Foraging activity of bumblebees (Bombus terrestris L.) on Bt-expressing eggplants

A greenhouse experiment was setup to study foraging behavior of the bumblebee Bombus terrestris L. on Cry3Bb-expressing genetically modified (GM) eggplants and their near-isogenic control. Commonly, more bumblebees visited GM eggplants compared to near-isogenic control, but this difference was only marginally significant. The mean length of feeding bouts was similar on the two treatments. Neither the number of flowers produced nor their size could explain bumblebees?? tendency to prefer GM eggplants. Volatile compounds were extracted from five plants per genotype and separated using gas chromatography. Thirteen compounds were identified and five of them appeared significantly more abundant in GM eggplants. Six of the identified compounds [(+)-limonene, Z-jasmone, p-cymene, ??-pinene, methyl-salicilate, and (?)-limonene] were tested in electrophysiological bioassays with antennas detached from young bumblebees, and a response was recorded in all six cases. Experimental results indicate that pollination activity of bumblebees is compatible with this GM eggplant event as a food source and that chemical cues may have an important role in plant identification. The implications for environmental risk assessment of GM plants are discussed.     

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.     

Polymorphisms in STK17A gene are associated with systemic lupus erythematosus and its clinical manifestations

Systemic lupus erythematosus (SLE) is an autoimmune disorder with several clinical manifestations. SLE etiology has a strong genetic component, which plays a key role in disease's predisposition, as well as participation of environmental factors, such and UV light exposure. In this regard, we investigated whether polymorphisms in STK17A, a DNA repair related gene, encoding for serine/threonine-protein kinase 17A, are associated with SLE susceptibility. A total of 143 SLE patients and 177 healthy controls from Southern Brazil were genotyped for five STK17A TagSNPs. Our results indicated association of rs7805969 SNP (A and G/A genotype, OR = 1.40 and OR = 1.73, respectively) with SLE predisposition and the following clinical manifestations: arthritis, cutaneous and immunological alterations. When analyzing haplotypes distribution, we found association between TGGTC, TAGTC and AAGAT haplotypes and risk to develop SLE. When considering clinical manifestations, the haplotypes TGGTT and TAGTC were associated with protection against cutaneous alterations and the haplotype TAGTC to hematological alterations. We also observed association between SLE clinical manifestations and ethnicity, with the European-derived patients being more susceptible to cutaneous and hematological alterations.     

DASH: a method for identical-by-descent haplotype mapping uncovers association with recent variation

Rare variants affecting phenotype pose a unique challenge for human genetics. Although genome-wide association studies have successfully detected many common causal variants, they are underpowered in identifying disease variants that are too rare or population-specific to be imputed from a general reference panel and thus are poorly represented on commercial SNP arrays. We set out to overcome these challenges and detect association between disease and rare alleles using SNP arrays by relying on long stretches of genomic sharing that are identical by descent. We have developed an algorithm, DASH, which builds upon pairwise identical-by-descent shared segments to infer clusters of individuals likely to be sharing a single haplotype. DASH constructs a graph with nodes representing individuals and links on the basis of such segments spanning a locus and uses an iterative minimum cut algorithm to identify densely connected components. We have applied DASH to simulated data and diverse GWAS data sets by constructing haplotype clusters and testing them for association. In simulations we show this approach to be significantly more powerful than single-marker testing in an isolated population that is from Kosrae, Federated States of Micronesia and has abundant IBD, and we provide orthogonal information for rare, recent variants in the outbred Wellcome Trust Case-Control Consortium (WTCCC) data. In both cohorts, we identified a number of haplotype associations, five such loci in the WTCCC data and ten in the isolated, that were conditionally significant beyond any individual nearby markers. We have replicated one of these loci in an independent European cohort and identified putative structural changes in low-pass whole-genome sequence of the cluster carriers.     

Advanced oxidation processes applied for color removal of textile effluent using a home-made peroxidase from rice bran

Bioprocess and Biosystems Engineering - Enzymes are becoming tools in industrial processes because of several advantages, including activity in mild environmental conditions, and high specificity....     

Nitric oxide content is associated with tolerance to bicarbonate-induced chlorosis in micropropagated Prunus explants

Iron (Fe) chlorosis is a common nutritional deficiency in fruit trees grown in calcareous soils. Grafting on tolerant rootstocks is the most efficient practice to cope with it. In the present work, three Prunus hybrid genotypes, commonly used as peach rootstocks, and one peach cultivar were cultivated with bicarbonate in the growth medium. Parameters describing oxidative stress and the metabolism of reactive nitrogen species were studied. Lower contents of nitric oxide and a decreased nitrosoglutathione reductase activity were found in the most sensitive genotypes, characterized by higher oxidative stress and reduced antioxidant defense. In the peach cultivar, which behaved as a tolerant genotype, a specifically nitrated polypeptide was found.     

Structure–activity relationships of various amino-hydroxy-benzenesulfonic acids and sulfonamides as tyrosinase substrates

Background

o-Aminophenols have been long recognised as tyrosinase substrates. However their exact mode of interaction with the enzyme's active site is unclear. Properly vic-substituted o-aminophenols could help gain some insight into tyrosinase catalytic mechanism.

