Precision-cut liver slices as a model for assess hepatic cellular response of chitosan-glutathione nanoparticles on cultures treated with zilpaterol and clenbuterol.

Zilpaterol and clenbuterol are two ß-adrenergic agonist drugs used in animal production. Both drugs have anabolic effects with advantages on carcass yield. Meanwhile, zilpaterol is approved for animal feed in authorized countries. Clenbuterol is a banned substance due to the risk of toxicity; however, it is still being used in unknown dose levels in many farm species. Therefore, the use and abuse of these substances should be closely monitored, considering the clenbuterol ability and the not proved yet of zilpaterol to produce reactive oxygen and nitrogen species. Regarding glutathione which is the main intracellular antioxidant plays detoxification functions on liver metabolism; in this work, it is our interest to know the capacity of chitosan-glutathione nanoparticles (CS/GSH-NP) as a complementary source of exogenous GSH to modify the oxide-reduction status on bovine precision-cut liver slice cultures (PCLS) exposed to clenbuterol and zilpaterol. A single drug assay was performed in first instance by adding clenbuterol, zilpaterol, chitosan nanoparticles (CS-NP), and CS/GSH-NP. Then combinate drug assay was carried out by testing clenbuterol and zilpaterol combined with CS-NP or CS/GSH-NP. The results showed that both ß-adrenergic agonists modify in a dose-dependent manner in oxide-reduction response through ROS generation. The activity or content of glutathione peroxidase activity, intracellular GSH, gamma glutamyl-transpeptidase, aspartate aminotrasnferase and alanine aminotrasnferase were modified. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.

Ultrastructural insights into the replication cycle of salmon pancreas disease virus (SPDV) using salmon cardiac primary cultures (SCPCs).

Salmon pancreas disease virus (SPDV) has been affecting the salmon farming industry for over 30 years, but despite the substantial amount of studies, there are still a number of recognized knowledge gaps, for example in the transmission of the virus. In this work, an ultrastructural morphological approach was used to describe observations after infection by SPDV of an ex vivo cardiac model generated from Atlantic salmon embryos. The observations in this study and those available on previous ultrastructural work on SPDV are compared and contrasted with the current knowledge on terrestrial mammalian and insect alphaviral replication cycles, which is deeper than that of SPDV both morphologically and mechanistically. Despite their limitations, morphological descriptions remain an excellent way to generate novel hypotheses, and this has been the aim of this work. This study has used a target host, ex vivo model and resulted in some previously undescribed features, including filopodial membrane projections, cytoplasmic stress granules or putative intracytoplasmic budding. The latter suggests a new hypothesis that warrants further mechanistic research: SPDV in salmon may have retained the capacity for non-cytolytic (persistent) infections by intracellular budding, similar to that noted in arthropod vectors of other alphaviruses. In the notable absence of a known intermediate host for SPDV, the presence of this pattern suggests that both cytopathic and persistent infections may coexist in the same host. It is our hope that the ultrastructural comparison presented here stimulates new research that brings the knowledge on SPDV replication cycle up to a similar level to that of terrestrial alphaviruses.

Detection and characterisation of haplosporidian parasites of the blue mussel Mytilus edulis, including description of the novel parasite Minchinia mytili n. sp.

The edible mussel Mytilus edulis is a major aquaculture commodity in Europe, with 168000 t produced in 2015. A number of abundant, well characterised parasites of the species are known, though none are considered to cause significant mortality. Haplosporida (Rhizaria, Endomyxa) is an order of protistan parasites of aquatic invertebrates, the best studied of which are the oyster pathogens Haplosporidium nelsoni and Bonamia ostreae. While these species are well characterised within their hosts, the diversity, life-cycle and modes of transmission of haplosporidians are very poorly understood. Haplosporidian parasites have previously been reported from Mytilus spp., however the majority of these remain uncharacterised, and no molecular data exist for any species. In this study, we identified 2 novel haplosporidian parasites of M. edulis present in the UK. The first of these, observed by light microscopy and in situ hybridisation infecting the gills, mantle, gonadal tubules and digestive connective tissues of mussels in the Tamar estuary, England, we describe as Minchinia mytili on the basis of 18S sequence data. The second, observed infecting a single archive specimen collected in Loch Spelve, Mull, Scotland, infects the foot muscle, gills and connective tissue of the digestive gland. Sequence data places this parasite in an uncharacterised clade of sequences amplified from tropical bivalve guts and water samples, sister to H. nelsoni. Screening of water and sediment samples collected at the sample site in the Tamar estuary revealed the presence of both sequence types in the water column, suggesting host-free or planktonic life stages.

