Integron-associated genes are reliable indicators of antibiotic resistance in wastewater despite treatment- and seasonality-driven fluctuations.

The present study aims to characterize the bacterial community, resistome and integron abundance of a municipal wastewater treatment plant (WWTP) over the course of 12 months and evaluate the year-long performance of integron-related genes as potential indicators of antibiotic resistance mechanisms in influents and effluents. For that, total DNA was extracted and subjected to 16S rRNA-targeted metabarcoding, high-throughput (HT) qPCR (48 targets) and standard qPCR (5 targets). Targets included integrase genes, antibiotic resistance genes (ARGs) and putative pathogenic groups. A total of 16 physicochemical parameters determined in the wastewater samples were also considered. Results revealed that the WWTP treatment significantly impacted the bacterial community, as well as the content in ARGs and integrase genes. Indeed, there was a relative enrichment from influent to effluent of 13 pathogenic groups (e.g., Legionella and Mycobacterium) and genes conferring resistance to sulphonamides, aminoglycosides and disinfectants. Effluent samples (n = 25) also presented seasonal differences, with an increase of the total ARGs' concentration in summer, and differences between winter and summer on relative abundance of sulphonamide and disinfectant resistance mechanisms. From the eight putative integron-related genes selected, all were positively correlated with the total ARGs' content in wastewater and the relative abundance of resistance to most of the specific antibiotic classes. The genes intI1, blaGES and qacE∆1 were the most strongly correlated with the total concentration of ARGs. Genes blaGES and blaVIM, were better correlated to resistance to beta-lactams, aminoglycosides and tetracyclines. This study supports the use of integron-related genes as powerful indicators of antibiotic resistance in wastewater, being robust despite the variability caused by wastewater treatment and seasonality.

Altered expression of Sialyl Lewis X in experimental models of Parkinson's disease.

The mechanisms underlying neurodegeneration in Parkinson's disease (PD) are still not fully understood. Glycosylation is an important post-translational modification that affects protein function, cell-cell contacts and inflammation and can be modified in pathologic conditions. Although the involvement of aberrant glycosylation has been proposed for PD, the knowledge of the diversity of glycans and their role in PD is still minimal. Sialyl Lewis X (sLeX) is a sialylated and fucosylated tetrasaccharide with essential roles in cell-to-cell recognition processes. Pathological conditions and pro-inflammatory mediators can up-regulate sLeX expression on cell surfaces, which has important consequences in intracellular signalling and immune function. Here, we investigated the expression of this glycan using in vivo and in vitro models of PD. We show the activation of deleterious glycation-related pathways in mouse striatum upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin-based model of PD. Importantly, our results show that MPTP triggers the presentation of more proteins decorated with sLeX in mouse cortex and striatum in a time-dependent manner, as well as increased mRNA expression of its rate-limiting enzyme fucosyltransferase 7. sLeX is expressed in neurons, including dopaminergic neurons, and microglia. Although the underlying mechanism that drives increased sLeX epitopes, the nature of the protein scaffolds and their functional importance in PD remain unknown, our data suggest for the first time that sLeX in the brain may have a role in neuronal signalling and immunomodulation in pathological conditions. KEY MESSAGES: MPTP triggers the presentation of proteins decorated with sLeX in mouse brain. MPTP triggers the expression of sLeX rate-limiting enzyme FUT 7 in striatum. sLeX is expressed in neurons, including dopaminergic neurons, and microglia. sLeX in the brain may have a role in neuronal signalling and immunomodulation.

NPC1-like phenotype, with intracellular cholesterol accumulation and altered mTORC1 signaling in models of Parkinson's disease.

