Short-and long-term exposure to heavy metals induced oxidative stress response in Pseudokirchneriella subcapitata

Algal cells can be exposed to toxicants for a short term due to accidental discharges or, more commonly, for a long term. The present work aimed to assess the ability of Cd, Cr, Cu and Zn to induce accumulation of reactive oxygen species (ROS) in the alga Pseudokirchneriella subcapitata after a short (6?h) or a long (72?h) exposure time. The relationship between the ROS induction and the content of reduced glutathione (GSH) was also examined. For this purpose, three nominal concentrations of each metal were used corresponding approximately to 72 h-EC10 and 72 h-EC50 values and a high concentration (>72 h-EC90 values). Intracellular ROS accumulation and GSH content were evaluated using a fluorescent-based approach. A long-term (chronic) exposure of algal cells to Cd, Cu and Zn, at the highest concentrations tested, induced an increase of intracellular ROS and GSH content. The increase of GSH content might be a form of algal cells to redress the imbalance caused by the oxidative stress. However, the increase of GSH was not enough to protect the algal cells against the long-term exposure to oxidative stress. The exposure of algal cells to low or intermediate metals concentrations induced a modification of GSH content; however, no increase of ROS production was detected, which indicates that the toxic symptoms exhibited by algal cells, under these conditions, cannot be attributed to intracellular ROS accumulation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic founding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER- 006684), and the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124- FEDER-027462). Manuela D. Machado gratefully acknowledges the post-doctoral grant from FCT (SFRH/BPD/72816/2010)

Targeted correction of a thalassemia-associated β-globin mutation induced by pseudo-complementary peptide nucleic acids

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction

Long homopurine•homopyrimidine sequences are characteristic of genes expressed in brain and the pseudoautosomal region

Homo(purine•pyrimidine) sequences (R•Y tracts) with mirror repeat symmetries form stable triplexes that block replication and transcription and promote genetic rearrangements. A systematic search was conducted to map the location of the longest R•Y tracts in the human genome in order to assess their potential function(s). The 814 R•Y tracts with ≥250 uninterrupted base pairs were preferentially clustered in the pseudoautosomal region of the sex chromosomes and located in the introns of 228 annotated genes whose protein products were associated with functions at the cell membrane. These genes were highly expressed in the brain and particularly in genes associated with susceptibility to mental disorders, such as schizophrenia. The set of 1957 genes harboring the 2886 R•Y tracts with ≥100 uninterrupted base pairs was additionally enriched in proteins associated with phosphorylation, signal transduction, development and morphogenesis. Comparisons of the ≥250 bp R•Y tracts in the mouse and chimpanzee genomes indicated that these sequences have mutated faster than the surrounding regions and are longer in humans than in chimpanzees. These results support a role for long R•Y tracts in promoting recombination and genome diversity during evolution through destabilization of chromosomal DNA, thereby inducing repair and mutation

Weak interpolation in Banach spaces

1. Trait-based approaches predict ecosystem functioning under environmental change by relating traits predicting changes in species densities (response traits) to traits driving ecosystem functioning (effect traits). Stressors can however affect ecosystem functioning not only by altering species densities, but also by directly changing species effect traits. 2. We first identified the response traits predicting the cell density of 18 marine benthic diatom strains along gradients of two chemical stressors (a pesticide and a metal, atrazine and copper). We then tested if response traits could predict stressor-induced changes in ecosystem functioning, i.e. changes in the effect traits driving the diatoms’ potential contribution to primary production, sediment stabilization and energy content in intertidal systems. Finally, we examined if changes in density and changes in ecosystem functioning were correlated, to assess whether species capable of growing under stressful conditions could maintain their contribution to ecosystem functioning. 3. The relationship between response traits and stressor-induced changes in density and ecosystem functioning was different depending on stressor type: a set of intercorrelated morphological traits predicted changes in both density and ecosystem functioning under metal stress, with large cells being more stress-resistant. Changes in density and changes in ecosystem functioning were positively related: diatoms whose density was least affected by the metal were also able to sustain functioning under metal exposure. 4. In contrast, the capacity for mixotrophic growth predicted changes in density, but not changes in ecosystem functioning under pesticide stress. Pesticide effects on density and on ecosystem functioning were negatively related for energy content and sediment stabilization, indicating a limited capacity of pesticide-tolerant diatoms to maintain their contribution to ecosystem functioning. Synthesis. Ecosystem functioning under stress can depend on whether response traits driving changes in density also predict direct stress effects on the species’ contribution to ecosystem functioning. Based on our results, we expect a disproportionate loss of functioning when traits driving species densities do not allow to maintain ecosystem functioning under stress

Relationship between Anaemia, Haemolysis, Inflammation and Haem Oxygenase-1 at Admission with Sepsis: a pilot study