Development of a method for the determination of 8-iso-PGF2α in sheep and goat plasma using solid-phase microextraction and ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Isoprostane 8-iso-PGF2α is a biomarker of lipid peroxidation in cell membranes. The method developed to measure plasma total levels (esterified + free) of 8-iso-PGF2α must be reproducible and be able to reduce the use of solvents in solid phase extraction. It should be useful to evaluate oxidative stress due to the excess of free radicals that are generated by some disorder or disease. METHODS: The method was developed using solid-phase microextraction with Oasis®MAX µElution plates and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). Electrospray ionization was performed in the negative mode (ESI-); the multiple reaction monitoring mode (MRM) was used. The development of the method included the optimization of the chromatographic conditions to achieve the separation of PGF2α and 8-iso-PGF2α as well as the optimization of the microextraction conditions of the analyte of interest in ovine and goat plasma. RESULTS: The developed method was validated with a calibration curve of plasma samples fortified with standards at five concentration levels in the range 49-639 pg/mL. The average recovery was 89% with a standard deviation of 10.73%. The inter-day precision was evaluated, obtaining a coefficient of variance (CV) less than 15%. The limit of quantification was 20 pg/mL and the limit of detection was 10 pg/mL. 8-iso-PGF2a was determined in the plasma of 14 sheep and 20 goats of 5 months of age and 6 goats of 24 months of age. The concentrations found were 50-300 pg/mL. CONCLUSIONS: The method developed is precise, accurate and reliable with low reagent consumption compared with conventional solid-phase extraction. The analysis time was decreased because, with the use of the microextraction plate, the step of the evaporation and reconstitution of the analyte was avoided. The method is applicable to quantify the plasma total levels (esterified + free) of 8-iso-PGF2α.

Use of Salmon Cardiac Primary Cultures (SCPCs) of different genotypes for comparative kinetics of mx expression, viral load and ultrastructure pathology, after infection with Salmon Pancreas Disease Virus (SPDV).

In vitro fish based models have been extensively applied in human biomedical research but, paradoxically, less frequently in the research of fish health issues. Farmed Atlantic salmon can suffer from several viral conditions affecting the heart. Therefore, species-specific, cardiac in vitro models may represent a useful tool to help further understanding and management of these diseases. The mechanisms underlying genotype based resistance are complex and usually rely on a combined effect of elements from both the innate and adaptive immune response, which are further complicated by external environmental factors. Here we propose that Salmon Cardiac Primary Cultures (SCPCs) are a useful tool to investigate these mechanisms as the basis for genotypic differences between Atlantic salmon families in susceptibility to cardiotropic viral disease. Using SCPCs produced from two different commercially available Atlantic salmon embryonated ova (Atlantic Ova IPN sensitive" (S) and "Atlantic QTL-innOva® IPN/PD" (R)), the influence of host genotype on the viral load and mx expression following Salmon Pancreas Disease Virus infection was assessed over a 15 day period. Both R and S SCPCs groups were successfully infected. A measurable difference between groups of viral nsP1 and host antiviral mx gene expression was observed (i.e. a later, but larger onset of mx expression in the R group). Mx expression peaks were followed by a decrease in viral nsP1 in both groups. Additionally, ultrastructural examination of infected SCPCs allowed the description of degenerative changes at the individual cell level. The SCPC model presents some advantages, over current fish cell culture monolayers and in vivo material, such as the presence of different cell components normally present in the target organ, as well as the removal of a layer of functional complexity (acquired immunity), making it possible to focus on tissue specific, early innate immune mechanisms. These preliminary results highlight the importance of considering genetic origin when selecting the fish source for the production of SCPCs, as well as their usefulness as screening tools for assessment of genotypic differences in disease resistance.