Disruption of brain cholesterol homeostasis has been implicated in neurodegeneration. Nevertheless, the role of cholesterol in Parkinson's Disease (PD) remains unclear. We have used N2a mouse neuroblastoma cells and primary cultures of mouse neurons and 1-methyl-4-phenylpyridinium (MPP+), a known mitochondrial complex I inhibitor and the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), known to trigger a cascade of events associated with PD neuropathological features. Simultaneously, we utilized other mitochondrial toxins, including antimycin A, oligomycin, and carbonyl cyanide chlorophenylhydrazone. MPP+ treatment resulted in elevated levels of total cholesterol and in a Niemann Pick type C1 (NPC1)-like phenotype characterized by accumulation of cholesterol in lysosomes. Interestingly, NPC1 mRNA levels were specifically reduced by MPP+. The decrease in NPC1 levels was also seen in midbrain and striatum from MPTP-treated mice and in primary cultures of neurons treated with MPP+. Together with the MPP+-dependent increase in intracellular cholesterol levels in N2a cells, we observed an increase in 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and a concomitant increase in the phosphorylated levels of mammalian target of rapamycin (mTOR). NPC1 knockout delayed cell death induced by acute mitochondrial damage, suggesting that transient cholesterol accumulation in lysosomes could be a protective mechanism against MPTP/MPP+ insult. Interestingly, we observed a negative correlation between NPC1 protein levels and disease stage, in human PD brain samples. In summary, MPP+ decreases NPC1 levels, elevates lysosomal cholesterol accumulation and alters mTOR signaling, adding to the existing notion that PD may rise from alterations in mitochondrial-lysosomal communication.

Cholesterol redistribution triggered by CYP46A1 gene therapy improves major hallmarks of Niemann-Pick type C disease but is not sufficient to halt neurodegeneration.

Cholesterol 24-hydroxylase (CYP46A1) is an exclusively neuronal cytochrome P450 enzyme responsible for converting cholesterol into 24S-hydroxycholesterol, which serves as the primary pathway for eliminating cholesterol in the brain. We and others have shown that increased activity of CYP46A1 leads to reduced levels of cholesterol and has a positive effect on cognition. Therefore, we hypothesized that CYP46A1 could be a potential therapeutic target in Niemann-Pick type C (NPC) disease, a rare and fatal neurodegenerative disorder, characterized by cholesterol accumulation in endolysosomal compartments. Herein, we show that CYP46A1 ectopic expression, in cellular models of NPC and in Npc1tm(I1061T) mice by adeno-associated virus-mediated gene therapy improved NPC disease phenotype. Amelioration in functional, biochemical, molecular and neuropathological hallmarks of NPC disease were characterized. In vivo, CYP46A1 expression partially prevented weight loss and hepatomegaly, corrected the expression levels of genes involved in cholesterol homeostasis, and promoted a redistribution of brain cholesterol accumulated in late endosomes/lysosomes. Moreover, concomitant with the amelioration of cholesterol metabolism dysregulation, CYP46A1 attenuated microgliosis and lysosomal dysfunction in mouse cerebellum, favoring a pro-resolving phenotype. In vivo CYP46A1 ectopic expression improves important features of NPC disease and may represent a valid therapeutic approach to be used concomitantly with other drugs. However, promoting cholesterol redistribution does not appear to be enough to prevent Purkinje neuronal death in the cerebellum. This indicates that cholesterol buildup in neurons might not be the main cause of neurodegeneration in this human lipidosis.

The H9c2(2-1) cell-based sulforhodamine B assay is a non-animal alternative to evaluate municipal wastewater quality over time.

The present study validates the potential of the in vitro H9c2(2-1) cell-based sulforhodamine B (SRB) assay to evaluate the temporal variability of wastewater quality. The impact of effluent disposal on water quality and the efficiency of the wastewater treatment process were also assessed. To correlate standard analytical method results with in vitro results, a total of 16 physicochemical parameters, such as nutrients, pH, chemical oxygen demand, total suspended solids and metals, were determined in both raw and treated wastewater samples. Results revealed that the H9c2(2-1) cell-based SRB assay has an enormous potential to evaluate municipal wastewater quality over time and to discriminate influent and effluent toxic characteristics, as well as for water quality monitoring and surveillance of the efficacy of treatment processes. Finally, the gathered results alerted to the impact of phosphates in a biological system, leading us to recommend the selection of this parameter as a potential environmental health indicator.

Development and repair of blood vessels in the zebrafish spinal cord.

The vascular system is inefficiently repaired after spinal cord injury (SCI) in mammals, resulting in secondary tissue damage and immune deregulation that contribute to the limited functional recovery. Unlike mammals, zebrafish can repair the spinal cord (SC) and restore motility, but the vascular response to injury has not been investigated. Here, we describe the zebrafish SC blood vasculature, starting in development with the initial vessel ingression in a body size-dependent manner, the acquisition of perivascular support and the establishment of ventral to dorsal blood circulation. The vascular organization grows in complexity and displays multiple barrier specializations in adulthood. After injury, vessels rapidly regrow into the lesion, preceding the glial bridge and axons. Vascular repair involves an early burst of angiogenesis that creates dysmorphic and leaky vessels. Dysfunctional vessels are later removed, as pericytes are recruited and the blood-SC barrier is re-established. This study demonstrates that zebrafish can successfully re-vascularize the spinal tissue, reinforcing the value of this organism as a regenerative model for SCI.

