Effects of Wind Mixing in a Stratified Water Column on Toxic Cyanobacteria and Microcystin-LR Distribution in a Subtropical Reservoir.

We analyzed the effects of stratification changes due to wind on the vertical cyanobacteria distribution and microcystin-LR concentrations in a reservoir and assessed the implications for water management. Under stratified conditions, the highest microcystin concentrations (up to 4.16 µg/L) and toxic cyanobacteria biovolume occurred in the epilimnion (~ 1 m). The lowest microcystin concentrations were between 0.02 and 1.28 µg/L and occurred in the hypolimnion (~ 20 m). A cold front passage associated with high wind velocities induced water column mixing, promoting the redistribution of microcystin-LR and cyanobacteria throughout the water column and increasing their concentrations in deeper zones. Microcystin-LR concentration was positively correlated with cyanobacteria biovolume (r = 0.747) and chlorophyll a concentration (r = 0.798). Changes in thermal profile due to wind would imply a greater challenge for drinking water treatment plants, since high cyanobacterial and microcystin concentrations could reach deep-water intakes.

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays.

Single domain heavychain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.

Oxidative effects and toxin bioaccumulation after dietary microcystin intoxication in the hepatopancreas of the crab Neohelice (Chasmagnathus) granulata.

We studied the accumulation and depuration of microcystin-LR (MCLR) in the hepatopancreas of the crab Neohelice granulata fed twice weekly with either non toxic or MCLR-producing Microcystis aeruginosa (strain NPDC1 or NPJB, respectively) during seven weeks. We also analyzed MCLR effects on the oxidative stress- and detoxification-related variables, superoxide dismutase and glutathione-S-transferase activities, and the levels of reduced glutathione and lipid peroxidation (as thiobarbituric acid reactive substances, TBARS). Hepatopancreas MCLR content slightly increased during the first three weeks, up to 8.81±1.84ngg(-1) wet tissue mass (WTM) and then started to decrease to a minimum of 1.57±0.74ngg(-1) WTM at the seventh week (p<0.05 with respect to that in the first week). TBARS levels were about 55% higher in treated than in control N. granulata (p<0.001 and p<0.05) during the first three weeks of the experimental period. GSH content became 50% lower than in control individuals (p<0.01) during weeks 6 and 7. SOD activity was increased by about 2-fold (p<0.05 or p<0.001) from week 3 to 7 in treated crabs with respect to control ones, while GST activity was about 70% higher in treated than in control crabs from week 4 to week 7 (p<0.05). Our data suggest that in the hepatopancreas of N. granulata MCLR accumulation and oxidative damage are limited and reversed by detoxification-excretion and antioxidant mechanisms. The activation of these defensive mechanisms becomes evident at 3-4 weeks after the start of the intoxication.

Limited analytical capacity for cyanotoxins in developing countries may hide serious environmental health problems: simple and affordable methods may be the answer.

In recent years, the international demand for commodities has prompted enormous growth in agriculture in most South American countries. Due to intensive use of fertilizers, cyanobacterial blooms have become a recurrent phenomenon throughout the continent, but their potential health risk remains largely unknown due to the lack of analytical capacity. In this paper we report the main results and conclusions of more than five years of systematic monitoring of cyanobacterial blooms in 20 beaches of Montevideo, Uruguay, on the Rio de la Plata, the fifth largest basin in the world. A locally developed microcystin ELISA was used to establish a sustainable monitoring program that revealed seasonal peaks of extremely high toxicity, more than one-thousand-fold greater than the WHO limit for recreational water. Comparison with cyanobacterial cell counts and chlorophyll-a determination, two commonly used parameters for indirect estimation of toxicity, showed that such indicators can be highly misleading. On the other hand, the accumulated experience led to the definition of a simple criterion for visual classification of blooms, that can be used by trained lifeguards and technicians to take rapid on-site decisions on beach management. The simple and low cost approach is broadly applicable to risk assessment and risk management in developing countries.

Microcystin accumulation and antioxidant responses in the freshwater clam Diplodon chilensis patagonicus upon subchronic exposure to toxic Microcystis aeruginosa.

We investigated the accumulation and toxicity of microcystin-LR (MCLR) in the digestive gland of the freshwater clam Diplodon chilensis patagonicus. Treated clams were fed with a toxic strain of Microcystis aeruginosa (NPJB1) during 6 weeks and control clams received the non-toxic strain NPDC1. Filtration rate was estimated for both groups. Toxic effects were evaluated through the hepatosomatic index (HSI) and different oxidative stress biomarkers, lipid peroxidation (content of thiobarbituric reactive substances-TBARS), protein oxidation (carbonyl groups) and reduced glutathione (GSH) levels, and enzymatic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST). The extractable MCLR measured by ELISA in digestive gland extracts showed little or no change during the first 3 weeks and increased significantly at weeks 5 and 6. HSI was reduced by 30% in treated clams at weeks 5 and 6. No significant oxidative damage to lipids or proteins was. All the antioxidant defense parameters analyzed were significantly increased at week 5 or 6. GSH increased in treated clams at week 5, reaching 62% increase at week 6. SOD, CAT and GST activities were significantly increased in treated clams by 50%, 66% and 60%, respectively, at the end of the experiment. D. chilensis patagonicus can be exposed to prolonged cyanobacterial blooms accumulating significant quantities of MCLR, which could be a risk for mammals and birds, which feed on this species and, in a lesser extent, to humans.