Modulating receptor-ligand binding in biorecognition by setting surface wettability.

Modulation of support wettability used for microarray format biosensing has led to an improvement of results. Hydrophobicity of glass chips was set by derivatizing with single vinyl organosilanes of different chain length and silane mixtures. Thiol-ene photochemical linking has been used as effective chemistry for covalent anchoring of thiolated probes. Lowest unspecific binding and highest signal intensity and SNR were obtained with large hydrocarbon chain (C22) silanes or a shorter one (C10) containing fluorine atoms. SNR resulting values are improved, reaching levels higher than 1500 in some cases, when using vinyl silanes modified with 1% C10 alkyl fluorinated one, because mild hydrophobicity was achieved (water contact angle ca. 110°) for all silanes, including the short C2 and C3, thus giving rise to smaller and better defined array spots. In addition, unspecific binding of reagents and targets was totally withdrawn. Hence, good-performing surfaces for biosensing applications can be built using appropriate organosilane reagent selection, including fluorinated ones. Graphical abstract ᅟ.

Effects of repeated anaesthesia on gill and general health of Atlantic salmon, Salmo salar.

Fish are the second most widely utilized vertebrate group used for scientific procedures in the United Kingdom, but the development and application of 3Rs (the principles of replacement, reduction, and refinement) in aquaculture disease research lags behind methodologies in place for mammalian studies. With a need for individual monitoring and non-lethal sampling, the effect of repeat anaesthesia on experimental fish needs to be better understood. This study analyses the effect of repeat anaesthesia with MS-222, metomidate and AQUI-S upon the gill and general health of post-smolt Atlantic salmon Salmo salar. A single, lethal dose of anaesthetic was compared with seven anaesthetizing time points over 28 days, terminating in a lethal dose. No anaesthetic showed significant differences in accumulation in the muscle tissue, or changes in plasma glucose after repeated or single dosing. Fish repeatedly anaesthetized with MS-222 or AQUI-S exhibited upregulation of osmoregulatory genes in the gill and AQUI-S-treated individuals showed, histologically, epithelial lifting from the lamellae capillary irrespective of whether they had a single or repeated dose history. No significant changes were seen in inflammatory or stress genes in the head kidney of fish repeatedly anaesthetized with AQUI-S or metomidate, however MS-222 treatment resulted in upregulation of tnfα3. Repeated anaesthesia with MS-222 and metomidate gave a significant decrease and increase in peripheral blood neutrophils, respectively. This study concludes that no increase in cumulative stress or inflammation is induced by the repeated anaesthetization of S. salar with any of the tested anaesthetics, however gill osmotic regulation and blood parameters may be affected.

Oxidative stress as a damage mechanism in porcine cumulus-oocyte complexes exposed to malathion during in vitro maturation.

Malathion is one of the most commonly used insecticides. Recent findings have demonstrated that it induces oxidative stress in somatic cells, but there are not enough studies that have demonstrated this effect in germ cells. Malathion impairs porcine oocyte viability and maturation, but studies have not shown how oxidative stress damages maturation and which biochemical mechanisms are affected in this process in cumulus-oocyte complexes (COCs). The aims of the present study were to determine the amount of oxidative stress produced by malathion in porcine COCs matured in vitro, to define how biochemical mechanisms affect this process, and determine whether trolox can attenuate oxidative damage. Sublethal concentrations 0, 750, and 1000 µM were used to evaluate antioxidant enzyme expressions, reactive oxygen species (ROS production), protein oxidation, and lipid peroxidation, among other oxidation products. COCs viability and oocyte maturation decreased in a concentration-dependent manner. Malathion increased Cu, Zn superoxide dismutase (SOD1), glutathione-S-transferase (GST), and glucose 6 phosphate dehydrogenase (G6PD) protein level and decreased glutathione peroxidase (GSH-Px) and catalase (CAT) protein level. Species reactives of oxygen (ROS), protein oxidation and Thiobarbituric acid reactive substances (TBARS) levels increased in COCs exposed to the insecticide, but when COCs were pre-treated with the trolox (50 µM) 30 min before and during malathion exposure, these parameters decreased down to control levels. This study showed that malathion has a detrimental effect on COCs during in vitro maturation, inducing oxidative stress, while trolox attenuated malathion toxicity by decreasing oxidative damage.