Microplastics accumulate priority antibiotic-resistant pathogens: Evidence from the riverine plastisphere.

Microplastics (MPs) might accumulate and transport antibiotic-resistant bacteria (ARB) in aquatic systems. We determined the abundance and diversity of culturable ciprofloxacin- and cefotaxime-resistant bacteria in biofilms covering MPs placed in river water, and characterized priority pathogens from these biofilms. Our results showed that the abundance of ARB colonizing MPs tends to be higher compared to sand particles. Also, higher numbers were cultivated from a mixture of polypropylene (PP), polyethylene (PE) and polyethylene terephthalate (PET), compared to PP and PET alone. Aeromonas and Pseudomonas isolates were the most frequently retrieved from MPs placed before a WWTP discharge while Enterobacteriaceae dominated the culturable plastisphere 200 m after the WWTP discharge. Ciprofloxacin- and/or cefotaxime-resistant Enterobacteriaceae (n = 54 unique isolates) were identified as Escherichia coli (n = 37), Klebsiella pneumoniae (n = 3), Citrobacter spp. (n = 9), Enterobacter spp. (n = 4) and Shigella sp. (n = 1). All isolates presented at least one of the virulence features tested (i.e. biofilm formation, haemolytic activity and production of siderophores), 70% carried the intI1 gene and 85% exhibited a multi-drug resistance phenotype. Plasmid-mediated quinolone resistance genes were detected in ciprofloxacin-resistant Enterobacteriaceae [aacA4-cr (40% of the isolates), qnrS (30%), qnrB (25%), and qnrVC (8%)], along with mutations in gyrA (70%) and parC (72%). Cefotaxime-resistant strains (n = 23) harbored blaCTX-M (70%), blaTEM (61%) and blaSHV (39%). Among CTX-M producers, high-risk clones of E. coli (e.g. ST10 or ST131) and K. pneumoniae (ST17) were identified, most of which carrying blaCTX-M-15. Ten out of 16 CTX-M producers were able to transfer blaCTX-M to a recipient strain. Our results demonstrated the occurrence of multidrug resistant Enterobacteriaceae in the riverine plastisphere, harboring ARGs of clinical concern and exhibiting virulence traits, suggesting a contribution of MPs to the dissemination of antibiotic-resistant priority pathogens. The type of MPs and especially water contamination (e.g. by WWTPs discharges) seem to determine the resistome of the riverine plastisphere.

It's a two-way street: Photoswitching and reversible changes of the protein matrix in photoswitchable fluorescent proteins and bacteriophytochromes.

Chromophore-bearing proteins that are (reversibly) altered after light illumination are major functional components of nature. They gained considerable attention in the last decades since the dynamic interactions of the chromophore and protein matrix can be used to control downstream effects altering the functionality of proteins, cells, or complete organisms with light (optogenetics). Additionally, the photophysical effects can be employed to add capabilities to optical imaging. For example, light can be used to reversibly switch the signal on or off (e.g., fluorescence). In this article, we review chromophore and protein matrix interactions, focusing on photoswitching fluorescent proteins of the GFP family (RSFPs) and natively photoswitching bacteriophytochromes (BphPs). This review aims to provide an in-depth understanding of the dynamic interplay between photoswitching photophysics and the protein matrix and a thorough discussion on how this connection has been harnessed for the development of optogenetic and imaging tools.

Are mercury levels in fishery products appropriate to ensure low risk to high fish-consumption populations?

