Microcystis aeruginosa is a cyanobacterium frequently associated with toxic blooms in eutrophic freshwater systems. Certain strains produce microcystins (MCs), a group of hepatotoxins with significant ecological and public health implications. In this study, we examined the quantitative response of a temperate native M. aeruginosa strain to combinations of temperature (26, 30, and 36 °C), light intensity (30, 50, and 70 µmol photons·m−2·s−1), and N:P ratio (10, 100, 150), using a full-factorial experimental design. Growth parameters (µ, lag phase duration, and maximum cell density), chlorophyll-a production, and MC-LR synthesis were modeled using Gompertz, linear, and dynamic approaches. High temperature and irradiance increased the specific growth rate but decreased final biomass, while elevated N:P ratios shortened the lag phase. MC-LR production peaked under low temperature, low irradiance, and low N:P ratio. Although MC-LR synthesis did not correlate positively with growth rate, and the environmental conditions maximizing growth differed from those enhancing toxin production, a population-level coupling between both processes was observed using the Long model. These findings suggest that MC-LR synthesis in M. aeruginosa is not merely a metabolic by-product of growth, but a context-dependent trait with potential adaptive significance.

Cyanotoxins are a significant concern due to their frequent presence in Southamerica waters. While numerous studies have investigated the toxic effects of MC-LR, knowledge regarding the toxicity of [D-Leu1]MC-LR remains limited. The aim of this study was to determine the impact of [D-Leu1]MC-LR and MC-LR administration on different brain structures in rats and the resulting modifications in oxidative stress. Male Sprague-Dawley rats were divided into two groups and administered 5 intraperitoneal injections of mixed MCs at doses of 2 and 15 µg kg-1 for each injection over a 21-day period, i.e. total doses of 10 and 75 µg kg-1.The MCs consisted on MC-LR (3 %), [D-Leu1]MC-LR (96.7 %) and others (0.3 %) isoforms. To evaluate the effect of treatments with different doses, the concentration of both MC isoforms, reactive oxygen species (ROS), lipid damage and antioxidant activity were measured in the cerebral cortex, hippocampus, striatum and cerebellum. The results revealed variability in MC concentration across brain regions. The accumulation rate of MC-LR was 2000 times higher than that of [D-Leu1]MC-LR, regardless of the dose administered at different areas. Taken together, our results highlighted that chronic exposure to MCs induced a mild oxidative stress in the rat brain characterized by increased ROS and antioxidant defense activation due to [D-Leu1]MC-LR in both the striatum and cortex at high dose. At low dose, the uptake of only MC-LR was determined in the cerebellum and hippocampus, resulting in increased ROS levels but no change in CAT activity in the hippocampus. In contrast, in the cerebellum, there was a decrease in ROS, possibly due to increased CAT consumption. However, the absence of detection of an MC variant under certain conditions does not allow for the exclusion of its metabolic effects. Chronic MC administration resulted in dose- and region-dependent distribution within the rat brain. Reactive species levels and cellular responses also varied by dose and region.

Different technologies have been evaluated to promote food safety and improve the microbiological quality and shelf life of food. The aim of this work was to determine the effect of gaseous ozone on beef carcass weight loss and indicator microorganism counts in an exporting abattoir. Two gaseous ozone concentrations (3 and 10 ppm) were applied on carcasses from Experiment 1 (Exp. 1, n = 100 test, n = 100 control) and Experiment 2 (Exp. 2, n = 100 test, n = 100 control), respectively. Cold chamber without ozone was used as control in both experiments. For mesophilic aerobic organism (MAO), coliform and Escherichia coli counts, 100 cm2 of each carcass was swabbed before and 10, 20 and 30 h after ozone application. In Exp. 1, the carcass entire surface was swabbed for Shiga-toxin (stx) gene detection. The counts of MAO were influenced by treatment and sampling time in both experiments. In Exp. 1, control carcasses had higher counts than ozonated carcasses at all sampling times, whereas the opposite occurred in Exp. 2. Coliform count was affected by sampling time in Exp. 2, whereas E. coli count was not affected in any experiment. All samples analyzed were stx-negative. Differences in carcass weight loss were not significant. In conclusion, gaseous ozone was not effective to reduce bacterial load or carcass weight loss. To our knowledge, this is the first study evaluating ozone effect on beef carcasses conducted in a commercial abattoir, not at laboratory scale. Future research would help demonstrate whether the use of ozone impacts on the quality and sensory characteristics of beef.