Leaf saponins of Quillaja brasiliensis enhance long-term specific immune responses and promote dose-sparing effect in BVDV experimental vaccines.

Saponin-based adjuvants are promising adjuvants that enhance both humoral and T-cell-mediated immunity. One of the most used natural products as vaccine adjuvants are Quillaja saponaria bark saponins and its fraction named Quil A®. Despite that, its use has been restricted for human use due to safety issues. As an alternative, our group has been studying the congener species Quillaja brasiliensis saponins and its performance as vaccine adjuvants, which have shown to trigger humoral and cellular immune responses comparable to Quil A® but with milder side effects. Here, we studied a semi purified aqueous extract (AE) and a previously little characterized saponin-enriched fraction (QB-80) from Q. brasiliensis as vaccine adjuvants and an inactivated virus (bovine viral diarrhea virus, BVDV) antigen co-formulated in experimental vaccines in mice model. For the first time, we show the spectra pattern of the Q. brasiliensis saponins by MALDI-TOF, a novel and cost-effective method that could be used to characterize different batches during saponins production. Both AE and QB-80 exhibited noteworthy chemical similarities to Quil A®. In addition, the haemolytic activity and toxicity were assessed, showing that both AE and QB-80 were less toxic than Quil A®. When subcutaneously inoculated in mice, both fractions promoted long-term strong antibody responses encompassing specific IgG1 and IgG2a, enhanced the avidity of IgG antibodies, induced a robust DTH reaction and significantly increased IFN-É£ production in T CD4+ and T CD8+ cells. Furthermore, we have proven herein that AE has the potential to promote dose-sparing, substantially reducing the dose of antigen required for the BVDV vaccines and still eliciting a mixed Th1/Th2 strong immune response. Based on these results, and considering that AE is a raw extract, easier and cheaper to produce than commercially available saponins, this product can be considered as candidate to be escalated from experimental to industrial uses.

Cellular transport of microcystin-LR in rainbow trout (Oncorhynchus mykiss) across the intestinal wall: possible involvement of multidrug resistance-associated proteins.

We studied Abcc mediated-transport in middle and posterior intestine of the rainbow trout, Oncorhynchus mykiss. Luminal and serosal transport were evaluated in everted and non-everted intestinal sacs, respectively, incubated with 1-chloro-2,4-dinitrobenzene (CDNB; 200 µM). CDNB enters the cells and is conjugated with glutathione via glutathione S-transferase (GST) to form 2,4-dinitrophenyl-S-glutathione (DNP-SG), a known Abcc substrate. DNP-SG concentration in the bath was recorded every 10 min, in order to calculate the mass-specific transport rate. For evaluating the possible involvement of Abcc proteins in microcystin-LR (MCLR) transport, 1.135 µM MCLR was added to the bath or inside the sacs, in everted or non-everted preparations, respectively. Both luminal and serosal DNP-SG efflux were significantly inhibited by MCLR. A concentration-response curve obtained using strips from middle intestine yielded an IC50 value of 1.33 µM MCLR. The Abcc inhibitor, MK571 produced concentration-dependent inhibition of DNP-SG similar to that produced by MCLR. Since competition of MCLR and CDNB as GST substrates could bias the DNP-SG transport results, we evaluated the effects of MCLR on calcein efflux, which does not depend on GST activity. We applied the non-fluorescent, cell-permeant compound calcein-AM (0.25 µM) to middle intestinal strips and recorded the efflux of its hydrolysis product, the fluorescent Abcc substrate calcein. 2.27 µM MCLR and 3 µM MK571 inhibited calcein efflux (17.39 and 20.2%, respectively). Finally, MCLR interaction with Abcc transporters was evaluated by measuring its toxic intracellular effects. Middle intestinal segments were incubated in saline solution with 1.135 µM MCLR (MC1), 2.27 µM MCLR (MC2), 3 µM MK571 (MK) or 1.135 µM MCLR+3 µM MK571 (MC1/MK). After 1h, GSH concentration, protein phosphatase 1 and 2A (PP1, PP2A) and GST activities were measured in each segment. MC1did not produce significant effect while MC1/MK and MC2 significantly inhibited PP1and PP2A in similar proportions (34-49%). MK alone significantly increased PP2A activity (40%) with no effect in any other variable. GST activity and GSH concentration were not affected by any treatment. Concentration-response curves for MCLR (1.135 to 13.62 µM) alone or plus 3 or 6 µM MK571 were obtained using PP1 activity as response variable. The IC50 values were 1.0, 0.52, and 0.37 µM, respectively. Our results suggest that O. mykiss enterocytes are capable of eliminating MCLR by GST-mediated conjugation and luminal excretion through an Abcc-like apical transporter. This mechanism would prevent toxic effects and reduce the toxin uptake into the blood, which is likely mediated by basolateral Abccs.