The main goal of the present study is to determine the sources of methylmercury (MeHg) for high fish-consumption populations with the Portuguese population as showcase, as Portugal is the EU country with the highest fish consumption per capita (2019: 59.91 kg year-1). Since limited information is available on the effective levels of mercury after culinary treatments, cooked and raw codfish (Gadus morhua), hake (Merluccius merluccius), octopus (Octopus vulgaris), horse mackerel (Trachurus trachurus) and sardine (Sardina pilchardus) were considered. The mercury concentration ranking Hake > Horse mackerel > Codfish > Octopus > Sardine was observed in all situations (cooked and raw samples) for both MeHg and total mercury (T-Hg). The gathered results reinforce the general assumption that the loss of moisture during cooking increases MeHg and T-Hg concentrations in fish, but the idea that MeHg in fish muscle tissue represents the bulk of T-Hg cannot be generalised, as our study determined a MeHg/T-Hg ratio of 0.43 for grilled sardines.

Validity of a technological rehabilitation nursing program for people undergoing knee arthroplasty / Validación del programa de enfermería tecnológica en rehabilitación de personas realizadas en artroplastia de rodilla / Validação do programa de enfermagem de reabilitação tecnológico para pessoas submetidas a artroplastia do joelho

ABSTRACT Objective: to validate a Technological Rehabilitation Nursing Program for people undergoing total knee arthroplasty. Methods: this is a qualitative study, carried out through a focus group, with 12 nurses, considered experts in the area of rehabilitation. The program was developed using digital technology, such as an application for a mobile device. Experts assessed the program structure, the content made available to people undergoing total knee arthroplasty pre-operatively and post-operatively and the follow-up and communication strategies with nurses. Results: after content validity by experts, the final version of the program integrated three thematic areas and their respective categories: Rehabilitation program (Program phases, Program operationalization, Exercise plans included in the program); Useful information (Preparation for surgery, Care to be taken during surgery recovery); and Communication channel with nurses (Talk to a rehabilitation nurse, Self-assessment of health condition and Help with decision-making). Conclusion: experts' contributions made it possible to achieve the content validity of the program and, consequently, improve patient literacy about the procedure, complication prevention and self-care; training patients to carry out exercise plans in the pre- and post-operative periods; and communication with nurses through application.

RESUMEN Objetivo: validar un Programa Tecnológico de Enfermería de Rehabilitación para personas sometidas a artroplastia total de rodilla. Métodos: estudio cualitativo, realizado a través de un focus group, con 12 enfermeros, considerados expertos en el área de rehabilitación. El programa fue desarrollado utilizando tecnología digital, como una aplicación para un dispositivo móvil. Los expertos evaluaron la estructura del programa, los contenidos puestos a disposición de las personas sometidas a artroplastia total de rodilla en el preoperatorio y postoperatorio, y las estrategias de seguimiento y comunicación con la enfermera. Resultados: luego de la validación de contenido por parte de expertos, la versión final del Programa integró tres áreas temáticas y sus respectivas categorías: Programa de rehabilitación (Fases del programa, Operacionalización del programa, Planes de ejercicio incluidos en el programa); Información útil (Preparación para la cirugía, Cuidados a tener durante la recuperación quirúrgica); y Canal de Comunicación con la Enfermera (Hablar con una enfermera de rehabilitación, Autoevaluación del estado de salud y Ayuda en la toma de decisiones). Conclusión: los aportes de los expertos permitieron lograr la validez de contenido del programa y, en consecuencia, mejorar la alfabetización de los pacientes sobre el procedimiento, la prevención de complicaciones y el autocuidado; entrenar al paciente para que lleve a cabo planes de ejercicios en los períodos pre y postoperatorios; y comunicación con la enfermera a través de la aplicación.

RESUMO Objetivo: validar um Programa de Enfermagem de Reabilitação Tecnológico para pessoas submetidas à artroplastia total do joelho. Métodos: estudo qualitativo, realizado por meio de focus group, com 12 enfermeiros, considerados peritos na área de reabilitação. O Programa foi desenvolvido com recurso de uma tecnologia digital, do tipo aplicativo para dispositivo móvel. Os peritos avaliaram a estrutura do Programa, os conteúdos disponibilizados às pessoas submetidas a artroplastia total do joelho no pré-operatório e pós-operatório e as estratégias de acompanhamento e comunicação com o enfermeiro. Resultados: após a validação de conteúdo pelos peritos, a versão final do Programa integrou três áreas temáticas e suas respectivas categorias: Programa de Reabilitação (Fases do Programa, Operacionalização do Programa, Planos de exercícios incluídos no Programa); Informação Útil (Preparação para a cirurgia, Cuidados a ter durante a recuperação cirúrgica); e Canal Comunicacional com o Enfermeiro (Fale com enfermeiro de reabilitação, Autoavaliação da condição de saúde e Ajuda na tomada de decisão). Conclusão: os contributos dos peritos permitiram alcançar a validade de conteúdo do Programa e, consequentemente, melhorar a literacia do paciente sobre o procedimento, prevenção de complicações e autocuidado; instrumentalização do paciente para a realização dos planos de exercícios nos períodos pré e pós-operatório; e a comunicação com o enfermeiro pelo aplicativo.