Methods

Eight vic-substituted o-aminophenols belonging to two isomeric series were systematically evaluated as tyrosinase substrates and/or activators and/or inhibitors, by means of spectrophotometric techniques and HPLC-MS analysis. Some relevant kinetic parameters have also been obtained.

Results

Four o-aminophenolic compounds derived from 3-hydroxyorthanilic acid (2-amino-3-hydroxybenzenesulfonic acid) and their four counterparts derived from the isomeric 2-hydroxymetanilic acid (3-amino-2-hydroxybenzenesulfonic acid) were synthesised and tested as putative substrates for mushroom tyrosinase. While the hydroxyorthanilic derivatives were quite inactive as both substrates and inhibitors, the hydroxymetanilic compounds on the contrary all acted as substrates for the enzyme, which oxidised them to the corresponding phenoxazinone derivatives.

General significance

Based on the available structures of the active sites of tyrosinases, the different affinities of the four metanilic derivatives for the enzyme, and their oxidation rates, we propose a new hypothesis regarding the interaction between o-aminophenols and the active site of tyrosinase that is in agreement with the obtained experimental results.     

Transport of animals between rooms: a little-noted aspect of laboratory procedure that may interfere with memory

This study investigated the effects of transporting animals from the experimental room to the animal facility in between experimental sessions, a procedure routinely employed in experimental research, on long-term social recognition memory. By using the intruder-resident paradigm, independent groups of Wistar rats exposed to a 2-h encounter with an adult intruder were transported from the experimental room to the animal facility either 0.5 or 6 h after the encounter. The following day, residents were exposed to a second encounter with either the same or a different (unfamiliar) intruder. Resident's social and non-social behaviors were carefully scored and subjected to Principal Component Analysis, thus allowing to parcel out variance and relatedness among these behaviors. Resident rats transported 6 h after the first encounter exhibited reduced amount of social investigation towards familiar intruders, but an increase of social investigation when exposed to a different intruder as compared to the first encounter. These effects revealed a consistent long-lasting (24 h) social recognition memory in rats. In contrast, resident rats transported 0.5 h after the first encounter did not exhibit social recognition memory. These results indicate that this common, little-noted, laboratory procedure disturbs long-term social recognition memory.     

External parasites of raptors (Falconiformes and Strigiformes): identification in an ex situ population from Mexico

Raptorial birds harbor a variety of ectoparasites and the mayority of them are host specific. The aim of this study was to identify the ectoparasites of captive birds of prey from Mexico, as well as to verify their impact in the health of infested birds. Raptorial birds were confiscated and kept in captivity at the Centro de Investigación y Conservación de Vida Silvestre (CIVS) in Los Reyes La Paz, Mexico State. Seventy-four birds of prey (66 Falconiformes and eigth Strigiformes) of 15 species were examined for the presence of ectoparasites. We examined both juvenile and adult birds from both sexes. The overall prevalence was 16.2%; 66.7% of raptors were infested with a single type of external parasite. Lice were the most prevalent ectoparasites (91.7%), followed by feather mites and fleas (8.3%). Degeeriella fulva (72.7%), Craspedorrhynchus sp. (45.4%) and Strigiphilus aitkeni (9.1%) (Ischnocera, Philopteridae) were recovered from wings, head and neck regions of red-tailed hawk (Buteo jamaicensis), Swainson's hawk (B. swainsoni), Harris's hawk (Parabuteo unicinctus) and Barn owl (Tyto alba). Low lice infestation level was observed. Nymphs and females of feather mites Kramerella sp. (Pterolichoidea, Kramerellidae) were recovered solely from Barn owl (T. alba); while one Caracara (Caracara cheriway) was infested by the sticktight flea Echidnophaga gallinacea (Siphonaptera, Pulicidae). No clinical signs were observed in any infested bird. Probably the periodic use of organophosphorates was responsible of the low prevalence and lice infestation levels. The diversity of external parasites illustrates the importance of detailed revision of incoming and long-term captive raptors as part of responsible captive management. Five new hosts and geographic records are presented.     

Chromosomal size conservation through the cell cycle supports karyotype stability in Trypanosoma cruzi

The Trypanosoma cruzi karyotype shows an extensive chromosomal size polymorphism. Absence of condensed mitotic chromosomes and chromatin fragility are characteristic features of T. cruzi which would allow DNA breaks and chromosomal rearrangements during cell proliferation. We have investigated by pulsed field gel electrophoresis (PFGE) eventual changes in chromosomal size during exponential and stationary phases of T. cruzi epimastigotes in culture, in G0 trypomastigotes and throughout the cell cycle in synchronized epimastigotes. T. cruzi molecular karyotype was stable throughout the cell cycle and during differentiation. Thus, the chromosomal size polymorphism previously reported in T. cruzi contrasts with the stability of the molecular karyotype observed here and suggests that chromosomal rearrangements leading to changes in chromosomal size are scarce events during the clonal propagation of this parasite.