Integrated metabolomics analysis of chill-stored rose shrimp (Parapenaeus longirostris) treated with different pressure levels of high hydrostatic pressure by 1H-NMR spectroscopy.

The antimicrobial effects of high hydrostatic pressure (HHP) treatments on chill-stored seafood are well-documented, while their impact on the metabolic profile of seafood, especially the metabolome of fish flesh, and remains underexplored. Addressing this gap, this study investigates the effects of HHP on the metabolome of chill-stored rose shrimp by conducting multivariate data analysis based on untargeted proton nuclear magnetic resonance observations. Vacuum-packed rose shrimp samples were subjected to HHP at 0, 400, 500, and 600 MPa for 10 min and then stored at 2-4°C. The microorganism analysis and metabolic analysis were carried out on days 1 and 14. HHP treatment effectively deactivated Lactobacillus spp., Escherichia coli, Pseudomonas spp., total Coliforms, and sulfite-reducing anaerobic bacteria. Consequently, HHP treatment significantly reduced the formation rate of decay-related metabolites, such as hypoxanthine, trimethylamine, and biogenic amines, which exhibited significant accumulation in untreated samples. Multivariate unsupervised analyses provided insights into the overall changes in the metabolite profile induced by HHP. Metabolic pathway analysis revealed several pathways underlying spoilage, including pyruvate metabolism, valine, leucine, and isoleucine biosynthesis, purine metabolism, methane metabolism, glycine, serine, and threonine metabolism, citrate cycle (TCA cycle), glycolysis/gluconeogenesis, alanine, aspartate, and glutamate metabolism, sulfur metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and glyoxylate and dicarboxylate metabolism. Importantly, these pathways underwent alterations due to the application of HHP, particularly at high-pressure levels. In summary, the results unveil the potential mechanisms of HHP effects on chill-stored rose shrimps.

Genetically engineered eucalyptus expressing pesticidal proteins from Bacillus thuringiensis for insect resistance: a risk assessment evaluation perspective.

Eucalyptus covers approximately 7.5 million hectares in Brazil and serves as the primary woody species cultivated for commercial purposes. However, native insects and invasive pests pose a significant threat to eucalyptus trees, resulting in substantial economic losses and reduced forest productivity. One of the primary lepidopteran pests affecting eucalyptus is Thyrinteina arnobia (Stoll, 1782) (Lepidoptera: Geometridae), commonly referred to as the brown looper caterpillar. To address this issue, FuturaGene, the biotech division of Suzano S.A., has developed an insect-resistant (IR) eucalyptus variety, which expresses Cry pesticidal proteins (Cry1Ab, Cry1Bb, and Cry2Aa), derived from Bacillus thuringiensis (Bt). Following extensive safety assessments, including field trials across various biomes in Brazil, the Brazilian National Technical Commission of Biosafety (CTNBio) recently approved the commercialization of IR eucalyptus. The biosafety assessments involved the analysis of molecular genomics, digestibility, thermostability, non-target organism exposure, degradability in the field, and effects on soil microbial communities and arthropod communities. In addition, in silico studies were conducted to evaluate allergenicity and toxicity. Results from both laboratory and field studies indicated that Bt eucalyptus is as safe as the conventional eucalyptus clone for humans, animals, and the environment, ensuring the secure use of this insect-resistant trait in wood production.

Five-years post commercial approval monitoring of eucalyptus H421.

Eucalyptus comprises the largest planted area of cultivated production forest in Brazil. Genetic modification of eucalyptus can provide additional characteristics for increasing productivity, protecting plant yield, and potentially altering fiber for various industrial uses. With this objective, a transgenic eucalyptus variety, event H421, received regulatory approval for commercial release after 6 years of approved risk assessment studies by the Brazilian National Technical Biosafety Commission (CTNBio) in 2015, becoming the first approved genetically modified (GM) eucalyptus in the world. GM event H421 enables increased plant biomass accumulation through overexpression of the Arabidopsis 1,4-ß-endoglucanase Cel1, which remodels the xyloglucan-cellulose matrix of the cell wall during development to promote cell expansion and growth. As required, in that time, by the current normative from CTNBio, a post-commercial release monitoring plan for H421 was submitted, incorporating general surveillance for five consecutive years with the submission of annual reports. The monitoring plan was conducted on fields of H421 progenies, with conventional clones as comparators, cultivated in representative regions where eucalyptus is cultivated in the states of São Paulo, Bahia, and Maranhão, representing Southeast, Northeast, and Northern Brazil. Over the course of the five-year general surveillance monitoring plan for the approved GM eucalyptus H421, no adverse effect that could impact the biosafety of the commercially approved event was identified. Additionally, the GM eucalyptus exhibited behavior highly consistent with that of conventional commercial clones. Therefore, there was no need for an extra risk assessment study of a case-specific monitoring plan. The results show the importance of continuously updating the regulation norms of governmental agencies to align with scientific advances.

