Bacteria causing chronic infections are generally observed living in cell aggregates suspended in polymer-rich host secretions, and bacterial phenotypes induced by aggregated growth may be key ...factors in chronic infection pathogenesis. Bacterial aggregation is commonly thought of as a consequence of biofilm formation; however the mechanisms producing aggregation in vivo remain unclear. Here we show that polymers that are abundant at chronic infection sites cause bacteria to aggregate by the depletion aggregation mechanism, which does not require biofilm formation functions. Depletion aggregation is mediated by entropic forces between uncharged or like-charged polymers and particles (e.g., bacteria). Our experiments also indicate that depletion aggregation of bacteria induces marked antibiotic tolerance that was dependent on the SOS response, a stress response activated by genotoxic stress. These findings raise the possibility that targeting conditions that promote depletion aggregation or mechanisms of depletion-mediated tolerance could lead to new therapeutic approaches to combat chronic bacterial infections.
Abstract
The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it ...can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: −0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%–2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.
Near-shore marine/estuarine environments play an important role in the functioning of the marine ecosystem and are extremely vulnerable to the presence of chemical pollution. The ability to ...investigate the effects of pollution is limited by a lack of model organisms for which sufficient ecotoxicological information is available, and this is particularly true for tropical regions. The circumtropical marine amphipod Parhyale hawaiensis has become an important model organism in various disciplines, and here we summarize the scientific literature regarding the emergence of this model within ecotoxicology. P. hawaiensis is easily cultured in the laboratory and standardized ecotoxicity protocols have been developed and refined (e.g., miniaturized), and effects of toxicants on acute toxicity (Cd, Cu, Zn, Ag, ammonia, dyes, pesticides, environmental samples), genotoxicity as comet assay/micronuclei, and gene expression (Ag ion and Ag nanoparticles) and regeneration (pesticides) have been published. Methods for determination of internal concentrations of metals (Cu and Ag) and organic substances (synthetic dye) in hemolymph were successfully developed providing sources for the establishment of toxicokinetics models in aquatic amphipods. Protocols to evaluate reproduction and growth, for testing immune responses and DNA damage in germ cells are under way. The sensitivity of P. hawaiensis, measured as 50% lethal concentration (LC50), is in the same range as other amphipods. The combination of feasibility to culture P. hawaiensis in laboratory, the recent protocols for ecotoxicity evaluation and the rapidly expanding knowledge on its biology make it especially attractive as a model organism and promising tool for risk assessment evaluations in tropical environments.
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•Parhyale hawaiensis is easily cultured in the laboratory.•Acute toxicity, genotoxicity, gene expression and regeneration protocols are available.•P.hawaiensis' sensitivity is in the same range as other amphipods.•Methods for determination of internal concentrations of toxicants were established.•P.hawaiensis is a promising model for risk assessment of marine environments.
The aim of this work was to examine the polyphenol content in Castanea sativa leaf extracts and to determine their antioxidant and antigenotoxic effect. The antioxidant activity of extracts and main ...components were determined by the application of spectophotometric and electronic spin resonance methods of capturing DPPH, superoxide and hydroxyl radical. The antigenotoxic effect was evaluated with genotoxic mycotoxins – aflatoxin B1 (AFB1) and ochratoxin A (OTA). Castanea leaf extract contained 6.74% of total polyphenols. Castanea extract was the richest in tannins and flavonoids (gallic and ellagic acid derivatives). Castanea leaf extract showed the ability to neutralize DPPH radicals (IC50 = 1.87 μg/mL). Similar results were also identified by testing the capture of hydroxyl and superoxide radicals. The strong antioxidant activity of the dominant components, that indicate phenolic acids as the ingredients that contribute the most to the antioxidant and antigenotoxic effect of castanea leaf extract, was also found. Genotoxic suppression of AFB1 and OTA was established by a comet test, establishing a significant reduction in tail, tail intensity and tail torque in leukocytes co-treated with mycotoxins and different concentrations of extracts. Our findings emphasize the potential of C. sativa to prevent genotoxics and also its antioxidant effects.
Imidacloprid (IMI), a systemic neonicotinoid insecticide widely used in worldwide scale, is reported in freshwater bodies. Nevertheless, there is a lack of information about IMI sublethal effects on ...freshwater fish. Thus, the aim of this study was to identify the potential hazard of this insecticide to the South American fish Prochilodus lineatus exposed for 120 h to four IMI concentrations (1.25, 12.5, 125, and 1250 μg L−1). A set of biochemical, genotoxic and physiological biomarkers were evaluated in different organs of the fish. IMI exposure induced significant changes in the enzymatic profiles of P. lineatus, with alterations in the activity of biotransformation and antioxidant enzymes in different tissues. Redox balance of the tissues was affected, since oxidative damage such as lipoperoxidation (LPO) and protein carbonylation (PCC) were evidenced in the liver, gills, kidney and brain of fish exposed to different IMI concentrations. Fish exposed to all IMI concentrations showed decreased blood glucose indicating an increase of energetic demand. DNA damage was evidenced by the comet test, in the erythrocytes of fish all the concentrations evaluated. We integrated these results in the Integrated Biomarker Response (IBR) index, which evidenced that the organs most affected by IMI exposure were the liver and kidney, followed by the gills. Our results highlight the importance of investigating different target tissues after IMI exposure and show the sublethal effects of IMI in some of them; they also warn to the possible consequences that fish living in freshwater ecosystems can suffer due to IMI exposure.