Upregulation of haem oxygenase-1 (HO-1), due to haemolysis and/or inflammation, can lead to impaired immune function. Anaemia is common among sepsis patients, but the consequences of sepsis-associated anaemia are poorly understood. Here, our objective was to determine the prevalence and extent of anaemia, haemolysis, inflammation, and HO-1 induction after early hospital admission. We hypothesised that inflammation- or infection-induced haemolysis contributes to sepsis-associated anaemia and that this will lead to expression of HO-1. In this study, plasma obtained from seventy adult patients within 12 hours of admission to intensive care due to sepsis were analysed for anaemia, haemolysis and inflammatory markers by ELISA and microbead array. The majority (82.6%) of patients were anaemic with evidence of haemolysis (raised haem, haptoglobin, haemopexin, and HO-1 concentrations). Interestingly, concentrations of both haemoglobin and IL-10 were moderately positively correlated with HO-1 concentration (Hb: r = 0.32, p = 0.007; IL-10 r = 0.39, p = 0.0008) whereas HO-1 concentration was weakly negatively correlated with haemopexin (r = -0.23, p = 0.055). Anaemia, while common, was not associated with HO-1 concentration. After adjusting for confounding, HO-1 induction appears to be associated primarily with IL-10 concentration rather than haemolysis. Disease severity at diagnosis was correlated with early plasma IL-10 (r = 0.35, p = 0.003) and HO-1 (r = 0.24, p = 0.048) concentrations. Notably, admission levels of haem, HO-1, and IL-10 were indicators of survival

Endocrine disruptors and obesity: a current review on environmental obesogens

Obesity represents an important public health concern because it substantially increases the risk of multiple chronic diseases and thereby contributing to a decline in both quality of life and life expectancy. Besides unhealthy diet, physical inactivity and genetic susceptibility, environmental pollutants also contribute to the rising prevalence of obesity epidemic. An environmental obesogen is defined as a chemical that can alter lipid homeostasis to promote adipogenesis and lipid accumulation whereas an endocrine disrupting chemical (EDC) is defined as a synthetic chemical that can interfere with the endocrine function and cause adverse health effects. Many obesogens are EDCs that interfere with normal endocrine regulation of metabolism, adipose tissue development and maintenance, appetite, weight, and energy balance. An expanding body of scientific evidence from animal and epidemiological studies has begun to provide links between exposure to EDCs and obesity. Despite the significance of environmental obesogens in the pathogenesis of metabolic diseases, the contribution of synthetic chemical exposure to obesity epidemic remains largely unrecognised. Hence, the purpose of this review is to provide a current update on the evidences from animal and human studies on the role of fourteen environmental obesogens in obesity, a comprehensive view of the mechanisms of action of obesogens and current green and sustainable chemistry strategies to overcome chemical exposure to prevent obesity. Designing of safer version of obesogens through green chemistry approaches requires a collaborative undertaking to evaluate the toxicity of endocrine disruptors using appropriate experimental methods, which will help in developing a new generation of inherently safer chemicals