Safety Assessment of the CP4 EPSPS and NPTII Proteins in Eucalyptus.

Glyphosate herbicide treatment is essential to sustainable Eucalyptus plantation management in Brazil. Eucalyptus is highly sensitive to glyphosate, and Suzano/FuturaGene has genetically modified eucalyptus to tolerate glyphosate, with the aim of both protecting eucalyptus trees from glyphosate application damage and improving weed management. This study presents the biosafety results of the glyphosate-tolerant eucalyptus event 751K032, which expresses the selection marker neomycin phosphotransferase II (NPTII) enzyme and CP4-EPSPS, a glyphosate-tolerant variant of plant 5-enolpyruvyl-shikimate-3-phosphate synthase enzyme. The transgenic genetically modified (GM) event 751K032 behaved in the plantations like conventional non-transgenic eucalyptus clone, FGN-K, and had no effects on arthropods and soil microorganisms. The engineered NPTII and CP4 EPSPS proteins were heat-labile, readily digestible, and according to the bioinformatics analyses, unlikely to cause an allergenic or toxic reaction in humans or animals. This assessment of the biosafety of the glyphosate-tolerant eucalyptus event 751K032 concludes that it is safe to be used for wood production.

Survivorship and food consumption of immatures and adults of Apis mellifera and Scaptotrigona bipunctata exposed to genetically modified eucalyptus pollen.

Eucalyptus comprises the largest planted area of cultivated production forest in Brazil. Genetic modification (GM) of eucalyptus can provide additional characteristics for increasing productivity and protecting wood yield, as well as potentially altering fiber for a diversity of industrial uses. However, prior to releasing a new GM plant, risk assessments studies with non-target organisms must be undertaken. Bees are prominent biological models since they play an important role in varied ecosystems, including for Eucalyptus pollination. The main goal of this study was to evaluate whether a novel event (Eucalyptus 751K032), which carries the cp4-epsps gene that encodes the protein CP4-EPSPS and nptII gene that encodes the protein NPTII, might adversely affect honey bees (Apis mellifera) and stingless bees (Scaptotrigona bipunctata). The experiments were performed in southern Brazil, as follows: (i) larvae and adults were separately investigated, (ii) three or four different pollen diets were offered to bees, depending on larval or adult status, and (iii) two biological attributes, i.e., survivorship of larvae and adults and food intake by adults were evaluated. The diets were prepared with pollen from GM Eucalyptus 751K032; pollen from conventional Eucalyptus clone FGN-K, multifloral pollen or pure larval food. The insecticide dimethoate was used to evaluate the sensitivity of bees to toxic substances. Datasets were analyzed with Chi-square test, survival curves and repeated measures ANOVA. Results indicated no evidence of adverse effects of Eucalyptus pollen 751K032 on either honey bees or stingless bees assessed here. Therefore, the main findings suggest that the novel event may be considered harmless to these organisms since neither survivorship nor food consumption by bees were affected by it.

Pulsed Electric Fields (PEF) and Accelerated Solvent Extraction (ASE) for Valorization of Red (Aristeus antennatus) and Camarote (Melicertus kerathurus) Shrimp Side Streams: Antioxidant and HPLC Evaluation of the Carotenoid Astaxanthin Recovery.

Shrimp side streams represent an important natural source of astaxanthin. Optimization of the astaxanthin extraction process from shrimp side streams is of great importance for the valorization of crustacean side streams and the development of astaxanthin-related products. The combined and independent effects of two innovative extraction technologies (pulsed electric fields (PEFs) and accelerated solvent extraction (ASE)) alone and/or combined in a sequential step, using two different solvents on astaxanthin extraction from two shrimp species, were evaluated. Astaxanthin content in the extracts of shrimp side streams was determined by both spectrophotometric and HPLC assays, being the determination of the carotenoid profiles performed by HPLC analysis. Compared to a solvent extraction control procedure, the astaxanthin content was increased after ASE and PEF treatments, for both shrimp species, independently of the solvent used. The highest recovery (585.90 µg/g) was obtained for the species A. antennatus, with the solvent DMSO when PEF and ASE were combined, while the increase in antioxidant capacity varied depending on the solvent used. HPLC analysis of the samples revealed the presence of unesterified (all-E) astaxanthin, four unesterified Z isomers of astaxanthin and many unresolved astaxanthin esters. Both technologies are useful tools to recover antioxidant valuable carotenoids such as astaxanthin from shrimp side streams.