Recently, the development of materials with antimicrobial properties has become a challenge under scrutiny. The incorporation of copper nanoparticles (NpCu) into a chitosan matrix appears to represent a viable strategy to contain the particles and prevent their oxidation. Regarding the physical properties, the nanocomposite films (CHCu) showed a decrease in the elongation at break (5 %) and an increase in the tensile strength of 10 % concerning chitosan films (control). They also showed solubility values lower than 5 % while the swelling diminished by 50 %, on average. The dynamical mechanical analysis (DMA) of nanocomposites revealed two thermal events located at 113° and 178 °C, which matched the glass transitions of the CH-enriched phase and nanoparticles-enriched phase, respectively. In addition, the thermogravimetric analysis (TGA) detected a greater stability of the nanocomposites. Chitosan films and the NpCu-loaded nanocomposites demonstrated excellent antibacterial capacity against Gram-negative and Gram-positive bacteria, proved through diffusion disc, zeta potential, and ATR-FTIR techniques. Additionally, the penetration of individual NpCu particles into bacterial cells and the leakage of cell content were verified by TEM. The mechanism of the antibacterial activity of the nanocomposites involved the interaction of chitosan with the bacterial outer membrane or cell wall and the diffusion of the NpCu through the cells. These materials could be applied in diverse fields of biology, medicine, or food packaging.

To identify the causal agent of the massive intoxication occurred in February 2022 in Buenos Aires, with 98 hospitalized victims and 24 deaths. The first victims presented the following signs: miosis, shock, sensory depression, respiratory distress (bradypnea), psychomotor excitement, seizures, and cardiorespiratory arrest. Some showed a typical opiomimetic sign: "wood chest". Given the parasympathetic signs, the victims were treated with Naloxone at high doses, responding favourably and with a continuous infusion drip of 10 mg in 250 mL at a flow rate of 11 mL/h. The latter to prevent them from deteriorating when they waked up. All the cases had in common having inhaled a line of cocaine acquired from the same dealer. Seized powder samples were preliminarily analysed by HPTLC and UPLC-DAD (Acquity System-Waters Corp, USA). A cocaine standard (LGC Std, Lot. 979337, USA) was used. Subsequently, for the detailed analysis of components, a fractionation was made by extracting the soluble components in chloroform and the soluble and insoluble fractions were analysed by 1H and 13 C NMR on a Bruker A Neo 500 spectrometer (1H 500 MHz, 13C 125 MHz). The chloroform insoluble fraction was further analysed by LC-MS with exact mass detection on a Bruker micrOTOF-Q II spectrometer coupled to an Agilent 1200 HPLC. Structure confirmation was performed by MRM (multiple reaction monitoring), and MS/MS fragmentation. UPLC-DAD analysis showed a purity of 51.6% for COC, the rest being mostly sorbitol as cutting material (confirmed by NMR, as noted below). In the soluble fraction, where the spectra were measured in deuterochloroform solution, the NMR spectra showed the characteristic signals of cocaine hydrochloride and small amounts of cinnamyl cocaine (CIN) that usually accompanies cocaine hydrochloride and truxillines. (The latter very important to infer the origin of cocaine). In the insoluble fraction the main component was identified as sorbitol from its 1H and 13C NMR spectra (in DMSO-d6). A small remnant of cocaine and cinnamyl cocaine (CIN) was also observed. On the other hand, this fraction was analysed by LC-MS, observing 7 relevant peaks: cocaine, benzoylecgonine, cinnamyl cocaine (cis and trans), truxillines and a minor component of m/z 395.2316 that corresponds to the formula C24H31N2O3 (calculated 395,2329) coincident with the [M + H]+ quasi-molecular ion of carfentanil. Structure confirmation was performed by MRM, and fragmentation by MS/MS of the M + H ion, observing the characteristic fragments of the carfentanil structure including the two confirmatory ions of m/z 335.2123 (calculated for C22H27N2O+ 335,2118) and m/z 246.1489 (calculated for C15H20NO2+ 246,1489). Quantitative analysis was performed by NMR for the COC/CIN ratio and from LC-MS for the CIN/Carfentanil ratio. Giving an estimated result of 30 μg/100 mg of pure cocaine. The toxic dose for humans is unknown, although would be less than 1 mg based on animal studies and known fentanyl lethal cases (estimated lethal dose is 20 μg) (Casale et al., 2017). The therapeutic treatment followed by the victims and the results observed allowed us to focus on opiomimetics, as the most relevant factor in the cause of the 24 deaths. The high-resolution methods applied allowed us to confirm cocaine in a percentage of purity close to half of the total powder. Carfentanil was confirmed by MRM analysis and in an amount of approximately 30 μg/100 mg of pure cocaine. Based on the data collected in the case, the consumption of cocaine was about 200 mg, therefore, in the proportion of purity, each line contained about 30 μg of carfentanil. So, for 400 mg of powder consumed, carfentanil would be found at 60 μg. Therefore, we estimated the toxic dose for this episode between 30–60 μg.