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•We evaluated acute effects of Imidacloprid (IMI) on the fish Prochilodus lineatus.•IMI promoted oxidative damage in different fish tissues.•IMI promoted hypoglycemia in the exposed fish.•DNA damage was detected in erythrocytes of fish exposed to IMI.•Liver, kidney and gill were the most affected organs by IMI exposures.
•Cytotoxicity and genotoxicity of SA, SDA and PS on HUVECs have been studied.•To distinguish apoptotic cells from necrotic cells, flow cytometry was used.•DAPI staining and DNA ladder assays were ...used for DNA damage assessment.•These additives did not show any significant cytotoxic and genotoxic effects.
Cytotoxicity and genotoxicity of sodium acetate (SA), sodium diacetate (SDA), and potassium sorbate (PS) was tested on Human Umbilical Vein Endothelial Cells (HUVEC). Cytotoxicity was investigated by MTT assay and flow cytometry analysis, while genotoxicity was evaluated using DNA fragmentation and DAPI staining assays. The growth of treated HUVECs with various concentrations of SA, SDA and PS decreased in a dose-and time-dependent manner. The IC50 of 487.71, 485.82 and 659.96 µM after 24 h and IC50 of 232.05, 190.19 and 123.95 µM after 48 h of treatment were attained for SA, SDA and PS, respectively. Flow cytometry analysis showed that early and late apoptosis percentage in treated cells was not considerable. Also neither considerable DNA fragmentation nor DNA smear was observed using DAPI staining and DNA ladder assays. Overall, it can be concluded that the aforementioned food additives can be used as safe additives at low concentration in food industry.
Tumor growth relies on efficient DNA repair to mitigate the detrimental impact of DNA damage associated with excessive cell division. Modulating repair factor function, thus, provides a promising ...strategy to manipulate malignant growth. Here, we identify the ubiquitin-specific protease USP21 as a positive regulator of BRCA2, a key mediator of DNA repair by homologous recombination. USP21 interacts with, deubiquitinates and stabilizes BRCA2 to promote efficient RAD51 loading at DNA double-strand breaks. As a result, depletion of USP21 decreases homologous recombination efficiency, causes an increase in DNA damage load and impairs tumor cell survival. Importantly, BRCA2 overexpression partially restores the USP21-associated survival defect. Moreover, we show that USP21 is overexpressed in hepatocellular carcinoma, where it promotes BRCA2 stability and inversely correlates with patient survival. Together, our findings identify deubiquitination as a means to regulate BRCA2 function and point to USP21 as a potential therapeutic target in BRCA2-proficient tumors.BRCA2 is essential for the repair of DNA damage; therefore, defects in BRCA2 are associated with tumorigenesis but also with increased susceptibility to genotoxic stress. Here the authors show that USP21 regulates the ability of tumor cells to repair damaged DNA by regulating BRCA2 stability.
Excessive use of pesticides and herbicides is a major environmental and health concern worldwide. Atrazine, a synthetic triazine herbicide commonly used to control grassy and broadleaf weeds in ...crops, is a major pollutant of soil and water ecosystems. Atrazine modifies the growth, enzymatic processes and photosynthesis in plants. Atrazine exerts mutagenicity, genotoxicity, defective cell division, erroneous lipid synthesis and hormonal imbalance in aquatic fauna and nontarget animals. It has threatened the sustainability of agricultural soils due to detrimental effects on resident soil microbial communities. The detection of atrazine in soil and reservoir sites is usually made by IR spectroscopy, ELISA, HPLC, UPLC, LC–MS and GC–MS techniques. HPLC/LC–MS and GC–MS techniques are considered the most effective tools, having detection limits up to ppb levels in different matrices. Biodegradation of atrazine by microbial species is increasingly being recognized as an eco-friendly, economically feasible and sustainable bioremediation strategy. This review presents the toxicity, analytical techniques, abiotic degradation and microbial metabolism of atrazine.
The class-selective molecular-imprinted polymers (MIPs) have shown the recognition ability to multiple targeted molecules through using one or multiple templates. However, choosing the right ...templates, the core problem, still lacks a systemic guide and decision-making. In this work, we propose a strategy of selecting templates through expanding the recognition width for the improvement of class-selectivity. First, three families of genotoxic impurity (GTI) were selected as model objects, and the spatial size and binding energy of each GTI-monomer complexes were obtained and compared by computational simulation. The two indexes of energy width (W E) and size width (W L) were introduced to compare the similarity and differences on the two recognition factors, binding strength and spatial size, among these GTIs in each family. Through shortening the width to increase similarity on binding energy and size, the dual templates in the aromatic amines (AI) family and sulfonic acid esters (SI) family were successfully selected. Correspondingly, the prepared dual-template MIPs in the two GTI families can simultaneously recognize all the GTIs comparing with that of single template MIP, respectively. Meanwhile, through comparing the adsorption capacity of the selected template and its analogues in one GTI family, the recognition efficiency of the dual-template MIPs was higher than that of the single-template MIP. This indicates that though using the selected right templates, the higher class-selectivity and the larger recognition width can be realized. Thus, this work can solve the problem of blind template selection, and provide the useful theoretical guidance for designing family-selective molecular imprinting.
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