Biochemical parameters, oxidative stress biomarkers, and anatomopathological changes in Wistar rats treated with 3'-demethoxy-6-O-demethylisoguaiacin and norisoguaiacin.

Artemisia cina (Ac) is a plant with anthelmintic compounds such as 3'-demethoxy-6-O-demethylisoguaiacin (D) and norisoguaiacin (N). Three major objectives were proposed: (1) To evaluate biochemical parameters in blood (2) to determine the tissue oxidative stress by biomarkers as TBARS and glutathione peroxidase activity, and (3) to evaluate anatomopathological changes in organs such as the brain, liver, kidney, and lung after oral administration of n-hexane extract of Ac and D and N. D and N were administrated following the OECD guides for acute oral toxicity evaluation (Guide 420). Fifty Wistar rats were distributed into ten groups as follows: Group 1 (G1): 4 mg/Kg; G2: 40 mg/Kg; G3: 240 mg/Kg; G4: 1600 mg/Kg of n-hexane extract of Ac. G5: 2 mg/Kg; G6: 20 mg/Kg; G7: 120 mg/Kg; G8: 800 mg/Kg of D and N, G9: water and G10: polyvinylpyrrolidone at 2000 mg/Kg. At 14 days, the rats were euthanized, and the blood, liver, brain, kidney, and lung were taken for biochemical analysis, anatomopathological changes, and TBARS and GSH evaluation. Glucose, cholesterol, and phosphorus were altered. Histopathological analysis showed multifocal neuronal degeneration in the brain (G2). The kidney and lungs had changes in G7. The GSH and TBARS increased in G6 and G7. The TBARS activity was higher in G1 and G2. In conclusion, extract and D and N of Ac did not have damage at therapeutic doses. D, N, and n-hexane extract of A. cina do not cause histopathological damage at pharmaceutical doses. Still, the brain, kidney, and liver are related to biochemical parameters at higher doses. However, compounds are proposed as antioxidant agents.

Study of the Early Effects of Chitosan Nanoparticles with Glutathione in Rats with Osteoarthrosis.

Due to cartilage's limited capacity for regeneration, numerous studies have been conducted to find new drugs that modify osteoarthrosis's progression. Some evidence showed the capability of chitosan nanoparticles with glutathione (Np-GSH) to regulate the oxide-redox status in vitro in human chondrocytes. This work aimed to evaluate the capacity of Np-GSH in vivo, using Wistar rats with induced surgical osteoarthritis. Radiographic, biochemical (GSH and TBARS quantification), histopathological, and immunohistochemical (Col-2 and MMP-13) analyses were performed to evaluate the progress of the osteoarthritic lesions after the administration of a single dose of Np-GSH. According to the results obtained, the GSH contained in the NPs could be vectored to chondrocytes and used by the cell to modulate the oxidative state reduction, decreasing the production of ROS and free radicals induced by agents oxidizing xenobiotics, increasing GSH levels, as well as the activity of GPx, and decreasing lipid peroxidation. These results are significant since the synthesis of GSH develops exclusively in the cell cytoplasm, and its quantity under an oxidation-reduction imbalance may be defective. Therefore, the results allow us to consider these nanostructures as a helpful study tool to reduce the damage associated with oxidative stress in various diseases such as osteoarthritis.

Carbon nanoparticles as detection labels in antibody microarrays. Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli.

