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”.     

Immunohistochemical expression of HER - 2 and Ki - 67 in granulosa cell tumor in bitches

Granulosa cell tumour, an ovarian neoplasm of stromal origin, is an important tumour related to oestrogenic dominance syndrome and cystic endometrial hyperplasia–pyometra complex. In order to analyse ovarian tumour´s malignant potential, immunohistochemical markers can be used, such as anti-HER2 and anti-Ki-67. The aim of this study was to evaluate the expression of immunohistochemical markers HER-2 and Ki-67 in granulosa cell tumour from bitches´ ovaries. In HER-2 immunomarker analysis using the HercepTest^® method, most tumours were classified as 2+ (moderate labelling). Concerning Ki-67 immunomarker, only one case was described as having a high proliferative index. An association was found between immunostained cell percentage by anti-HER-2 antibodies and high pleomorphism, represented by the pattern of follicular/trabecular tumour arrangement. There was no correlation between anti-Ki-67 and anti-HER-2 antibody immunostaining intensities, probably due to only one case with a high Ki-67 index. With an effective protocol for HER-2 and Ki-67 immunohistochemical identification in granulosa cell tumours in bitches, it was possible to characterize this neoplasm proliferation profile.     

Evaluation of single and comparative intradermal tuberculin tests for tuberculosis eradication in caprine flocks in Castilla y León (Spain)

Goats can act as reservoirs for tuberculosis (TB) infection. The main etiological agents of TB in goats are Mycobacterium caprae and Mycobacterium bovis and they infect also a wide range of domestic and wild animals and humans. Control programmes based mainly on the application of single and comparative intradermal tuberculin (SIT and SCIT respectively) tests are being implemented in certain regions of Spain with a high density of caprine flocks as Castilla y León, including goats with epidemiological relationship with cattle. The aim of this study was to evaluate the performance of the intradermal tests in naturally TB-infected caprine flocks from this region. The study was performed using data from 17,450 goats in 54 different flocks that were classified as TB-infected in the control programmes executed in 2010 and 2011. Data from 1237 goats from 7 dairy flocks depopulated after the first intradermal testing were used to estimate the sensitivity (Se) using bacteriology as the gold-standard. Overall Se of the SIT test using the severe interpretation was 43.9% (CI 95%, 40.4–47.4) and decreased to 38.8% (CI 95%, 35.5–42.3) using the standard interpretation. Overall Se of the SCIT test ranged between 21.3% (CI 95%, 17.6–25.4) and 7% (CI 95%, 4.9–9.8) depending of the interpretation criteria. A significant weak positive correlation was found between age and skin fold thickness (Spearman’s test p < 0.05). Results from this study yielded, in general, low Se values probably due the systematic detection and slaughter of reactors as a consequence of the eradication programme in previous years or the presence of factors that may interfere in the diagnosis. Therefore, these results suggest the necessity of including ancillary diagnostic tools and/or strict interpretation criteria to maximize detection of positive animals in infected settings.     

Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice

Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.     

Sperm subpopulations influence the pregnancy rates in cattle

This study aimed to study the characteristics and subpopulations of spermatozoa from bulls with low and high reproductive performance based on pregnancy rates. Based on historical records of pregnancy rate from four farms, 24 bulls were selected. Two groups were established, with low pregnancy rates (n = 12; LOW), including bulls that presented pregnancy rates <52.27% (33.33% to 51.81%); and a group with high pregnancy rates (n = 12; HIGH), with pregnancy rates >52.27% (52.27% to 69.64%), after fixed-time artificial insemination (FTAI). The thawed sperm straws were analysed to sperm kinetics, morphology, plasma membrane integrity and sperm subpopulations. The LOW group exhibited a higher proportion of static cells (p < .05). In contrast, the HIGH group showed greater percentages for membrane integrity and total and progressive motility, and cells with fast and medium velocity (p < .05). In the cluster procedures, four sperm subpopulations were established. The low-fertility bulls presented the highest percentage of subpopulation 2 (41.46%), characterized by slow and progressive spermatozoa. The high-fertility bulls exhibited the highest percentage of subpopulation 3 (37.17%), characterized by fast and nonlinear spermatozoa. Results from this study indicated that bulls with greater percentages of fast and nonlinear spermatozoa seem to have greater fertilization capacity and the subpopulations analysis can be considered a tool to identify ejaculates with high fertility.     

Free amino acid composition of quince (Cydonia oblonga Miller) fruit (pulp and peel) and jam

Twenty-one free amino acids present in several samples of quince fruit (pulp and peel) and quince jam (homemade and industrially manufactured) were analyzed by GC/FID. The analyses showed some differences between quince pulps and peels. Generally, the highest content in total free amino acids and in glycine was found in peels. As a general rule, the three major free amino acids detected in pulps were aspartic acid, asparagine, and hydroxyproline. For quince peels, usually, the three most abundant amino acids were glycine, aspartic acid, and asparagine. Similarly, for quince jams the most important free amino acids were aspartic acid, asparagine, and glycine or hydroxyproline. This study suggests that the free amino acid analysis can be useful for the evaluation of quince jam authenticity. It seems that glycine percentage can be used for the detection of quince peel addition while high alanine content can be related to pear addition.     

