Evaluation of Blast Furnace Slag as a Means of Reducing Metal Availability in a Contaminated Sediment for Beneficial Use Purposes

An attractive option for the management of dredged sediment involves the use of dredged sediment for beneficial use purposes, such as for fill material. Treatment (chemical amendment) of contaminated sediment may be necessary to limit the environmental and human availability (bioaccessibility, leachability, plant uptake) of heavy metals associated with the contaminated sediment before it is placed. A laboratory study was conducted to investigate the effect of admixing a specific chemical amendment (blast furnace slag) with slightly contaminated fresh-water sediment for reducing metal availability. Initial characterization tests of the un-amended sediment showed that the some of the metals analyzed were present in relatively available (non-residual) forms. Although sulfide was present in the un-amended sediment, the amount was not sufficient to bind all of the available metals. A series of metal availability testing methods indicated that the amendment of the sediment with blast furnace slag (4% on a dry weight ratio basis) had the potential to slightly reduce the availability of some, but not all of the available metals associated with the sediment. Results of the column and batch leaching tests showed that leachability of certain metals, such as barium, nickel and zinc, was reduced by the amendment, but the leachability of copper increased. The effect of the amendment for decreasing bioaccessibility for lead and arsenic was not demonstrated. The amended soil had a detrimental effect on most of the plant species that were evaluated. The metal availability results for the plant uptake tests were also mixed, with slightly lower uptake of certain metals by corn grown within the amended sediment.     

Action Potential Morphology of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Does Not Predict Cardiac Chamber Specificity and Is Dependent on Cell Density

Previous studies investigating human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have proposed the distinction of heart chamber-specific (atrial, ventricular, pacemaker) electrophysiological phenotypes based on action potential (AP) morphology. This suggestion has been based on data acquired using techniques that allow measurements from only a small number of cells and at low seeding densities. It has also been observed that density of culture affects the properties of iPSC-CMs. Here we systematically analyze AP morphology from iPSC-CMs at two seeding densities: 60,000 cells/well (confluent monolayer) and 15,000 cells/well (sparsely-seeded) using a noninvasive optical method. The confluent cells (n = 360) demonstrate a series of AP morphologies on a normally distributed spectrum with no evidence for specific subpopulations. The AP morphologies of sparsely seeded cells (n = 32) displayed a significantly different distribution, but even in this case there is no clear evidence of chamber-specificity. Reduction in gap junction conductance using carbenoxolone only minimally affected APD distribution in confluent cells. These data suggest that iPSC-CMs possess a sui generis AP morphology, and when observed in different seeding densities may encompass any shape including those resembling chamber-specific subtypes. These results may be explained by different functional maturation due to culture conditions.     

Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis

We previously reported that i) a Western diet increased levels of unsaturated lysophosphatidic acid (LPA) in small intestine and plasma of LDL receptor null (LDLR^−/−) mice, and ii) supplementing standard mouse chow with unsaturated (but not saturated) LPA produced dyslipidemia and inflammation. Here we report that supplementing chow with unsaturated (but not saturated) LPA resulted in aortic atherosclerosis, which was ameliorated by adding transgenic 6F tomatoes. Supplementing chow with lysophosphatidylcholine (LysoPC) 18:1 (but not LysoPC 18:0) resulted in dyslipidemia similar to that seen on adding LPA 18:1 to chow. PF8380 (a specific inhibitor of autotaxin) significantly ameliorated the LysoPC 18:1-induced dyslipidemia. Supplementing chow with LysoPC 18:1 dramatically increased the levels of unsaturated LPA species in small intestine, liver, and plasma, and the increase was significantly ameliorated by PF8380 indicating that the conversion of LysoPC 18:1 to LPA 18:1 was autotaxin dependent. Adding LysoPC 18:0 to chow increased levels of LPA 18:0 in small intestine, liver, and plasma but was not altered by PF8380 indicating that conversion of LysoPC 18:0 to LPA 18:0 was autotaxin independent. We conclude that i) intestinally derived unsaturated (but not saturated) LPA can cause atherosclerosis in LDLR^−/− mice, and ii) autotaxin mediates the conversion of unsaturated (but not saturated) LysoPC to LPA.     

Highly active antiretroviral therapy drug combination induces oxidative stress and mitochondrial dysfunction in immortalized human blood-brain barrier endothelial cells

The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, the prevalence of HIV-1-associated neurocognitive disorders has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3'-azido-2',3'-deoxythymidine (AZT) and indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating this condition. The viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72h treatment, in a dose-dependent manner. Reactive oxygen species were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione and malondialdehyde, were altered in the treated groups. Loss of mitochondrial membrane potential, as assessed by fluorescence microscopy and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT+IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with the thiol antioxidant N-acetylcysteine amide reversed some of the pro-oxidant effects of AZT+IDV. Results from our in vitro studies indicate that the AZT+IDV combination may affect the BBB in HIV-infected individuals treated with HAART drugs.     