The present study demonstrates that carbon nanoparticles (CNPs) can be used as labels in microarrays. CNPs were used in nucleic acid microarray immunoassays (NAMIAs) for the detection of different Shiga toxin-producing Escherichia coli (STEC) virulence factors: four genes specific for STEC (vt1, vt2, eae, and ehxA) and the gene for E. coli 16S (hui). Optimization was performed using a Box-Behnken design, and the limit of detection for each virulence factor was established. Finally, this NAMIA using CNPs was tested with DNA from 48 field strains originating from cattle feces, and its performance was evaluated by comparing results with those achieved by the reference method q-PCR. All factors tested gave sensitivity and specificity values higher than 0.80 and efficiency values higher than 0.92. Kappa coefficients showed an almost perfect agreement (k > 0.8) between NAMIA and the reference method used for vt1, eae, and ehxA, and a perfect agreement (k = 1) for vt2 and hui. The excellent agreement between the developed NAMIA and q-PCR demonstrates that the proposed analytical procedure is indeed fit for purpose, i.e., it is valuable for fast screening of amplified genetic material such as E. coli virulence factors. This also proves the applicability of CNPs in microarrays.

Modification of Proliferation and Apoptosis in Breast Cancer Cells by Exposure of Antioxidant Nanoparticles Due to Modulation of the Cellular Redox State Induced by Doxorubicin Exposure.

In this report, we investigated whether the use of chitosan-carrying-glutathione nanoparticles (CH-GSH NPs) can modify proliferation and apoptosis, and reduce cell damage induced by doxorubicin on breast cancer cells. Doxorubicin is a widely used antineoplasic agent for the treatment of various types of cancer. However, it is also a highly toxic drug because it induces oxidative stress. Thus, the use of antioxidant molecules has been considered to reduce the toxicity of doxorubicin. CH-GSH NPs were characterized in size, zeta potential, concentration, and shape. When breast cancer cells were treated with CH-GSH nanoparticles, they were localized in the cellular cytoplasm. Combined doxorubicin exposure with nanoparticles increased intracellular GSH levels. At the same time, decreasing levels of reactive oxygen species and malondialdehyde were observed and modified antioxidant enzyme activity. Levels of the Ki67 protein were evaluated as a marker of cell proliferation and the activity of the Casp-3 protein related to cell apoptosis was measured. Our data suggests that CH-GSH NPs can modify cell proliferation by decreasing Ki67 levels, induce apoptosis by increasing caspase-3 activity, and reduce the oxidative stress induced by doxorubicin in breast cancer cells by modulating molecules associated with the cellular redox state. CH-GSH NPs could be used to reduce the toxic effects of this antineoplastic. Considering these results, CH-GSH NPs represent a novel delivery system offering new opportunities in pharmacy, material science, and biomedicine.

Extracellular and intracellular zilpaterol and clenbuterol quantification in Hep G2 liver cells by UPLC-PDA and UPLC-MS/MS.

Zilpaterol and Clenbuterol are ß-adrenergic agonists that have been widely used to feed cattle. Although the use of Zilpaterol has been approved, Clenbuterol is still used illegally at unknown doses. However, the research of both substances has been based mainly on the evaluation of residues. To our knowledge, this is the first time that a cellular model using Hep G2 cells treated with Zilpaterol and Clenbuterol is presented as an alternative approach to quantify both drugs at the cellular level. Thus, a complete analytical methodology has been developed for the accurate quantitation of these ß-adrenergic agonists in both cellular compartments. We propose the use of ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA) for extracellular determinations while UPLC coupled to a tandem mass spectrometer (UPLC-MS/MS) for intracellular analysis. The methods were fully validated in terms of selectivity, linearity, accuracy, and precision, limits of detection and quantitation (LOD and LOQ, respectively), stability, carryover, and matrix effect. The method for intracellular content was linear ranging from 0.25 to 8 ng/mL while for extracellular content, the concentration of Zilpaterol and Clenbuterol ranged from 0.125 to 4 µg/mL, with correlation coefficients of R > 0.98 and >0.99, respectively. The combination of the two methodologies in the cellular model showed intracellular concentrations of 0.344 ± 0.06 µg/mL and 2.483 ± 0.36 µg/mL for Zilpaterol and Clenbuterol, respectively. Extracellular concentration was 0.728 ± 0.14 µg/mL and 0.822 ± 0.11 µg/mL for Zilpaterol and Clenbuterol, respectively. This work shows the potential applications of cellular modelling in the study of toxicity for the mentioned drugs.