Climate Variability and Oceanographic Settings Associated with Interannual Variability in the Initiation of Dinophysis acuminata Blooms

In 2012, there were exceptional blooms of D. acuminata in early spring in what appeared to be a mesoscale event affecting Western Iberia and the Bay of Biscay. The objective of this work was to identify common climatic patterns to explain the observed anomalies in two important aquaculture sites, the Galician Rías Baixas (NW Spain) and Arcachon Bay (SW France). Here, we examine climate variability through physical-biological couplings, Sea Surface Temperature (SST) anomalies and time of initiation of the upwelling season and its intensity over several decades. In 2012, the mesoscale features common to the two sites were positive anomalies in SST and unusual wind patterns. These led to an atypical predominance of upwelling in winter in the Galician Rías, and increased haline stratification associated with a southward advection of the Gironde plume in Arcachon Bay. Both scenarios promoted an early phytoplankton growth season and increased stability that enhanced D. acuminata growth. Therefore, a common climate anomaly caused exceptional blooms of D. acuminata in two distant regions through different triggering mechanisms. These results increase our capability to predict intense diarrhetic shellfish poisoning outbreaks in the early spring from observations in the preceding winter.     

Nutrient Loading by Precipitation in the Maracaibo Lake Basin, Venezuela

Nutrient content and pH of rain samples collected at nine sites during 1988-1996 were studied to determine the amounts of N, P and S added by precipitation over Maracaibo Lake basin. The basin is a wide depression occupied by Lake Maracaibo and extensive alluvial plains and delimited by a mountainous zone of variable physiolgraphic characteristics with different cattle ranching and industrial activities. Rain pHs averaged ≈ 4 to 6, individual samples occasionally reached as low as pH 3 in the south basin. NH₄ ⁺ levels were significantly higher (up to 2.6 mg/l NH₄-N) than NO₃ ^? plus NO₂ ^?. Phosphorus levels were low, ranging from undetectable to 1.5 mg/l. The amounts of inorganic N/ha/yr added ranged from ≈ 5.4 Kg in the north to ≈ 8 Kg in the south basin; around the lakeshore, it was estimated that ≈ 9.8–16.8 Kg TN/ha (≈ 55 % as organic-N) are annually deposited. The annual amounts of SO₄-S/ha added range from ≈ 10 to 13 Kg. The contribution of precipitation to P in the ecosystem is very small; ≈ 0.02 – 0.8 Kg of PO₄-P/ha/yr was added in basin. The results of this study show that precipitation represents a significant factor for the total nitrogen and sulfur loading to Maracaibo Lake Basin.     

Vitamin E composition of walnuts (Juglans regia L.): a 3-year comparative study of different cultivars

The tocopherol and tocotrienol composition of walnuts (Juglans regia L.) was determined for nine cultivars (cvs. Arco, Franquette, Hartley, Lara, Marbot, Mayette, Mellanaise, Parisienne, and Rego). Walnuts were harvested over three consecutive years from two different geographical origins (Bragan?a and Coimbra, Portugal), for a total of 26 samples. The methodology employed was a normal-phase high-performance liquid chromatography coupled to a series arrangement of a diode array detector followed by a fluorescence detector, allowing the simultaneous analysis of all tocopherols and tocotrienols. The analyses showed that all samples presented a similar qualitative profile composed of five compounds: alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, and gamma-tocotrienol. gamma-Tocopherol was the major compound in all samples, ranging from 172.6 to 262.0 mg/kg, followed by alpha- and delta-tocopherols, ranging from 8.7 to 16.6 mg/kg and from 8.2 to 16.9 mg/kg, respectively. Multivariate analysis of the data obtained showed the existence of significant differences in composition among cultivars. These differences were also significant when cultivars were grouped by year of production, showing that besides genetic factors, the vitamin E composition was influenced by environmental factors.     

Chemical composition and glycemic index of Brazilian pine (Araucaria angustifolia) seeds

The seeds of Parana pine (Araucaria brasiliensis syn. Araucaria angustifolia), named pinh?o, are consumed after cooking and posterior dehulling, or they are used to prepare a flour employed in regional dishes. Native people that live in the South of Brazil usually consume baked pinh?o. As a result of cooking, the white seeds become brown on the surface due to the migration of some tinted compounds present in the seed coat. In this work, the proximate composition, minerals, flavonoids, and glycemic index (GI) of cooked and raw pinh?o seeds were compared. No differences in moisture, lipids, soluble fiber, and total starch after boiling were found. However, the soluble sugars and P, Cu, and Mg contents decreased, probably as a consequence of leaching in the cooking water. Also, the boiling process modified the profile of the phenolic compounds in the seeds. No flavonols were detected in raw pinh?o seeds. The internal seed coat had a quercetin content five times higher than that of the external seed coat; also, quercetin migrated into the seed during cooking. The internal seed coat had a high content of total phenolics, and seeds cooked in normal conditions (with the seed coat) showed a total phenolics content five times higher than that of seeds cooked without the seed coat. Cooking was then extremely favorable to pinh?o seeds bioactive compounds content. The carbohydrate availability was evaluated in a short-term assay in humans by the GI. The GI of pinh?o seeds cooked with the coat (67%) was similar to that of the seeds cooked without a coat (62%) and lower than bread, showing that cooking does not interfere with starch availability. The low glycemic response can be partly due to its high content of resistant starch (9% of the total starch).