Glutathione signaling acts through NPR1-dependent SA-mediated pathway to mitigate biotic stress

Glutathione (GSH) has widely been known to be a multifunctional molecule especially as an antioxidant up until now, but has found a new role in plant defense signaling. Research from the past three decades indicate that GSH is a player in pathogen defense in plants, but the mechanism underlying this has not been elucidated fully. We have recently shown that GSH acts as a signaling molecule and mitigates biotic stress through non-expressor of PR genes 1 (NPR1)-dependent salicylic acid (SA)-mediated pathway. Transgenic tobacco with enhanced level of GSH (NtGB lines) was found to synthesize more SA, was capable of enhanced expression of genes belonging to NPR1-dependent SA-mediated pathway, were resistant to Pseudomonas syringae, the biotrophic pathogen and many SA-related proteins were upregulated. These results gathered experimental evidence on the mechanism through which GSH combats biotic stress. In continuation with our previous investigation we show here that the expression of glutathione S-transferase (GST), the NPR1-independent SA-mediated gene was unchanged in transgenic tobacco with enhanced level of GSH as compared to wild-type plants. Additionally, the transgenic plants were barely resistant to Botrytis cinerea, the necrotrophic pathogen. SA-treatment led to enhanced level of expression of pathogenesis-related protein gene (PR1) and PR4 as against short-chain dehydrogenase/reductase family protein (SDRLP) and allene oxide synthase (AOS). These data provided significant insight into the involvement of GSH in NPR1-dependent SA-mediated pathway in mitigating biotic stress.Key words: GSH, signaling molecule, biotrophic pathogen, NPR-1, PR-1, PR-4, transgenic tobaccoPlant responses to different environmental stresses are achieved through integrating shared signaling networks and mediated by the synergistic or antagonistic interactions with the phytohormones viz. SA, jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and reactive oxygen species (ROS).¹ Previous studies have shown that in response to pathogen attack, plants produce a highly specific blend of SA, JA and ET, resulting in the activation of distinct sets of defense-related genes.^2,3 Regulatory functions for ROS in defense, with a focus on the response to pathogen infection occur in conjunction with other plant signaling molecules, particularly with SA and nitric oxide (NO).^4–6 Till date, numerous physiological functions have been attributed to GSH in plants.^7–11 In addition to previous studies, recent study has also shown that GSH acts as a signaling molecule in combating biotic stress through NPR1-dependent SA-mediated pathway.^12,13Our recent investigation involved raising of transgenic tobacco overexpressing gamma-glutamylcysteine synthetase (γ-ECS), the rate-limiting enzyme of the GSH biosynthetic pathway.¹² The stable integration and enhanced expression of the transgene at the mRNA as well as protein level was confirmed by Southern blot, quantitative RT-PCR and western blot analysis respectively. The transgenic plants of the T₂ generation (Fig. 1), the phenotype of which was similar to that of wild-type plants were found to be capable of synthesizing enhanced amount of GSH as confirmed by HPLC analysis.Open in a separate windowFigure 1Transgenic tobacco of T₂ generation, (A) three-week-old plant, (B) mature plant.In the present study, the expression profile of GST was analyzed in NtGB lines by quantitative RT-PCR (qRT-PCR) and found that the expression level of this gene is unchanged in NtGB lines as compared to wild-type plants (Fig. 2). GST is known to be a NPR1-independent SA-related gene.¹⁴ This suggests that GSH does not follow the NPR1-independent SA-mediated pathway in defense signaling.Open in a separate windowFigure 2Expression pattern of GST in wild-type and NtGB lines.Disease test assay with NtGB lines and wild-type plants was performed using B. cinerea and the NtGB lines showed negligible rate of resistance to this necrotrophic pathogen (Fig. 3). SA signaling has been known to control defense against biotrophic pathogen in contrast, JA/ET signaling controls defense against necrotrophic pathogen.^1,15 Thus it has again been proved that GSH is not an active member in the crosstalk of JA-mediated pathway, rather it follows the SA-mediated pathway as has been evidenced earlier.¹²Open in a separate windowFigure 3Resistance pattern of wild-type and NtGB lines against Botrytis cinerea.Additionally, the leaves of wild-type and NtGB lines were treated with 1 mM SA and the expression of PR1, SDRLP, AOS and PR4 genes were analyzed and compared to untreated plants to simulate pathogen infection. The expression of PR1 increased after exogenous application of SA. In case of PR4, the ET marker, the expression level increased in NtGB lines. On the other hand, the level of SDRLP was nearly the same. However, the expression of AOS was absent in SA-treated leaves (Fig. 4). PR1 has been known to be induced by SA-treatment¹⁶ which can be corroborated with our results. In addition, ET is known to enhance SA/NPR1-dependent defense responses,¹⁷ which was reflected in our study as well. AOS, the biosynthetic pathway gene of JA, further known to be the antagonist of SA, was downregulated in SA-treated plants.Open in a separate windowFigure 4Gene expression pattern of PR1, SDRLP, PR4 and AOS in untreated and SA-treated wildtype and NtGB lines.Taken together, it can be summarized that this study provided new evidence on the involvement of GSH with SA in NPR1-dependent manner in combating biotic stress. Additionally, it can be claimed that GSH is a signaling molecule which takes an active part in the cross-communication with other established signaling molecules like SA, JA, ET in induced defense responses and has an immense standpoint in plant defense signaling.     

Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.     

Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies

Background

The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains.

Methods and Findings

Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC₅₀ 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC₅₀ of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro.

Conclusions

The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.