Integration of Transcriptome, Gross Morphology and Histopathology in the Gill of Sea Farmed Atlantic Salmon (Salmo salar): Lessons From Multi-Site Sampling.

The gill of teleost fish is a multifunctional organ involved in many physiological processes such as gas exchange, osmotic and ionic regulation, acid-base balance and excretion of nitrogenous waste. Due to its extensive interface with the environment, the gill plays a key role as a primary mucosal defense tissue against pathogens, as manifested by the presence of the gill-associated lymphoid tissue (GIALT). In recent years, the prevalence of multifactorial gill pathologies has increased significantly, causing substantial losses in Atlantic salmon aquaculture. The transition from healthy to unhealthy gill phenotypes and the progression of multifactorial gill pathologies, such as proliferative gill disease (PGD), proliferative gill inflammation (PGI) and complex gill disorder (CGD), are commonly characterized by epithelial hyperplasia, lamellar fusion and inflammation. Routine monitoring for PGD relies on visual inspection and non-invasive scoring of the gill tissue (gross morphology), coupled with histopathological examination of gill sections. To explore the underlying molecular events that are associated with the progression of PGD, we sampled Atlantic salmon from three different marine production sites in Scotland and examined the gill tissue at three different levels of organization: gross morphology with the use of PGD scores (macroscopic examination), whole transcriptome (gene expression by RNA-seq) and histopathology (microscopic examination). Our results strongly suggested that the changes in PGD scores of the gill tissue were not associated with the changes in gene expression or histopathology. In contrast, integration of the gill RNA-seq data with the gill histopathology enabled us to identify common gene expression patterns associated with multifactorial gill disease, independently from the origin of samples. We demonstrated that the gene expression patterns associated with multifactorial gill disease were dominated by two processes: a range of immune responses driven by pro-inflammatory cytokines and the events associated with tissue damage and repair, driven by caspases and angiogenin.

Red vent syndrome in wild Atlantic salmon Salmo salar in Scotland is associated with Anisakis simplex sensu stricto (Nematoda: Anisakidae).

Simultaneous reports were received between June and July 2007 of wild Atlantic salmon Salmo salar with red, swollen, bloody vents returning to geographically diverse rivers in Scotland. By the end of September the condition, colloquially known as 'red vent syndrome' (RVS), was reported from >50 rivers across Scotland. Fish were generally in good overall condition but the vent area showed mild to severe lesions. External characteristics of the syndrome included a swollen, raised, haemorrhagic vent and surrounding tissues, with erosion of the skin, scale loss and moderate to severe bleeding in more advanced cases. Predominantly, the fish affected were 1-sea-winter grilse; however, RVS was also recorded in 2-sea-winter salmon and sea trout S. trutta. High numbers of the nematode Anisakis Type I larvae were found infesting the discrete region of the vent, a localisation that is reported as novel for the parasite. The hypothesis that this is a different species than that commonly found in the body cavity and viscera was investigated through molecular studies. These studies failed to show evidence that the parasites infesting the vent were different from those in the body cavity, i.e. all were identified as A. simplex sensu stricto. No other disease agent was found associated with the lesions or was isolated systemically, and no mortality or prevention of spawning was recorded during the 2007 season. Possible causes, including warming environments in the North Atlantic, are hypothesised as playing a role in the development of RVS in Atlantic salmon.

Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation.

Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.

Lateral flow immunoassay for on-site detection of Xanthomonas arboricola pv. pruni in symptomatic field samples.

