Effect of a new thermal treatment in combination with saprobic fungal incubation on the phytotoxicity level of alperujo

Byproducts generated from food industries, such as olive oil mills, have been studied to decrease harmful pollution and their environmental consequences. In this work, a new thermal pretreatment and saprobic fungal incubation to detoxify alperujo (two-phase olive mill waste) have been evaluated in view of its use as fertilizer in agriculture. The sequential use of both methods simplifies the thermal conditions and incubation times of the fungal treatment. Optimization of the thermal treatment from 150 to 170 °C for 45 and 15 min, respectively, reduced the incubation time with Coriolpsis rigida from 20 to 10 weeks needed to reduce phytotoxic effects on tomato plants. Therefore, the combination of thermal and biological treatments will allow the development of the potential benefits of alperujo to improve nutrients in agricultural soil.     

Radical scavenging capacity and antimutagenic properties of purified proteins from Solanum betaceum fruits and Solanum tuberosum tubers

In this study, antioxidant activities in free-radical-mediated oxidative systems and the genotoxic/antigenotoxic effects of two proteins with molecular mass around 17 kDa, purified from Solanum betaceum fruits (cyphomine) and Solanum tuberosum tubers (solamarine), were investigated. Both proteins inhibited uric acid formation with IC(50) values between 55 and 60 μg/mL, and both proteins were able to reduce oxidative damage by scavenging hydroxyl radicals and superoxide anion in a dose-dependent manner. Furthermore, the DPPH? reduction assay showed SC(50) values of 55-73 μg/mL. Cyphomine and solamarine were able to retain their antioxidant activity after heat treatment at 80 °C for 15 min. Allium cepa and Salmonella /microsome assays showed no genotoxic and mutagenic effects. Solamarine showed an antimutagenic effect against a direct mutagen (4-nitro-o-phenylenediamine). Consequently, the present study showed that the investigated proteins are promising ingredients for the development of functional foods with a beneficial impact on human health and an important source for the production of bioactive peptides.     

Expression of 14-3-3 σ protein in normal and neoplastic canine mammary gland

14-3-3 σ protein is a negative cell cycle regulator, with both reduced and elevated levels associated with cancer in humans. This study assessed the expression of this protein in canine mammary tissues using immunohistochemistry and Western blotting. 14-3-3 σ was detected in 97% of the mammary tissue samples examined and was found in both myoepithelial (MECs) and epithelial (ECs) cells. Expression levels were elevated and reduced in neoplastic ECs and MECs, respectively (P < 0.001). Intense expression of 14-3-3 σ was detected in neoplastic ECs infiltrating blood vessels and lymph nodes and suggests a possible role for this protein in the malignant transformation of mammary neoplasms. Moreover, double immunostaining for 14-3-3 σ and the MEC – specific marker p63, confirmed that 14-3-3 σ is a highly sensitive marker of MECs since all p63 – positive cells were also positive for 14-3-3 σ. However, this protein is not exclusive to MECs as ECs also labelled positively.     

Stability of Saxitoxin in 50% Methanol Fecal Extracts and Raw Feces from Bowhead Whales (Balaena mysticetus)

In recent decades, harmful algal blooms (HABs) producing paralytic shellfish toxins (including saxitoxin, STX) have become increasingly frequent in the marine waters of Alaska, USA, subjecting Pacific Arctic and subarctic communities and wildlife to increased toxin exposure risks. Research on the risks of HAB toxin exposures to marine mammal health commonly relies on the sampling of marine mammal gastrointestinal (GI) contents to quantify HAB toxins, yet no studies have been published testing the stability of STX in marine mammal GI matrices. An understanding of STX stability in test matrices under storage and handling conditions is imperative to the integrity of toxin quantifications and conclusions drawn thereby. Here, STX stability is characterized in field-collected bowhead whale feces (stored raw in several treatments) and in fecal extracts (50% methanol, MeOH) over multiple time points. Toxin stability, as the percent of initial concentration (T0), was reported for each storage treatment and time point. STX was stable (mean 99% T0) in 50% MeOH extracts over the 8-week study period, and there was no significant difference in STX concentrations quantified in split fecal samples extracted in 80% ethanol (EtOH) and 50% MeOH. STX was also relatively stable in raw fecal material stored in the freezer (mean 94% T0) and the refrigerator (mean 93% T0) up to 8 weeks. STX degraded over time in the room-temperature dark, room-temperature light, and warm treatments to means of 48 ± 1.9, 38 ± 2.8, and 20 ± 0.7% T0, respectively, after 8 weeks (mean ± standard error; SE). Additional opportunistically analyzed samples frozen for ≤4.5 years also showed STX to be relatively stable (mean 97% T0). Mean percent of T0 was measured slightly above 100% in some extracts following some treatments, and (most notably) at some long-term frozen time points, likely due to evaporation from samples causing STX to concentrate, or variability between ELISA plates. Overall, these results suggest that long-term frozen storage of raw fecal samples and the analysis of extracts within 8 weeks of extraction in 50% MeOH is sufficient for obtaining accurate STX quantifications in marine mammal fecal material without concerns about significant degradation.     