APC couples neuronal mRNAs to multiple kinesins, EB1, and shrinking microtubule ends for bidirectional mRNA motility.

Understanding where in the cytoplasm mRNAs are translated is increasingly recognized as being as important as knowing the timing and level of protein expression. mRNAs are localized via active motor-driven transport along microtubules (MTs) but the underlying essential factors and dynamic interactions are largely unknown. Using biochemical in vitro reconstitutions with purified mammalian proteins, multicolor TIRF-microscopy, and interaction kinetics measurements, we show that adenomatous polyposis coli (APC) enables kinesin-1- and kinesin-2-based mRNA transport, and that APC is an ideal adaptor for long-range mRNA transport as it forms highly stable complexes with 3'UTR fragments of several neuronal mRNAs (APC-RNPs). The kinesin-1 KIF5A binds and transports several neuronal mRNP components such as FMRP, PURα and mRNA fragments weakly, whereas the transport frequency of the mRNA fragments is significantly increased by APC. APC-RNP-motor complexes can assemble on MTs, generating highly processive mRNA transport events. We further find that end-binding protein 1 (EB1) recruits APC-RNPs to dynamically growing MT ends and APC-RNPs track shrinking MTs, producing MT minus-end-directed RNA motility due to the high dwell times of APC on MTs. Our findings establish APC as a versatile mRNA-kinesin adaptor and a key factor for the assembly and bidirectional movement of neuronal transport mRNPs.

Determination of intestinal absorption of the paralytic shellfish toxin GTX-5 using the Caco-2 human cell model.

Contributing to the human health risk assessment, the present study aims to evaluate the ability of paralytic shellfish toxins (PSTs) to cross the human intestinal epithelium by using the Caco-2 permeability assay. A crude extract prepared from the PST producer dinoflagellate Gymnodinium catenatum strain, GCAT1_L2_16, and the PST analogue gonyautoxin-5 (GTX-5) prepared from a certified reference material (CRM) were tested. In the conditions of the assay, none of the compounds altered Caco-2 viability, or the integrity of cell monolayers. The GTX-5 apparent permeability coefficients are 0.9×10-7 and 0.6×10-7 cm s-1 for the crude extract and CRM, respectively, thus, <10-6 cm s-1, which indicates that humans absorb this PST analogue poorly. The present study also reveals that, during a 90-min exposure, GTX-5 is not metabolised to a high extent by Caco-2 or retained in the Caco-2 cytoplasm. Since it is known that GTX-5 can be found in the spleen, liver or kidney of the victims, as well as in the urine samples of patients who consumed contaminated seafood, further research is needed to clarify the transport mechanisms involved, permeation time and dose-dependence, and the possible role of intestinal microflora.

Mammalian Neuronal mRNA Transport Complexes: The Few Knowns and the Many Unknowns.

Hundreds of messenger RNAs (mRNAs) are transported into neurites to provide templates for the assembly of local protein networks. These networks enable a neuron to configure different cellular domains for specialized functions. According to current evidence, mRNAs are mostly transported in rather small packages of one to three copies, rarely containing different transcripts. This opens up fascinating logistic problems: how are hundreds of different mRNA cargoes sorted into distinct packages and how are they coupled to and released from motor proteins to produce the observed mRNA distributions? Are all mRNAs transported by the same transport machinery, or are there different adaptors or motors for different transcripts or classes of mRNAs? A variety of often indirect evidence exists for the involvement of proteins in mRNA localization, but relatively little is known about the essential activities required for the actual transport process. Here, we summarize the different types of available evidence for interactions that connect mammalian mRNAs to motor proteins to highlight at which point further research is needed to uncover critical missing links. We further argue that a combination of discovery approaches reporting direct interactions, in vitro reconstitution, and fast perturbations in cells is an ideal future strategy to unravel essential interactions and specific functions of proteins in mRNA transport processes.