Toxicity of pesticides and their mixture to primary producers

The earth’s aquatic ecosystems are threatened by the contamination with a multitude of anthropogenic chemical pollutants. Pesticides are one important group of environmental contaminants. They are frequently detected in our surface waters and occur as single substances and in mixtures of various compositions. Copper (Cu) is often used as fungicide and herbicide in orcharding and viniculture. Cu toxicity is generally linked to the bioavailable fraction, i.e. to the concentration of the free Cu2+. In phototrophic organisms, one toxic mode of action of Cu is due to the inhibition of photosynthesis. Phytotoxicity of Cu was also found to be related to the generation of reactive oxygen species (ROS). However, until now, it is not clear, whether ROS are a mere consequence of Cu toxicity or the primary cause. One objective of this dissertation thesis was thus to investigate the role of ROS in the toxicity of Cu to phototrophic organisms to gain a better understanding of its toxicity mechanism. The two freshwater green algal species Pseudokirchneriella subcapitata and Chlorella vulgaris were chosen as test organisms. Cu-induced ROS formation was investigated in relation to short-term effects on photosynthetic activity and long-term effects on growth of P. subcapitata and C. vulgaris. Photosynthetic activity was determined as in vivo chlorophyll fluorescence. Exposure to 30 nM and 300 nM Cu resulted in a light and time dependent increase in ROS concentrations in P. subcapitata and C. vulgaris. The potential of Cu to induce ROS was comparable in both algae but the effect on photosynthesis differed with 300 nM Cu leading to a 12 % reduction of photosynthetic activity in P. subcapitata but not C. vulgaris after 24 h. This indicates that species-specific sensitivities were not caused by differences in ROS content but more likely resulted from differences in each ROS defence systems. The ROS scavenger N-tert-butyl-α-phenylnitrone (BPN) diminished Cu induced ROS production to control levels and completely restored Cu inhibiting effects on photosynthetic activity of P. subcapitata. This implies that ROS may play a primary role in the mechanism of copper toxicity to photosynthesis in algal cells. Further experiments revealed a time-dependent ROS release process across the plasma membrane. More than 90 % of total ROS were determined to be extracellular in P. subcapitata, indicating an efficient way of cellular protection against oxidative stress. Besides Cu, the triazine atrazine and the phenyl urea herbicides isoproturon and diuron are frequently detected in our surface waters. In contrast to Cu, phytotoxicity of these three pesticides is due to one specific mode of action which has been thoroughly investigated. Atrazine, isoproturon, and diuron inhibit photosynthesis by interrupting electron transport through photosystem II (PSII). Laboratory studies demonstrated that mixture toxicity of triazine and phenylurea herbicides to single species and communities is predictable by the concept of concentration addition. However, there is a lack of studies that verify the applicability of these concepts for natural communities exposed under complex environmental conditions. A further objective of this thesis was thus to verify if the concept of concentration addition can be applied also for natural communities exposed under realistic environmental conditions. Therefore, a phytoplankton community and the three submersed macrophytes Myriophyllum spicatum, Elodea canadensis, and Potamogeton lucens were studied in freshwater outdoor mesocosms. The three PSII inhibitors atrazine, isoproturon, and diuron were chosen as test substances and applied as single substances and in a mixture. In the single treatments the 30 % hazardous concentrations (HC30) of the three substances derived from species sensitivity distribution (SSD) curves were used. The SSD curves were established on the basis of EC50 growth inhibition data obtained from laboratory tests with different algal and plant species. The herbicide mixture comprised one third of the HC30 of each individual herbicide. If the concept of concentration holds true the herbicide mixture was expected to elicit the same toxic effects as the HC30 of three herbicides alone. Herbicide concentrations and effects on phytoplankton and macrophytes were investigated during a five-week period of constant concentrations and a subsequent fivemonth post-treatment period when the herbicides dissipated from the water phase. Photosynthetic efficiency of phytoplankton and the three macrophytes was selected as an endpoint directly linked to the mode of action of the three test substances. Moreover, effects on abundance, diversity, and species composition of phytoplankton as well as on growth of the two macrophytes E. canadensis and M. spicatum were examined. In the period of constant concentrations averaged herbicide water concentrations were determined to be in the range of target concentrations ± 20 %. In the post-treatment period the dissipation of the herbicides was described by first order kinetics. Half-lives corresponded to 107 d for atrazine, 35 d for isoproturon, and 43 d for diuron. At the end of the experiment atrazine concentrations in the water phase still reached approximately 40 % of the target concentration whereas isoproturon and diuron had nearly completely disappeared. In the constant exposure period the single herbicides were shown to be equitoxic due to comparable effects on photosynthetic efficiency, abundance, and diversity of phytoplankton. Furthermore, the herbicides were found to act concentration additive since the effects of the mixture on photosynthetic efficiency, abundance, and diversity were similar to those of the single substances. Because of different sensitivities of a few algal species towards the herbicides, species composition of the communities in the various treatments developed differently during the post-treatment period. Diuron and isoproturon treated algal communities did not differ considerably from the untreated communities concerning photosynthetic efficiency, diversity, and species composition already a short time after the end of the period of constant exposure. This might be linked to the rapid decrease in herbicide concentrations in the water phase. However, tolerance of single algal species towards the herbicides might have also contributed to the recovery of diuron and isoproturon treated phytoplankton. Photosynthetic efficiency and species composition of atrazine and mixture treated phytoplankton was found to be adversely affected and considerably different compared to the untreated communities until the end of the post-treatment period. Continuous exposure to persisting atrazine concentrations resulted in a different succession of phytoplankton in these two treatments. In the three submersed macrophytes inhibition of photosynthesis was determined in a short time window from day 2 to 5 after first application only indicating a rapid adaptation of the macrophytes towards herbicide stress. The observed short term effects of the herbicides and their mixture on photosynthesis might also explain that growth of M. spicatum and E. canadensis was not affected. In addition, the single herbicide concentrations turned out to be equitoxic in M. spicatum since they elicited similar effects on photosynthetic efficiency of this macrophyte. Concentration addition of atrazine, isoproturon, and diuron could also be verified for this macrophyte since the mixture inhibited photosynthetic efficiency comparable to the single substances. In E. canadensis and P. lucens the HC30 of atrazine, isoproturon, and diuron did not stimulate a similar inhibition of photosynthesis and were thus found to be not equitoxic. For this reason, any conclusions on concentration addition of the herbicides in these two macrophytes could not be drawn. This case study confirmed the applicability of the concept of concentration addition for three PSII inhibitors when considering their effects on a natural algal community and on the macrophyte M. spicatum under environmental conditions. The results can thus contribute to the current discussion concerning the incorporation of mixture toxicity in the regulation of surface water quality to adequately protect aquatic communities from pesticide impact and to guarantee a sustained management of the aquatic ecosystems