The increase in cyanobacterial blooms linked to climate change and the eutrophication of water bodies is a global concern. The harmful cyanobacterium Microcystis aeruginosa is one of the most common bloom-forming species whose removal from fresh water and, in particular, from that used for water treatment processes, remains a crucial goal. Different biodegradable and environmentally friendly coagulants/flocculants have been assayed, with chitosan showing a very good performance. However, chitosan in its original form is of limited applicability since it is only soluble in acid solution. The objective of this work was therefore to test the coagulant/flocculant capacity of trimethylchitosan (TMC), a chitosan derivative produced from residues of the fishing industry. TMC has a constitutively net positive charge enabling it to remain in solution regardless of the pH. Results show that even at alkaline pHs, common during cyanobacterial blooms, TMC is effective in removing buoyant cyanobacteria from the water column, both in test tube and Jar-Test experiments. Cell integrity was confirmed by fluorescent stain and electron microscopy. Our findings lead us to conclude that the use of TMC to remove bloom cells early in the treatment of drinking water is both feasible and promising.

Among the bloom-forming cyanobacteria, Microcystis aeruginosa is one of the most harmful species [...].

This study assessed the acid-adaptation of pathogenic and non-pathogenic strains of Escherichia coli in orange juice and the microbial resistance to the subsequent UV-C radiation treatment. Nine Shiga toxin-producing E. coli (STEC) and one strain of a non-pathogenic surrogate E. coli were used in this study. Each E. coli strain was inoculated in orange juice, following pre-exposure during 0, 1, 2, and 3 h at 10 degrees C. Then, the inoculated juices with the ten different strains separately were exposed to 0 and 2 J/cm(2) of UV-C radiation. The D value (i.e., the UV-C dose in J/cm(2) required to cause a one-log reduction in the target microorganism) was calculated. Further, the resistance coefficient [RC; i.e., the ratio between the D-values for the control condition (D-0h) and each pre exposure tested time (D-1h, D-2h, D-3h)] were determined. The results indicated that the resistance of E. coli was influenced by the pre-exposure period in the orange juice, with increased resistance to UV-C observed for periods >2 h. Furthermore, the sensitivity of cells to subsequent UV-C treatment was found to be strain-dependent. The results may allow the development of more reliable UV-C radiation processes for orange juice processing aiming the inactivation of pathogenic E. coli.