H9c2(2-1)-based sulforhodamine B assay as a possible alternative in vitro platform to investigate effluent and metals toxicity on fish.

To overcome restrictions on the use of fish in toxicity testing, the present study proposes to compare the 50% growth inhibition potential (EC50) of four types of effluents on the rat cardiomyoblast H9c2(2-1) cell line by using the sulforhodamine B (SRB) cell mass colorimetric assay, with the corresponding fish lethal test results. Our objective was to evaluate if H9c2(2-1) cells shows comparable sensitivities, in both relative and absolute terms, to those provided by fish. In parallel, this study also compared the results of the chemical characterization with the legislation in force for environmental protection against effluent release into the receiving environment. Moreover, we tested the H9c2(2-1)-based SRB assays with the metals of concern found in the effluent samples. Both fish and cell assays showed the same toxicity rank for effluents: Metal > Oil > Municipal > Paper, and it should be stressed that the complementarity of using chemical and biological data represents a step forward to guarantee both environmental and human safety, since the chemical characterization showed a different toxicity rank: Metal > Municipal > Oil > Paper. Regarding metal elements, the short-term fish results showed a toxicity rank non-comparable with the rank obtained for cells. Nevertheless, the gathered results reveal the potentiality of the in vitro H9c2(2-1) platform as an alternative for fish lethal testing to assess, in absolute terms, the toxicity of effluents, particularly municipal effluents, and metals.

Foliar application of 3-hydroxy-4-pyridinone Fe-chelate [Fe(mpp)3 ] induces responses at the root level amending iron deficiency chlorosis in soybean.

Iron (Fe) deficiency chlorosis (IDC) affects the growth of several crops, especially when growing in alkaline soils. The application of synthetic Fe-chelates is one of the most commonly used strategies in IDC amendment, despite their associated negative environmental impacts. In a previous work, the Fe-chelate tris(3-hydroxy-1-(H)-2-methyl-4-pyridinonate) iron(III) [Fe(mpp)3 ] has shown great potential for alleviating IDC in soybean (Glycine max) in the early stages of plant development under hydroponic conditions. Herein, its efficacy was verified under soil conditions in soybean grown from seed to full maturity. Chlorophyll levels, plant growth, root and shoot mineral accumulation (K, Mg, Ca, Na, P, Mn, Zn, Ni, and Co) and FERRITIN expression were accessed at V5 phenological stage. Compared to a commonly used Fe chelate, FeEDDHA, supplementation with [Fe(mpp)3 ] led to a 29% higher relative chlorophyll content, 32% higher root biomass, 36% higher trifoliate Fe concentration, and a twofold increase in leaf FERRITIN gene expression. [Fe(mpp)3 ] supplementation also resulted in increased accumulation of P, K, Zn, and Co. At full maturity, the remaining plants were harvested and [Fe(mpp)3 ] application led to a 32% seed yield increase when compared to FeEDDHA. This is the first report on the use of [Fe(mpp)3 ] under alkaline soil conditions for IDC correction, and we show that its foliar application has a longer-lasting effect than FeEDDHA, induces efficient root responses, and promotes the uptake of other nutrients.

Exposure to marine benthic dinoflagellate toxins may lead to mitochondrial dysfunction.

Even though marine dinoflagellates are important primary producers, many toxic species may alter the natural equilibrium of aquatic ecosystems and even generate human intoxication incidents, as they are the major causative agents of harmful algal blooms. In order to deepen the knowledge regarding benthic dinoflagellate adverse effects, the present study aims to clarify the influence of Gambierdiscus excentricus strain UNR-08, Ostreopsis cf. ovata strain UNR-03 and Prorocentrum lima strain UNR-01 crude extracts on rat mitochondrial energetic function and permeability transition pore (mPTP) induction. Our results, expressed in number of dinoflagellate cell toxic compounds tested in a milligram of mitochondrial protein, revealed that 934 cells mg prot-1 of G. excentricus, and 7143 cells mg prot-1 of both O. cf. ovata and P. lima negatively affect mitochondrial function, including by decreasing ATP synthesis-related membrane potential variations. Moreover, considerably much lower concentrations of dinoflagellate extracts (117 cells mg prot-1 of G. excentricus, 1429 cells mg prot-1 of O. cf. ovata and 714 cells mg prot-1 of P. lima) produced mPTP-induced swelling in Ca2+-loaded isolated mitochondria. The present study clearly demonstrates the toxicity of G. excentricus, O. cf. ovata and P. lima extracts at the mitochondrial level, which may lead to mitochondrial failure and consequent cell toxicity, and that G. excentricus always provide much more severe effects than O. cf. ovata and P. lima.

