Predictive Factors of Sensitivity to Elisidepsin,a Novel Kahalalide F-Derived Marine Compound

Elisidepsin (PM02734, Irvalec^®) is a synthetic marine-derived cyclic peptide of the Kahalalide F family currently in phase II clinical development. Elisidepsin was shown to induce rapid oncosis in ErbB3-expressing cells. Other predictive factors of elisidepsin sensitivity remained unknown. A panel of 23 cancer cell lines of different origin was assessed for elisidepsin cytotoxicity and correlated with mutational state, mRNA and protein expression of selected genes. Elisidepsin showed potent and broad cytotoxic effects in our cancer cell line panel, being active at concentrations ranging from 0.4 to 2 μM that may be relevant for clinical settings. We have shown that elisidepsin is more active in cells harboring epithelial phenotype with high E-cadherin and low vimentin expression. In addition, high ErbB3 and Muc1 expression was correlated with sensitivity to elisidepsin, whereas the presence of KRAS activating mutations was associated with resistance. In DU-PM cells with acquired resistance to elisidepsin, ErbB3 expression was decreased, while Bcl2 was increased. DU-PM cells displayed higher sensitivity to ErbB1-inhibitors suggesting possible cross-talk of ErbB1 and ErbB3 signaling pathways. Combinations of elisidepsin with lapatinib and several chemotherapies including 5-FU and oxaliplatin resulted in synergistic effects that offer the potential of clinical use of elisidepsin in combination settings.     

Anti HSV-1 Activity of Halistanol Sulfate and Halistanol Sulfate C Isolated from Brazilian Marine Sponge Petromica citrina (Demospongiae)

The n-butanol fraction (BF) obtained from the crude extract of the marine sponge Petromica citrina, the halistanol-enriched fraction (TSH fraction), and the isolated compounds halistanol sulfate (1) and halistanol sulfate C (2), were evaluated for their inhibitory effects on the replication of the Herpes Simplex Virus type 1 (HSV-1, KOS strain) by the viral plaque number reduction assay. The TSH fraction was the most effective against HSV-1 replication (SI = 15.33), whereas compounds 1 (SI = 2.46) and 2 (SI = 1.95) were less active. The most active fraction and these compounds were also assayed to determine the viral multiplication step(s) upon which they act as well as their potential synergistic effects. The anti-HSV-1 activity detected was mediated by the inhibition of virus attachment and by the penetration into Vero cells, the virucidal effect on virus particles, and by the impairment in levels of ICP27 and gD proteins of HSV-1. In summary, these results suggest that the anti-HSV-1 activity of TSH fraction detected is possibly related to the synergic effects of compounds 1 and 2.     

The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia

Background

Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.

Results

Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.

Conclusions

Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.     

Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine

Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.     

Optimal Management Practices for Nitrogen Application in Corn Cultivated During Summer and Fall in the Tropics

Optimal management practices for nitrogen (N) fertilization is well defined for corn (Zea mays) cultivated during summer (“summer” corn), but not for corn cultivated during the fall (“fall” corn) in the tropics. Two experiments were carried out to evaluate N rates (50, 100, 200, and 300 kg N ha^?1), N application timing (pre-planting – PP, V2–V3, and V5–V6) and N split application, once (at PP, V3, and V6), two (at V3+ V6) and three times (at PP+ V3+ V6) in corn cultivated during summer (2014/2015) and fall (2015/2016). Data on corn grain yield (CGY), weight of 1000-grains, leaf N content and values of soil-plant analyses development (SPAD) were collected and analyzed using univariate, multivariate (principal component analysis, PCA) and regression analysis. Results showed that corn growth was affected by N rates and splitting. Corn cultivated during summer presented higher CGY and weight-1000 g than corn cultivated during the fall. The highest yields were obtained with higher N rates on “summer” corn (125 kg N ha^?1) than “fall” corn (50 kg N ha^?1). Split N-application at vegetative growth stages, V3+ V6, or PP+ V2+ V6, provided higher yields for “summer” corn, while only PP application was a reliable period of N fertilization for “fall” corn. The finding is that corn cultivated during the fall presented a lower response to N and no obvious advantages to split N fertilization when compared to corn cultivated during summer. These optimal management practices for N fertilization in corn production in the tropics depend on soil water availability.     

Recombinant human lactoferrin and iron transport across Caco-2 monolayers: effect of heat treatment on the binding to cells

Recombinant human lactoferrin (rhLF) from Aspergillus awamori bound to Caco-2 cell membranes in a saturable manner. The dissociation constant for the apo form was (Kd)=2.2 x 10(-7) M; however, the specific binding of the iron-saturated rhLF and of lactoferrin from human milk (hLF) was too low to calculate the binding parameters. Recombinant human lactoferrin subjected to heat treatment did not lose the ability to bind to cell membranes except at high temperature and long time treatments (85 and 89 degrees C for 40 min) for which there was a slight decrease in the binding. No significant differences have been found in the transport of iron bound to rhLF or to hLF across Caco-2 cell monolayers. Nevertheless, the amount of iron-saturated hLF transported across Caco-2 monolayers was significantly higher than that of rhLF. For both lactoferrins, the amount of intact protein in the lower chamber was about 4.5% of the total radioactivity transported, indicating the degradation of lactoferrin in the passage across Caco-2 cells.     