Rheological and Viscoelastic Properties of Chitosan Solutions Prepared with Different Chitosan or Acetic Acid Concentrations.

Chitosan (Ch) is a partially crystalline biopolymer, insoluble in pure water but soluble in acid solutions. It has attracted interest from researchers to prepare solutions using different acid types and concentrations. This research aims to study both the effect of chitosan (Ch) or acetic acid (Ac) concentrations, at different temperatures, on rheological and viscoelastic properties of Ch solutions. To study the effect of Ch, solutions were prepared with 0.5−2.5 g Ch/100 g of solution and Ac = 1%, whereas to study the effect of Ac, the solutions were prepared with 2.0 g of Ch/100 g of solution and Ac = 0.2−1.0%. Overall, all analyzed solutions behaved as pseudoplastic fluid. The Ch strongly affected rheological properties, the consistency index (K) increased and the index flow behavior (n) decreased as a function of Ch. The activation energy, defined as the energy required for the molecule of a fluid to move freely, was low for Ch = 0.5%. The effect of Ac was less evident. Both K and n varied according to a positive and negative, respectively, parabolic model as a function of Ac. Moreover, all solutions, irrespective of Ch and Ac, behaved as diluted solutions, with G" > G'. The relaxation exponent (n") was always higher than 0.5, confirming that these systems behaved as a viscoelastic liquid. This n" increased with Ch, but it was insensitive to Ac, being slightly higher at 45 °C.

Characterization and Genomic Analysis of a New Phage Infecting Helicobacter pylori.

Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.

Innovative Non-Thermal Technologies for Recovery and Valorization of Value-Added Products from Crustacean Processing By-Products-An Opportunity for a Circular Economy Approach.

The crustacean processing industry has experienced significant growth over recent decades resulting in the production of a great number of by-products. Crustacean by-products contain several valuable components such as proteins, lipids, and carotenoids, especially astaxanthin and chitin. When isolated, these valuable compounds are characterized by bioactivities such as anti-microbial, antioxidant, and anti-cancer ones, and that could be used as nutraceutical ingredients or additives in the food, pharmaceutical, and cosmetic industries. Different innovative non-thermal technologies have appeared as promising, safe, and efficient tools to recover these valuable compounds. This review aims at providing a summary of the main compounds that can be extracted from crustacean by-products, and of the results obtained by applying the main innovative non-thermal processes for recovering such high-value products. Moreover, from the perspective of the circular economy approach, specific case studies on some current applications of the recovered compounds in the seafood industry are presented. The extraction of valuable components from crustacean by-products, combined with the development of novel technological strategies aimed at their recovery and purification, will allow for important results related to the long-term sustainability of the seafood industry to be obtained. Furthermore, the reuse of extracted components in seafood products is an interesting strategy to increase the value of the seafood sector overall. However, to date, there are limited industrial applications for this promising approach.

Encapsulated Pine Bark Polyphenolic Extract during Gastrointestinal Digestion: Bioaccessibility, Bioactivity and Oxidative Stress Prevention.

Polyphenolic extracts from pine bark have reported different biological actions and promising beneficial effects on human health. However, its susceptibility to environmental stresses (temperature, storage, etc.) and physiological human conditions prequires the development of efficient protection mechanisms to allow effective delivering of functionality. The aim of this work was to encapsulate pine bark extract rich phenolic compounds by spray-drying using maltodextrin, and understand the influence of encapsulation on the antioxidant and antimicrobial activity and bioaccessibility of phenolic compounds during gastrointestinal digestion. The optimized process conditions allowed good encapsulation efficiency of antioxidant phenolic compounds. The microencapsulation was effective in protecting those compounds during gastrointestinal conditions, controlling their delivery and enhancing its health benefits, decreasing the production of reactive oxygen species implicated in the process of oxidative stress associated with some pathologies. Finally, this encapsulation system was able to protect these extracts against acidic matrices, making the system suitable for the nutritional enrichment of fermented foods or fruit-based beverages, providing them antimicrobial protection, because the encapsulated extract was effective against Listeria innocua. Overall, the designed system allowed protecting and appropriately delivering the active compounds, and may find potential application as a natural preservative and/or antioxidant in food formulations or as bioactive ingredient with controlled delivery in pharmaceuticals or nutraceuticals.