Xanthomonas arboricola pv. pruni is a quarantine pathogen and the causal agent of the bacterial spot disease of stone fruits and almond, a major threat to Prunus species. Rapid and specific detection methods are essential to improve disease management, and therefore a prototype of a lateral flow immunoassay (LFIA) was designed for the detection of X. arboricola pv. pruni in symptomatic field samples. It was developed by producing polyclonal antibodies which were then combined with carbon nanoparticles and assembled on nitrocellulose strips. The specificity of the LFIA was tested against 87 X. arboricola pv. pruni strains from different countries worldwide, 47 strains of other Xanthomonas species and 14 strains representing other bacterial genera. All X. arboricola pv. pruni strains were detected and cross-reactions were observed only with four strains of X. arboricola pv. corylina, a hazelnut pathogen that does not share habitat with X. arboricola pv. pruni. The sensitivity of the LFIA was assessed with suspensions from pure cultures of three X. arboricola pv. pruni strains and with spiked leaf extracts prepared from four hosts inoculated with this pathogen (almond, apricot, Japanese plum and peach). The limit of detection observed with both pure cultures and spiked samples was 104 CFU ml-1. To demonstrate the accuracy of the test, 205 samples naturally infected with X. arboricola pv. pruni and 113 samples collected from healthy plants of several different Prunus species were analyzed with the LFIA. Results were compared with those obtained by plate isolation and real time PCR and a high correlation was found among techniques. Therefore, we propose this LFIA as a screening tool that allows a rapid and reliable diagnosis of X. arboricola pv. pruni in symptomatic plants.

Atlantic salmon cardiac primary cultures: An in vitro model to study viral host pathogen interactions and pathogenesis.

Development of Salmon Cardiac Primary Cultures (SCPCs) from Atlantic salmon pre-hatch embryos and their application as in vitro model for cardiotropic viral infection research are described. Producing SCPCs requires plating of trypsin dissociated embryos with subsequent targeted harvest from 24h up to 3 weeks, of relevant tissues after visual identification. SCPCs are then transferred individually to chambered wells for culture in isolation, with incubation at 15-22°. SCPCs production efficiency was not influenced by embryo's origin (0.75/ farmed or wild embryo), but mildly influenced by embryonic developmental stage (0.3 decline between 380 and 445 accumulated thermal units), and strongly influenced by time of harvest post-plating (0.6 decline if harvested after 72 hours). Beating rate was not significantly influenced by temperature (15-22°) or age (2-4 weeks), but was significantly lower on SCPCs originated from farmed embryos with a disease resistant genotype (F = 5.3, p<0.05). Two distinct morphologies suggestive of an ex vivo embryonic heart and a de novo formation were observed sub-grossly, histologically, ultra-structurally and with confocal microscopy. Both types contained cells consistent with cardiomyocytes, endothelium, and fibroblasts. Ageing of SCPCs in culture was observed with increased auto fluorescence in live imaging, and as myelin figures and cellular degeneration ultra-structurally. The SCPCs model was challenged with cardiotropic viruses and both the viral load and the mx gene expression were measurable along time by qPCR. In summary, SCPCs represent a step forward in salmon cardiac disease research as an in vitro model that partially incorporates the functional complexity of the fish heart.

Relación entre trastorno del espectro autista y epilepsia en una población pediátrica en Quito-Ecuador / Relationship between autism spectrum disorder and epilepsy in a pediatric population in Quito-Ecuador

Introducción: No existe un mecanismo plausible del trastorno del espectro autista (TEA) como causa de epilepsia, sin embargo, su coocurrencia es seguramente el resultado de factores predisponentes para ambas condiciones, incluyendo factores genéticos y ambien-tales. El objetivo de este estudio es establecer la prevalencia de epilepsia en pacientes con TEA y encontrar asociación con otros factores. Métodos: Se realizó un estudio longitudinal retrospectivo basado en las historias clínicas del Centro de Enfermedades Neurológicas y Nutricionales en Niños y Adolescentes (CENNA) de 81 pacientes (3-19 años) con diagnóstico de TEA, en donde se identificaron a los pacientes con coexistencia epilepsia durante un periodo de 6 años, y las diferentes variables en este grupo. Resultados: Se identificaron 81 pacientes con diagnóstico de TEA, de los cuales 12 pacientes (15%) presentaban coexistencia de epilepsia. Al analizar el grado de TEA, se evidenció que la comorbilidad en ambas entidades es más común en el TEA grado 3 (58.33%). La edad inicio de la epilepsia en el rango entre 5 a 10 años (42%). Se evidencio que el 25% de los pacientes tienen antecedentes familiares de epilepsia, mientras que sólo el 8% tiene ante-cedente familiar de TEA. Todos los tipos de crisis epiléptica se presentaron en los pacientes con TEA, pero las más comunes fueron las crisis de tipo focal (58%), específicamente moto-ras con alteración de la conciencia (33%). Además, existió un 100% de mejoría en el comportamiento autista en los pacientes que recibieron su tratamiento antiepiléptico, y sólo el 8% presentó epilepsia de difícil control. Conclusiones: El estudio mostró una prevalencia significativa de epilepsia en la población con diagnóstico de TEA. El estudio logró observar la distribución de población con comorbilidad de TEA y epilepsia, para en un futuro encontrar una variable común entre ambas patologías. A nuestro conocimiento, este es el primer estudio retrospectivo en Ecuador que analiza la comorbilidad de TEA y epilepsia en la población ecuatoriana