Fatty acid effect on hydroxypropyl methylcellulose-beeswax edible film properties and postharvest quality of coated 'Ortanique' mandarins

The objective of this work was to investigate the effect of fatty acid (FA) type and content on mechanical properties, water vapor permeability and oxygen permeability of hydroxypropyl methycellulose (HPMC)-beeswax (BW) stand-alone edible films. The effect of these films formed as coatings on the postharvest quality of 'Ortanique' mandarins was also studied. Selected FAs were stearic acid (SA), palmitic acid (PA), and oleic acid (OA), using BW/FA ratios of 1:0.5 and 1:0.2 (w/w). HPMCBW coatings reduced weight and firmness loss of 'Ortanique' mandarins, without compromising flavor quality compared to uncoated mandarins. Coatings containing OA provided the best weight loss control at both concentrations tested; however, when the BW/OA ratio was 1:0.5, the coatings increased fruit internal CO2, ethanol, and acetaldehyde contents of 'Ortanique' mandarins, therefore reducing flavor compared to the rest of the coatings studied. Although barrier and mechanical properties might be used to understand coating performance, differences observed between film oxygen permeability and coating permeability indicate that permeance should be measured on the coated fruit.     

Phenoloxidase activity of hemocyanin in whiteleg shrimp Penaeus vannamei: conversion, characterization of catalytic properties, and role in postmortem melanosis

Latent phenoloxidase activity of hemocyanin (Hc) in whiteleg shrimp Penaeus vannamei was assayed to determine its potential involvement in postmortem melanosis. Conversion of pure 12-mer, but not 6-mer, hemocyanin to phenoloxidase by endogenous (serine proteinases) and exogenous (SDS) effectors demonstrated the need of complex aggregation for displaying enzyme activity. Because Hc was converted to Hc-phenoloxidase (HcPO) by hemocytes extracts, the mechanism of conversion seems to be the same for polyphenoloxidases. HcPO has similar biochemical and kinetic properties as real polyphenoloxidases and uses mono- and diphenols as substrates. The kinetics of hydroxygenation of monophenols has a lag phase, typical for tyrosinases, contrary to oxidation of diphenols. Regardless of the structure of the substrate, melanin is finally formed. Because of the abundance, distribution, and resistance of Hc to freezing-thawing, involvement of Hc in black spot formation postmortem is suggested. This has important implications for commercialization of shrimp and related seafood.     

Growth and survival of juvenile red drum,Sciaenops ocellatus,acclimated to freshwater at three different stocking densities in a partial recirculation system

Preliminary technical baseline information for the inland culture of juvenile red drum, Sciaenops ocellatus, in a freshwater fish hatchery is presented. Three initial stocking densities (d₁ = 83, d₂ = 167, and d₃ = 250 fish/m³) were tested in a semirecirculation system for 575 days using a commercially available feed (48.1% protein, 25% lipids, and 0.13% fiber). Initial and final mean fish weight were 7.11 ± 0.02 g and 287.6 ± 27 g. As carrying capacity appeared to have been reached, only data from Days 1–333 were analyzed. One‐way ANOVA analyses indicated that the survival rate of the smaller density (d₁ =40 ± 3.7%) was significantly different from the other two densities (d₃ = 21 ± 1.1% and d₂ = 23 ± 1.0%), which did not differ between them. Absolute growth rate (0.87 ± 0.02 g/day) was not significantly different between densities (F = 0.23. p = .801). Neither was weight significantly different between treatments. There were significant differences in the feed conversion rate (FCR) between densities (F = 8.54; p = .02). FCR for d₁ was significantly lower than for the two other densities, which did not differ from each other. A von Bertalanffy growth model was adjusted (R² = 0.95), and weight–length relationship presented negative allometric values (b = 2.85, R² = 0.98).     

Nutritional Contribution of Biofloc within the Diet of Growout and Broodstock of Litopenaeus vannamei,Determined by Stable Isotopes and Fatty Acids

The relative contributions of feed sources were determined through the isotopic signal (δ¹³C and δ¹⁵N) and fatty acid profile of feed items, shrimp muscle, and eggs of Litopenaeus vannamei reared in a biofloc system. In the growout phase, the isotope analysis showed the biofloc particle size class ≥250 μm contributed 55–100%; size ≥50 < 250 μm contributed 0–22%; and artificial feed contributed 0–45%. Principal component analysis applied to fatty acid profiles showed that biofloc ≥250 μm and artificial feed were the most important items in shrimp growout. For the egg production, isotope analysis suggested that the most important feed sources according to their relative contributions were polychaetes (0–100%), followed by artemia biomass (0–86%) and semi‐moist feed (0–66%), with lower contributions from squid, mussel, and the muscle of L. vannamei broodstock that had been cultured in biofloc. In terms of fatty acids, the most important items were artemia, polychaetes, and semi‐moist feeds. This work clarified the importance of feed sources for shrimp during culture in biofloc systems and during reproduction. Analysis of stable isotopes and fatty acids can be successfully used to trace the assimilation of nutrients during the nutrition of shrimp.