The treatment of beef with gaseous ozone (GO) was analyzed and the physicochemical characteristics (texture, lipid oxidation, surface color) and food safety (microbial growth of heterotrophic microflora and inoculated Listeria monocytogenes) were evaluated. Transient changes in ozone concentration within the chamber were mathematically modeled using a non stationary mass balance. Ozone pulses were applied and treatment intensities were evaluated in each case. GO treatment prolonged refrigerated storage of vacuum packed beef. Long periods of exposure to ozone (>10 min) negatively affected the red color and oxidative rancidity. The most effective treatments to control microbial flora were ozone pulses ranging between 5 and 10 min duration every 30 min for 5 h using 280 mg O-3 m(-3); these treatments allowed the reduction of more than 1 logarithmic cycle the counts of the natural flora in beef (lactic acid bacteria, mesophilic and enterobacteriaceae), maintaining oxidative stability and adequate physicochemical parameters. Additionally these low doses of GO decreased the counts of inoculated L. monocytogenes (10(2) CFU g tissue(-1)) to values below the detection limit for 16 days at 4 degrees C limiting its growth during refrigerated storage. The use of GO in beef products allows the control of microbial growth during refrigerated storage maintaining high quality parameters.

Regulated cell death (RCD) encompasses the activation of cellular pathways that initiate and execute a self-dismissal process. RCD occur over a range of stressors doses that overcome pro-survival cellular pathways, while higher doses cause excessive damage leading to passive accidental cell death (ACD). Hydrogen peroxide (HP) has been proposed as a potential tool to control harmful cyanobacterial blooms, given its capacity to remove cyanobacterial cells and oxidize cyanotoxins. HP is a source of hydroxyl radicals and is expected to induce RCD only within a limited range of concentrations. This property makes this compound very useful to better understand stress-driven RCD. In this work, we analyzed cell death in microcystin-producing Microcystis aeruginosa by means of a stochastic dose response model using a wide range of HP concentrations (0, 0.29, 1.76, 3.67, 7.35, 14.70, and 29.5 mM). We used flow cytometry and unsupervised classification to study cell viability and characterize transitional cell death phenotypes after exposing cells to HP for 48 and 72 h. Non-linear regression was used to fit experimental data to a logistic cumulative distribution function (cdf) and calculate the half maximal effective concentration (EC50). The EC50 of M. aeruginosa exposed to HP were 3.77 ± 0.26 mM and 4.26 ± 0.22 mM at 48 and 72 h, respectively. The derivative of cdf (probability density function; pdf) provided theoretical and practical demonstration that EC50 is the minimal dose required to cause RCD in 50% of cells, therefore maximizing the probability of RCD occurrence. 1.76 mM HP lead to an antioxidant stress response characterized by increased reactive oxygen species (ROS) levels and HP decomposition activity. The exposure of 3.67 mM HP induced a dose-related transition in cell death phenotype, and produced several morphological changes (a less dense stroma, distortion of the cell membrane, partial disintegration of thylakoids, extensive cytoplasmic vacuolation and highly condensed chromatin). The EC50 and the stochastic cdf and pdf together with the multidimensional transitional phenotypic analysis of single cells contribute to further characterize cell death pathways in cyanobacteria.

Department of Environmental Medicine, Poznan University of Medical Sciences, Poznań, Poland, 2 Integrated Science Association (ISA), Universal Scientific, Education and Research Network (USERN), Poznań, Poland, 3 Instituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC-CONICET), Fundación para Investigaciones Biológicas Aplicadas (CIB-FIBA), Mar del Plata, Argentina, Centro de Investigación y Desarrollo en Criotecnología de Alimentos, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de La Plata, La Plata, Argentina, 5 Área de Toxicología General, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina

Cyanobacteria are globally widespread photosynthetic prokaryotes and are major contributors to global biogeochemical cycles. One of the most critical processes determining cyanobacterial eco-physiology is cellular death. Evidence supports the existence of controlled cellular demise in cyanobacteria, and various forms of cell death have been described as a response to biotic and abiotic stresses. However, cell death research in this phylogenetic group is a relatively young field and understanding of the underlying mechanisms and molecular machinery underpinning this fundamental process remains largely elusive. Furthermore, no systematic classification of modes of cell death has yet been established for cyanobacteria. In this work, we analyzed the state of knowledge in the field of cyanobacterial cell death. Based on that, we propose unified criterion for the definition of accidental, regulated, and programmed forms of cell death in cyanobacteria based on molecular, biochemical, and morphologic aspects following the directions of the Nomenclature Committee on Cell Death (NCCD). With this, we aim to provide a guide to standardize the nomenclature related to this topic in a precise and consistent manner, which will facilitate further ecological, evolutionary, and applied research in the field of cyanobacterial cell death.