Introduction: Compared to the general population, there is a higher prevalence of epilepsy in children with autism spectrum disorder (ASD), with an indecency of approximately 20%. There is no plausible mechanism for ASD as a cause of epilepsy; however, its cooccurrence is surely the result of predisposing factors for both conditions, including genetic and environmental factors. The objective of this study was to establish the prevalence of epilepsy in patients with ASD and find a correlation with other factors, such as sex, etiology, type of seizure or epileptic syndrome, age of onset of epilepsy, EEG abnormalities, and therapeutic response. Methods: A retrospective longitudinal study was carried out based on the clinical records of the Center for Neurological and Nutritional Diseases in Children and Adolescents (CENNA) of 81 patients (3-19 years) with a diagnosis of ASD, where patients coexisted with epilepsy for a period of 6 years, and the different variables in this group. Results: Eighty-one patients with a diagnosis of ASD were identified, of whom 12 patients (15%) had coexisting epilepsy. When analyzing the degree of ASD, it was evidenced that comorbidity in both entities is more common in ASD grade 3 (58.33%). The age of onset of epilepsy ranged between 5 and 10 years (42%). Twenty-five percent of patients had a family history of epilepsy, while only 8% had a family history of ASD. All types of epileptic seizures occurred in patients with ASD, but the most common were focal-type seizures (58%), specifically motor seizures with altered consciousness (33%). In addition, there was a 100% improvement in autistic behavior in the patients who received their antiepileptic treatment, and only 8% had difficult-to-control epilepsy. Conclusion: The study showed a significant prevalence of epilepsy in the population diagnosed with ASD. The study managed to observe the distribution of the population with comorbidities of ASD and epilepsy to find a common variable between both pathologies in the future. To our knowledge, this is the first retrospective study in Ecuador that analyzes the comorbidity of ASD and epilepsy in the Ecuadorian population.

Genotoxicity and cytotoxicity assessment of new ethyl-carbamates with ixodicidal activity.

The mammalian erythrocyte micronucleus test was used on the peripheral blood of Wistar rats exposed to two new ethyl-carbamates: ethyl-4-bromophenyl-carbamate (LQM 919) and ethyl-4-chlorophenyl-carbamate (LQM 996) to analyze their genotoxic potential. The mitotic index and cell proliferation kinetics in human lymphocyte cultures in the presence of these ethyl-carbamates were used to evaluate cytotoxicity and cytostaticity respectively. Exposure to greater acute doses (300mg/kg) and to all of the subchronic doses (12.5, 25 and 50mg/kg daily for 90 days) of these ethyl-carbamates induced an increased frequency (p<0.05) of micro-nucleated polychromatic erythrocytes (MN-PCE) compared with rats not exposed to the ethyl-carbamates. Increases in MN-PCE was higher in males than in females exposed to LQM 996 50mg/Kg (p<0.05). All observed changes in rats return 21days after suspending ethyl-carbamate exposure. The highest concentration (0.3mM) of both ethyl-carbamates in lymphocyte cultures increased the percentage of cells in first division metaphase and decreased the percentage of cells in third division metaphase, indicating an increase in cell cycle length or a possible cell cycle arrest in metaphase (cytostatic effect). The results of this study show that the evaluated ethyl-carbamates may induce genotoxic damage in rats and alterations in the human lymphocyte cell cycle.