Influence of the addition of different ingredients on the bioaccessibility of glucose released from rice during dynamic in vitro gastrointestinal digestion.

Rice represents a primary source of carbohydrates in human nutrition. Upon its consumption, the released sugars are mostly absorbed, categorising rice as a high glycemic index food. Addition of ingredients is common practice when cooking rice, which may affect rice digestibility and influence nutrients absorption in the gastrointestinal (GI) tract, enabling a controlled glucose release. In this sense, rice formulations were submitted to a dynamic in vitro GI model, constituted by reactors that simulates peristalsis coupled to filtration membranes, to evaluate carbohydrates hydrolysis and bioaccessibility. Addition of quinoa and wholegrains reduced carbohydrates hydrolysis (i.e. 38.5 ± 5.08% and 57.98 ± 1.91%, respectively) and glucose bioaccessibility (i.e. 25.92 ± 5.70% and 42.56 ± 1.39%, respectively) when compared with brown rice (i.e. 63.86 ± 2.96% hydrolysed and 44.33 ± 1.88% absorbed). Addition of vegetables significantly decreased sample chewiness and resulted in superior hydrolysis (71.75 ± 7.44%) and glucose absorption (51.61 ± 6.25%).

Quality Changes during Frozen Storage of Mechanical-Separated Flesh Obtained from an Underutilized Crustacean.

Despite their high nutritional value, high quantities of fish caught in the Adriatic Sea are underused or discarded for their insignificant economic value. Mechanical separation of flesh represents an opportunity for developing innovative semi-finished products, even if it can promote an increased quality degradation rate. The aim of this study was to evaluate physico-chemical modifications of mechanically separated mantis shrimp flesh during deep-freezing storage. Flesh samples obtained using a belt-drum separator, frozen and vacuum-packed, were stored at 3 temperatures (industrial: -26 °C; domestic: -18 °C and abuse: -10 °C) for 12 months. During storage, qualitative (color, water content, pH, fatty acids (FA) and lipid oxidation) were evaluated. Fish freshness parameters (e.g., trimethylamine (TMA), dimethylamine (DMA) and amino acids) were assessed using nuclear magnetic resonance (1H-NMR). The mechanical separation process accelerated the initial oxidation phenomena, promoting color alterations, compared to manual separation. The main degradation phenomena during storage were significantly affected by temperature and were related to changes in luminosity, oxidation of n-3 polyunsaturated fatty acids (PUFA), increased lipolysis with release of free FA, production of TMA and DMA by residual enzymatic activity, and changes in amino acids due to proteolysis. The inter-disciplinary approach permitted important findings to be made, in terms of the extent of different degradative phenomena, bound to processing and storage conditions of mechanically separated mantis flesh.

Characterization of the behavior of carotenoids from pitanga (Eugenia uniflora) and buriti (Mauritia flexuosa) during microemulsion production and in a dynamic gastrointestinal system.

Uncommon tropical fruits are emerging as raw-material for new food products with health benefits. This work aimed at formulating and processing microemulsions from pitanga (Eugenia uniflora) and buriti (Mauritia flexuosa) fruits, since they are very rich in carotenoids (particularly lycopene and ß-carotene), in order to encapsulate and increase carotenoids' bioaccessibility. Pitanga and buriti microemulsions were produced by applying a direct processing (high-speed homogenization at 15,000 rpm and ultrasound with 20 kHz probe at 40% amplitude) of the whole pulp together with surfactant (Tween 80 or Whey Protein Isolate at 2%) and corn oil (5%). All treatments (HSH-US for 0-4, 4-0, 4-4, 4-8 min-min) applied were able to increase the amount of carotenoid released. However, the processing also decreased the total amount of carotenoids in the whole pulp of studied fruits. The impact of processing during microemulsion production was not severe. The overall data suggest that the presence of surfactant and oil during processing may protect the carotenoids in fruits and microemulsions. Final recovery of total carotenoids, after passing the samples through a dynamic gastrointestinal system that simulates the human digestion, was higher for microemulsions than for whole pulps. High losses of total carotenoids in buriti and ß-carotene and lycopene in pitanga occurred during jejunum and ileum phases. The present work confirms that it is possible to increase ß-carotene and lycopene bioaccessibility from fruits by directly processing microemulsions (p < 0.01).