pH-Dependent Solution Structure and Activity of a Reduced Form of the Host-Defense Peptide Myticin C (Myt C) from the Mussel Mytilus galloprovincialis

Myticin C (Myt C) is a highly variable host-defense peptide (HDP) associated to the immune response in the mediterranean mussel (Mytilus galloprovincialis), which has shown to be active across species due to its strong antiviral activity against a fish rhabdovirus found in fish cells overexpressing this HDP. However, the potential antimicrobial properties of any synthetic analogue of Myt C has not yet been analysed. Thus, in this work we have synthesised the sequence of the mature peptide of Myt C variant c and analysed the structure activity relationships of its reduced (non-oxidized) form (red-MytCc). In contrast to results previously reported for oxidized isoforms of mussel myticins, red-MytCc was not active against bacteria at physiological pH and showed a moderate antiviral activity against the viral haemorrhagic septicaemia (VHS) rhabdovirus. However, its chemotactic properties remained active. Structure/function studies in neutral and acid environments by means of infrared spectroscopy indicated that the structure of red-MytCc is pH dependent, with acid media increasing its alpha-helical content. Furthermore, red-MytCc was able to efficiently aggregate artificial phospholipid membranes at low pH, as well as to inhibit the Escherichia coli growth, suggesting that this activity is attributable to its more structured form in an acidic environment. All together, these results highlight the dynamic and environmentally sensitive behavior of red-Myt C in solution, and provide important insights into Myt C structure/activity relationships and the requirements to exert its antimicrobial/immunomodulatory activities. On the other hand, the pH-dependent direct antimicrobial activity of Myt C suggests that this HDP may be a suitable template for the development of antimicrobial agents that would function selectively in specific pH environments, which are sorely needed in this “antibiotic-resistance era”.     

Salmonid fish viruses and cell interactions at early steps of the infective cycle

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.     

Genetic parameter estimation and gene association analyses for meat quality traits in open-air free-range Iberian pigs

Meat quality of Iberian pigs is defined by the combination of their genetic characteristics and the particular production system. To carry out a genetic analysis of the main meat quality traits, we estimated their heritabilities, genetic correlations and the association effects of 32 selected SNPs of 12 candidate genes. A total of ten traits were measured in longissimus dorsi samples from 1,199 Iberian pigs fattened in the traditional free-range system: water holding capacity (thawing, cooking and centrifuge force water losses), instrumental colour (lightness L*, redness a* and yellowness b*), myoglobin content, shear force on cooked meat, and shear force and maximum compression force on dry-cured loin. Estimated heritability values were low to moderate (0.01 to 0.43) being the lowest for L* and the highest for cooking loss. Strong genetic correlations between water holding capacity traits (0.93 to 0.96) and between myoglobin content and a* (0.94) were observed. The association analyses revealed 19 SNPs significantly associated with different traits. Consistent and strong effects were observed between PRKAG3 SNPs (rs319678464G > C and rs330427832C > T), MYH3_rs81437544T > C, CASP3_rs319658214G > T and CTSL_rs332171512A > G and water losses. Also for CAPN1_rs81358667G > A and CASP3_rs319658214G > T and shear force. The SNPs mapping on PRKAG3 showed the highest effects on Minolta colour traits. Genotyping of these SNPs could be useful for the selection of Iberian young boars with similar estimated breeding values for productive traits.     

Vegetative compatibility of cotton-defoliating <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in Israel and its pathogenicity to various crop plants

Verticillium dahliae isolates recovered from a new focus of severe Verticillium wilt of cotton in the northeast of Israel were tested for vegetative compatibility using nitrate non-utilizing (nit) mutants and identified as VCG1, which is a new record in Israel. Other cotton isolates of V. dahliae from the northern and southern parts of the country were assigned to VCG2B and VCG4B, respectively. VCG1 isolates induced severe leaf symptoms, stunting and defoliation of cotton cv. Acala SJ-2, and thus were characterized as the cotton-defoliating (D) pathotype, whereas isolates of VCG2B and VCG4B were confirmed as the earlier described defoliating-like (DL) and non-defoliating (ND) pathotypes, respectively. This is the first record of the D-pathotype in Israel. The host range of representative isolates of each VCG-associated pathotype was investigated using a number of cultivated plants. Overall, the D isolates were more virulent than DL isolates on all tested host plants, but the order of hosts (from highly susceptible to resistant) was the same: okra (Hibiscus esculentus local cultivar), cotton (Gossypium hirsutum cv. Acala SJ2), watermelon (Citrullus lanatus cv. Crimson Sweet), safflower (Carthamus tinctorius cv. PI 251264), sunflower (Helianthus annuum cv. 2053), eggplant (Solanum melongena cv. Black Beauty), and tomato (Lycopersicon esculentum cv. Rehovot 13). The pattern of virulence of ND isolates differed from that of D and DL isolates, so that the former were highly virulent on eggplant but mildly virulent on cotton. Tomato was resistant to all cotton V. dahliae isolates tested. RAPD and specific PCR assays confirmed that the D isolates from Israel were similar to those originating from other countries.