[D-Leu1]MC-LR and MC-LR, two microcystins differing in one amino acid, constitute a sanitary and environmental problem owing to their frequent and concomitant presence in water bodies of the Americas and their association with human intoxication during recreational exposure to cyanobacterial bloom. Present in reservoirs used for irrigation as well, they can generate problems in the development of crops such as Phaseolus vulgaris, of nutritional and economic interest to the region. Although numerous works address the toxic effects of MC-LR, information on the toxicity of [D-Leu1]MC-LR is limited. Our objective was to study the toxic effects of [D-Leu1]MC-LR and MC-LR (3.5 µg/ml) on P. vulgaris after a single contact at the imbibition stage. Our findings indicate that 10 days post treatment, [D-Leu1]MC-LR generates morphological and physiological alterations more pronounced than those caused by MC-LR. In addition to the alterations produced by [D-Leu1]MC-LR in the development of seedlings and the structure of the leaves, roots and stems, we also found alterations in leaf stomatal density and conductivity, a longer delay in the phototropic response and a decrease in the maximum curvature angles achieved with respect to that observed for MC-LR. Our findings indicate that these alterations are linked to the greater inhibition of phosphatase activity generated by [D-Leu1]MC-LR, rather than to oxidative damage. We observed that 30 days after treatment with MC-LR, plants presented better development and recovery than those treated with [D-Leu1]MC-LR. Further studies are required on [D-Leu1]MC-LR and MC-LR toxicity and their underlying mechanisms of action.

Two microcystins, MC-LR and [D-Leu1]MC-LR, present in La Plata Basin blooms, are differentiated by substitution of D-Alanine for D-Leucine at position 1. Our objective was to evaluate acute toxicity of [D-Leu1]MC-LR and MC-LR in mice (N:NIH Swiss) and beans (Phaseolus vulgaris). We observed variations in [D-Leu1]MC-LR lethal doses with respect to those reported for MC-LR (100 μg/kg), with an increased liver/body weight ratio and intrahepatic hemorrhages in mice exposed to 50–200 μg [D-Leu1]MC-LR/kg and slight steatosis after a single 25 μg [D-Leu1]MC-LR/kg i.p. dose. Our study in the plant model showed alterations in germination, development, morphology and TBARs levels after a single contact with the toxins during imbibition (3.5 and 15 µg/mL), those treated with [D-Leu1]MC-LR being more affected than those treated with the same concentration of MC-LR. Protein phosphatase 1 (PP1) IC50 values were 40.6 nM and 5.3 nM for [D-Leu1]MC-LR and MC-LR, respectively. However, the total phosphatase activity test in root homogenate showed 60% inhibition for [D-Leu1]MC-LR and 12% for MC-LR. In mouse liver homogenate, 50% inhibition was observed for [D-Leu1]MC-LR and 40% for MC-LR. Our findings indicate the need for further research into [D-Leu1]MC-LR toxicity since together with oxidative stress, the possible inhibition of other phosphatases could explain the differences detected in the potency of the two toxins.

The success of Limnoperna fortunei as an invasive freshwater bivalve species is related to its physiological plasticity to endure changes in environmental conditions. The aim of this study was to investigate the physiological responses of L. fortunei after feeding on Microcystis aeruginosa grown at 26 °C (control) and 29 °C during 10 days. At the beginning, we measured biomass, fatty acids (FAs) composition on Cyanobacteria grown at both temperatures at different time intervals. Afterwards, mussels were fed with the thawed M. aeruginosa cells and their FA profile was measured after 15 days of feeding. M. aeruginosa exposed to 29 °C had the highest content of the FAs 18:2ω6 and cis-18:1ω9. The FA profile of the consumer L. fortunei fed with M. aeruginosa cultures grown at 29 °C was also significantly different to those fed with cultures grown at 26 °C, with a significant increased Eicosapentaenoic acid (EPA, 20:5ω3) and Arachidonic acid (ARA, 20:4ω6) concentrations. L. fortunei was already known to be physiologically adapted to live at 29 °C, but our results also shown a high biosynthesis of EPA and ARA (increase of 70 and 40% respectively, compared with 26 °C) and avoided the lipid peroxidation of both FAs. This increased EPA and ARA biosynthesis may be an important source of ω3 and ω6 polyunsaturated FAs (PUFAs) for higher trophic levels, such as the pelagic fishes or birds that mainly prey on these mussels. The transfer of the cyanobacterial response at higher temperature to higher trophic levels will influence the overall functioning of freshwater bodies.