In vitro gastrointestinal evaluation of a juçara-based smoothie: effect of processing on phenolic compounds bioaccessibility.

In the present work, the bioaccessibility of the main phenolic compounds of a juçara, banana and strawberry homogenized smoothie (control), subjected to pasteurization and sonication, was evaluated. The smoothie was also evaluated in terms of its main chemical and physical characteristics. Pasteurized smoothie showed higher apparent viscosity, as well as higher initial shear stress when compared to the control and sonicated samples. The increase in the apparent viscosity of the pasteurized smoothie was associated with the smaller particle size of this sample (68 µm). These characteristics conferred to the pasteurized smoothie higher physical stability than the control and sonicated smoothies. Phenolic compounds bioaccessibility was higher in the pasteurized and sonicated smoothies than in the control sample, which confirmed the positive effect of the treatments for the preservation of these compounds after gastrointestinal digestion. Compared to the sonication process, the pasteurization provided higher total phenolic compounds bioaccessibility (47%), as well as of ferulic (16%) and ellagic (80%) acids. Antioxidant capacity was higher in gastric digest for all the samples evaluated by ABTS assay. These results confirm the importance of processing on the physical stability and phenolic compounds bioaccessibility of the juçara-based smoothie, standing out the thermally treated product.

The impact of gas mixtures of Argon and Nitrous oxide (N2O) on quality parameters of sardine (Sardina pilchardus) fillets during refrigerated storage.

The effect of modified atmosphere packaging (MAP) with unconventional gas mixtures on the main qualitative parameters of sardine fillets during refrigerated storage was investigated. Four different atmospheres conditions were tested: air; 30% CO2 + 70% N2; 30% CO2 + 70% N2O and 30% CO2 + 70% Ar. All samples were packaged in polypropylene trays sealed with a high barrier film and stored at 2-4 °C for 12 days. The quality and the freshness of sardine fillets packed in MAP were evaluated by microbiological, physical and chemical analyses after 0, 1, 2, 5, 6, 8 and 12 days of the storage period. The 2-thiobarbituric acid-reactive substances (TBARS) values for MAP samples were lower compared to air samples, reaching a final value of 1.09 mg malonaldehyde (MA)/kg and 3.39 mg MA/kg, respectively. The samples packed in Ar reached the fixed threshold for total mesophilic and psychrotrophic bacteria after 12 days of storage, resulting the best MAP condition adopted, able to increase the sardine shelf-life of 3 days with respect to the other tested conditions. Air packed samples showed significantly higher (p < 0.05) Hx content (50 mg/kg) compared to the rest of the MAP samples (20 mg/kg). At the end of the storage period, the sample packed in Ar showed a significantly lower value (p < 0.05) (around 40 mg/kg), than the other MAP conditions.

Influence of Butyrylcholinesterase in Progression of Mild Cognitive Impairment to Alzheimer's Disease.

BACKGROUND: Several demographic and genetic prognostic factors of conversion from mild cognitive impairment (MCI) to Alzheimer's disease (AD) have been recognized so far. The most frequent polymorphism of butyrylcholinesterase (BuChE), the K-variant, has been proposed as a risk factor for AD, but data regarding its influence on early disease progression is still limited. OBJECTIVE: To investigate the influence of the BuChE-K variant in MCI progression to AD. METHODS: 96 MCI patients were included in the study and were genotyped for BuChE-K variant and Apolipoprotein E (ApoE). Cerebrospinal fluid (CSF) BuChE activity, as well as the levels of AD biomarkers amyloid-ß 42 (Aß42), total and hyperphosphorylated tau (t-tau and p-tau) were also determined. RESULTS: No significant differences were found in either BuChE-K variant or BuChE activity between MCI patients that progressed to AD (MCI-AD) and patients that remained stable during clinical follow-up (MCI-St). As expected, baseline CSF levels of Aß42 were significantly lower and t-Tau, p-Tau, and ApoE ɛ4 allele frequency were significantly higher in MCI-AD patients. An association between the ApoE ɛ4 allele and the BuChE-K variant in MCI-AD, but not in MCI-St patients, was found with patients carrying both alleles presenting the highest incidence of progression and the lowest estimated time of progression to AD. CONCLUSION: Although BuChE-K alone does not seem to play a major role in progression to AD in MCI patients, a synergistic effect with the ApoE ɛ4 allele was found, highlighting the importance of assessing these combined genotypes for evaluating risk progression in MCI patients.