Rat cardiomyocyte H9c2(2-1)-based sulforhodamine B assay as a promising in vitro method to assess the biological component of effluent toxicity.

The treatment of wastewaters is crucial to maintain the ecological status of receiving waters, and thereby guarantee the protection of aquatic life and human health. Wastewater quality evaluation is conventionally based on physicochemical parameters, but increasing attention has been paid to integrate physicochemical and biological data. Nevertheless, the regulatory use of fish in biological testing methods has been subject to various ethical and cost concerns, and in vitro cell-based assays have thus become an important topic of interest. Hence, the present study intends: (a) to evaluate the efficiency of two different sample pre-concentration techniques (lyophilisation and solid phase extraction) to assess the toxicity of municipal effluents on rat cardiomyoblast H9c2(2-1) cells, and (b) maximizing the use of the effluent sample collected, to estimate the environmental condition of the receiving environment. The gathered results demonstrate that the H9c2(2-1) sulforhodamine B-based assay is an appropriate in vitro method to assess biological effluent toxicity, and the best results were attained by lyophilising the sample as pre-treatment. Due to its response, the H9c2(2-1) cell line might be a possible alternative in vitro model for fish lethal testing to assess the toxicity of municipal effluents. The physicochemical status of the sample suggests a high potential for eutrophication, and iron exceeded the permissible level for wastewater discharge, possibly due to the addition of ferric chloride for wastewater treatment. In general, the levels of carbamazepine and sulfamethoxazole are higher than those reported for other countries, and both surpassed the aquatic protective values for long-term exposure.

High sensitivity of rat cardiomyoblast H9c2(2-1) cells to Gambierdiscus toxic compounds.

Ciguatera fish poisoning is a frequently reported non-bacterial food-borne illness related to the consumption of seafood contaminated with ciguatoxins, and possibly maitotoxins. These toxins are synthesized by marine dinoflagellate species of Gambierdiscus and Fukuyoa genera, and their abundance is a matter of great concern due to their adverse effects to aquatic life and human health. The present study aims to assess the sensitivity of rat cardiomyoblast H9c2(2-1) cells to Gambierdiscus toxic compounds using concentration- and time-dependent sulforhodamine B (SRB) colorimetric assays. Low concentrations of Gambierdiscus extracts (corresponding to 1.3-2.3 cells mL-1) induced a concentration-dependent response. Specificity in time-dependent response of H9c2(2-1) cells was demonstrated for G. excentricus after a 180 min exposure compared to both G. cf. belizeanus and G. silvae species, with EC50s obtained after 720 and 360 min, respectively. The sensitivity of H9c2(2-1) cells to dinoflagellate toxic compounds was also tested with other genera from benthic (Coolia malayensis, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and P. lima) and planktonic (Amphidinium carterae and Lingulodinium polyedrum) habitats. Amphidinium, Coolia and Lingulodinium data did not present any concentration-response relationships, and EC50 values could only be obtained after 720 and 1440 min of exposure to both Prorocentrum species and O. cf. ovata, respectively. This study demonstrated that the H9c2(2-1) SRB assay represents a promising and sensitive tool for the detection of Gambierdiscus toxic compounds present in water samples, particularly of G. excentricus at very low cell abundances.

Mitochondrial impairment and cytotoxicity effects induced by the marine epibenthic dinoflagellate Coolia malayensis.

Mitochondria was used to clarify the effects of Coolia malayensis strain UNR-02 crude extract by studying mitochondrial membrane potential (ΔΨm) generation and the fluctuations of ΔΨm associated with the induction of mitochondrial permeability transition (MPT). The cytoxicity of C. malayensis was also determined using both HepG2 and H9c2(2-1) cells. C. malayensis extract significantly depressed the oxidative phosphorylation efficiency, as was inferred from the perturbations in ΔΨm and in the phosphorylative cycle induced by ADP. Increased susceptibility to Ca2+-induced MPT was also observed. At the cellular level, the extract significantly decreased cell mass of both cell lines.