Summary The effect of chemically modified resistant starch ( RS ) included in bread formulation on bone and gut health of growing male Wistar rats was studied. In order to determine the functional properties and the prebiotic potential of RS , three groups of rats were assayed: a control group ( CD ), a group fed with a diet including RS ( RSD ) and a group fed with a diet containing bread with RS ( BRSD ). Rats that consumed BRSD exhibited lower daily intake indicating that more satiety is reached with this food and this group presented similar calcium absorption than control rats. In addition, an increase in bone mineral content and bone mineral density and better intestinal balance as reflected by higher lactobacilli/enterobacteria ratio was observed in rats fed with both sources of RS . Thus, these results show a prebiotic role of RS that is maintained when RS is included in a bread formulation.

Chitosan-based emulsions functionalized with cabreuva essential oil (Myrocarpus fastigiatus) assembled with poly(vinyl alcohol) (PVA) were tailor-made to obtain a smooth, flexible and thin wound dressings capable for acting as a delivery vehicle. The cabreuva essential oil is composed mainly of trans-nerolidol, a natural compound with antimicrobial capacity that acts as a skin penetration enhancer. During the application of the dressing on skin explants, the penetration profile of cabreuva was analyzed through ex-vivo diffusion assays. Furthermore, the in-vivo essential oil delivery was followed by means of ATR-FTIR. The addition of chitosan-based emulsions produced modifications in the properties of the composite dressings associated with the water affinity and gas permeability as well as in the mechanical behavior. The dressing functionalized with cabreuva essential oil combined with chitosan proved its effectiveness against microorganisms such as S. aureus and S. epidermidis, capacity to produce cell regeneration after 24 h of contact time, and no cytotoxicity in HaCaT cells. The material developed could be used in superficial burns or minor wounds such as dressings due to its demonstrated functionality as controlled release devices of the cabreuva essential oil.

Blooms of the cyanobacterium Microcystis aeruginosa are common in many eutrophic freshwater bodies and pose a serious threat to water quality, potentially giving rise to high turbidity, food web alterations, increased production of toxic microcystin (MC-LR) and odorous compounds. The comparative effectiveness of oxidant treatment of M. aeruginosa cells in culture media was evaluated by applying a mathematical model of chlorophyll-a (Chl-a), cells and MC removal. The oxidants were chlorine (1–5 mg∙L-1), hydrogen peroxide (HP: 50–150 mg∙L-1), percitric acid (PCA: 10–50 mg∙L-1), and peracetic acid (PAA: 1.5–7.5 mg∙L-1). The Weibull distribution model was applied to assess the degree of inactivation of M. aeruginosa viability under different oxidant treatments. First-order kinetics was successfully applied to the experimental data for Chl-a decay. Using the Weibull model, it was possible to predict the required exposure time (Tr) for oxidants to achieve a 99.9% reduction in viable M. aeruginosa cells with respect to the initial value. 5 mg∙L-1 chlorine produced a 81% degradation of [D-Leu1] MC-LR after 72 h, with an exposure time (Tr) of 141 h. Among the peroxide treatments (HP, PCA and PAA), PCA (10–50 mg∙L-1) produced the highest level of [D-Leu1] MC-LR degradation (39–79%), with low exposure times (Tr = 119–125 h). Chl-a concentration and M. aeruginosa counts for each oxidant treatment were highly correlated and successfully linked by a cubic polynomial. This is the first modelling report of M. aeruginosa decay by oxidant treatments.