In vitro digestion of oil-in-water emulsions stabilized by whey protein nanofibrils.

The effect of pH (3 and 7) and varied energy density of a high-pressure homogenization process on the stability of oil-in-water (O/W) emulsions stabilized by whey protein fibrils was evaluated. A dynamic digestion model comprising the simulation of stomach, duodenum, jejunum and ileum, has been used to evaluate O/W emulsions' behavior under gastrointestinal (GI) conditions. The emulsions did not separate phases during the storage period (7 days). The emulsions stabilized by whey protein fibrils were stable under simulated gastric conditions but were destabilized in the simulated intestinal conditions. In a similar way, the whey protein fibrils' dispersion showed a high resistance to proteolytic in vitro digestion by pepsin (gastric stage) but was more readily degraded by pancreatin (intestinal stage). This fact confirms the significant impact of the interfacial characteristics on emulsions' digestion. The percentage of free fatty acids (FFA) absorbed in the simulated intestinal conditions (jejunum and ileum) was much lower than the total percentage of FFA released due to the use of WPI fibrils as emulsifier. This work contributes to a better understanding about the behavior of O/W emulsions stabilized by whey protein fibrils within the GI tract; this knowledge is fundamental when considering the final application of this protein in food products.

Association between butyrylcholinesterase and cerebrospinal fluid biomarkers in Alzheimer's disease patients.

The deficit of cholinergic activity is one of the main findings in Alzheimer's disease (AD), and is related to the synthesis of acetylcholine, and the hydrolysing enzymes, acetylcholinesterase and butyrylcholinesterase (BuChE). Together with the Apolipoprotein E-ε4 allele (ApoE-ε4), the BuChE-K variant has been proposed to increase AD risk in certain populations. In addition, this polymorphism has been associated with a lower capacity to attenuate ß-amyloid aggregation. In the present study we explored the interaction of the BuChE-K variant with its activity in CSF, conventional AD biomarkers and ApoE genotype. 217 AD patients and 200 age-matched controls were genotyped for the ApoE and the BuChE-K variant. BuChE activity in CSF, as well as the levels of the CSF-AD biomarkers amyloid-beta 42 (Aß42), total and hyperphosphorylated tau (t-tau and p-tau) were determined in 88 of these patients. The results showed no significant differences in the BuChE-K variant distribution between patients and controls. No influence of the BuChE-K variant was seen neither in CSF BuChE activity, nor in the levels of Aß42, t-tau and p-tau in AD patients. ApoE genotype also did not seem to influence CSF BuChE activity. Interestingly, in AD patients, an association between high CSF BuChE activity and increased levels of CSF Aß42 was shown, particularly in ApoE-ε4 allele carriers. In our population, the BuChE-K variant does not seem to confer risk for AD or to influence the activity of the enzyme in CSF. However, we demonstrated an association between BuChE activity, ApoE-ε4 genotype and CSF Aß42 levels, highlighting the importance of assessing BuChE activity as a possible modulator of Aß load in the brain.

PhTx3-4, a Spider Toxin Calcium Channel Blocker, Reduces NMDA-Induced Injury of the Retina.

The in vivo neuroprotective effect of PhTx3-4, a spider toxin N-P/Q calcium channel blocker, was studied in a rat model of NMDA-induced injury of the retina. NMDA (N-Methyl-D-Aspartate)-induced retinal injury in rats reduced the b-wave amplitude by 62% ± 3.6%, indicating the severity of the insult. PhTx3-4 treatment increased the amplitude of the b-wave, which was almost equivalent to the control retinas that were not submitted to injury. The PhTx3-4 functional protection of the retinas recorded on the ERG also was observed in the neuroprotection of retinal cells. NMDA-induced injury reduced live cells in the retina layers and the highest reduction, 84%, was in the ganglion cell layer. Notably, PhTx3-4 treatment caused a remarkable reduction of dead cells in the retina layers, and the highest neuroprotective effect was in the ganglion cells layer. NMDA-induced cytotoxicity of the retina increased the release of glutamate, reactive oxygen species (ROS) production and oxidative stress. PhTx3-4 treatment reduced glutamate release, ROS production and oxidative stress measured by malondialdehyde. Thus, we presented for the first time evidence of in vivo neuroprotection from NMDA-induced retinal injury by PhTx3-4 (-ctenitoxin-Pn3a), a spider toxin that blocks N-P/Q calcium channels.

Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus.

Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/µl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.