decreased protein expression of the transforming gene Cripto-1 and the reduced capability of the exposed NT2 cells to form colonies in soft agar supported these last results. In conclusion, our findings demonstrate that the Ca²⁺-ICR frequency is able to induce differentiation and reduction of tumorigenicity in NT2 exposed cells suggesting a new potential therapeutic use in regenerative medicine.     

Impact of Oil on Bacterial Community Structure in Bioturbated Sediments

Oil spills threaten coastlines where biological processes supply essential ecosystem services. Therefore, it is crucial to understand how oil influences the microbial communities in sediments that play key roles in ecosystem functioning. Ecosystems such as sediments are characterized by intensive bioturbation due to burrowing macrofauna that may modify the microbial metabolisms. It is thus essential to consider the bioturbation when determining the impact of oil on microbial communities. In this study, an experimental laboratory device maintaining pristine collected mudflat sediments in microcosms closer to true environmental conditions – with tidal cycles and natural seawater – was used to simulate an oil spill under bioturbation conditions. Different conditions were applied to the microcosms including an addition of: standardized oil (Blend Arabian Light crude oil, 25.6 mg.g⁻¹ wet sediment), the common burrowing organism Hediste (Nereis) diversicolor and both the oil and H. diversicolor. The addition of H. diversicolor and its associated bioturbation did not affect the removal of petroleum hydrocarbons. After 270 days, 60% of hydrocarbons had been removed in all microcosms irrespective of the H. diversicolor addition. However, 16S-rRNA gene and 16S-cDNA T-RFLP and RT-PCR-amplicon libraries analysis showed an effect of the condition on the bacterial community structure, composition, and dynamics, supported by PerMANOVA analysis. The 16S-cDNA libraries from microcosms where H. diversicolor was added (oiled and un-oiled) showed a marked dominance of sequences related to Gammaproteobacteria. However, in the oiled-library sequences associated to Deltaproteobacteria and Bacteroidetes were also highly represented. The 16S-cDNA libraries from oiled-microcosms (with and without H. diversicolor addition) revealed two distinct microbial communities characterized by different phylotypes associated to known hydrocarbonoclastic bacteria and dominated by Gammaproteobacteria and Deltaproteobacteria. In the oiled-microcosms, the addition of H. diversicolor reduced the phylotype-richness, sequences associated to Actinobacteria, Firmicutes and Plantomycetes were not detected. These observations highlight the influence of the bioturbation on the bacterial community structure without affecting the biodegradation capacities.