Cytotoxic Effects of Loperamide Hydrochloride on Canine Cancer Cells

Loperamide is a peripheral opiate agonist that can cause apoptosis and G2/M arrest in human cancer cell lines and may sensitize cells to chemotherapy. The objectives of this study were to investigate the effects of loperamide on viability, apoptosis and cell cycle kinetics in canine cancer cells and to establish whether the drug sensitizes cells to doxorubicin. Cell viability was assessed using Alamar Blue. Cell death and cell cycle were studied using flow cytometry with 7-Aminoactinomycin-D (7-AAD) and propidium iodide (PI), respectively. Loperamide decreased cell viability in a dose-dependent fashion and was most effective against canine osteosarcoma cells. In all cell lines, it induced a dose and time dependent apoptosis and resulted in accumulation in G0/G1. When co-incubated with doxorubicin, loperamide induced a synergistic cell kill in canine carcinoma cells. Investigation is warranted into the role of loperamide in the treatment of canine cancer.     

Using social media as a cost‐effective resource in the photo‐identification of a coastal bottlenose dolphin community

1. Bottlenose dolphins encountered around the Irish coast are considered part of a wide‐ranging coastal community; however, knowledge on the significance of the north of Ireland for this species is limited by a lack of dedicated effort.
2. Through social media, the opportunity now exists to gather large volumes of citizen science data in the form of high‐quality images, potentially extending the spatial and temporal scope of photo‐identification studies.
3. The purpose of this study was to investigate social media as a data resource for photo‐identification studies and to provide a preliminary assessment of bottlenose dolphins in the north of Ireland. Specifically, the study sought to examine the photo‐identification data for spatial clustering.
4. The study identified 54 well‐marked individuals and provided evidence of potential year‐round occurrence, with successful re‐sightings throughout the study period (2007–2016). There was a geographic concentration of re‐sightings along the north of Ireland, suggestive of interannual site fidelity. These results provide scientific rationale for strategically targeting the north of Ireland in future research on the Irish coastal community.
5. For effective conservation of the bottlenose dolphin it is imperative that scientific research, and resultant management objectives, consider wide‐ranging communities such as the Irish coastal community. Our research highlights data collection via social media as a cost‐effective and scientifically valuable tool in the photo‐identification of coastal cetaceans. We recommend that this method is used in research on low‐density and wide‐ranging coastal cetaceans.

     

Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, <Emphasis Type="Italic">Notemigonus crysoleucas</Emphasis>

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R ²: 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; inter-assay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R ²: 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.     

Short-term competition between crop plants and soil microbes for inorganic N fertilizer

Agricultural systems that receive high amounts of inorganic nitrogen (N) fertilizer in the form of either ammonium (NH₄⁺), nitrate (NO₃⁻) or a combination thereof are expected to differ in soil N transformation rates and fates of NH₄⁺ and NO₃⁻. Using ¹⁵N tracer techniques this study examines how crop plants and soil microbes vary in their ability to take up and compete for fertilizer N on a short time scale (hours to days). Single plants of barley (Hordeum vulgare L. cv. Morex) were grown on two agricultural soils in microcosms which received either NH₄⁺, NO₃⁻ or NH₄NO₃. Within each fertilizer treatment traces of ¹⁵NH₄⁺ and ¹⁵NO₃⁻ were added separately. During 8 days of fertilization the fate of fertilizer ¹⁵N into plants, microbial biomass and inorganic soil N pools as well as changes in gross N transformation rates were investigated. One week after fertilization 45-80% of initially applied ¹⁵N was recovered in crop plants compared to only 1-10% in soil microbes, proving that plants were the strongest competitors for fertilizer N. In terms of N uptake soil microbes out-competed plants only during the first 4 h of N application independent of soil and fertilizer N form. Within one day microbial N uptake declined substantially, probably due to carbon limitation. In both soils, plants and soil microbes took up more NO₃⁻ than NH₄⁺ independent of initially applied N form. Surprisingly, no inhibitory effect of NH₄⁺ on the uptake and assimilation of nitrate in both, plants and microbes, was observed, probably because fast nitrification rates led to a swift depletion of the ammonium pool. Compared to plant and microbial NH₄⁺ uptake rates, gross nitrification rates were 3-75-fold higher, indicating that nitrifiers were the strongest competitors for NH₄⁺ in both soils. The rapid conversion of NH₄⁺ to NO₃⁻ and preferential use of NO₃⁻ by soil microbes suggest that in agricultural systems with high inorganic N fertilizer inputs the soil microbial community could adapt to high concentrations of NO₃⁻ and shift towards enhanced reliance on NO₃⁻ for their N supply.     

Photostabilization of biocontrol agents by berberine

We studied the photostabilizing effect of the isoquinoline alkaloid berberine on the following three model photounstable biocontrol agents: the bacterium Bacillus thuringiensis ( Bt ), the baculovirus Spodoptera littoralis nucleopolyhedrosis virus ( SlNPV ), and the entomopathogenic fungus Beauveria bassiana . The photoprotection provided by berberine was evaluated following various periods of simulated sunlight-UV irradiation. Insecticidal activity of Bt and SlNPV , as well as the rate of B. bassiana spore germination served as parameters for the photostabilizing effects. Only a berberine- Bt complex is effective, as practically no photostabilization was evident by just mixing the two components. The UV-sensitive SlNPV and B. bassiana spores were considerably photostabilzed by berberine. The role of the cationic nature of the berberine molecule and the importance of its absorbance spectrum in photostabilization of biocontrol agents are discussed.     

Effects of Amylopectin/Amylose Starch Ratio on Growth, Body Composition and Glycemic Response of Sunshine Bass Morone chrysops × M. saxatilis

Manipulation of the ratio of amylopectin (α‐[1,4] and α‐[1,6] linked glucose) to amylose (α‐[1,41 linked glucose) starches in the carbohydrate fraction of the diet has been used to improve carbohydrate and lipid metabolism in mammalian models. A 10‐wk feeding trial was conducted to determine the effect of dietary amylopectin/amylose ratio on growth and composition of growth of advanced sunshine bass (Morone chrysops × M. saxatilis) fingerlings (60 g, initial weight). Fish were fed cold‐pelleted, semipurified, isonitrogenous (35% crude protein), isocaloric (3.6 kcaVg protein), isolipidic (5%) diets containing 25% carbohydrate. The carbohydrate fraction of the diets was composed of either glucose, dextrin, 100% amylopectin/0% amylose, 70% amylopectin/30% amylose, or 30% amylopectin/70% amylose. Diets differing in ratios of amylopectin/amylose were achieved by adjusting the proportion of high‐amylopectin (100% amylopectin) to high‐amylose (70% amylose) corn starch. Diets were fed to fish in quadruplicate 76‐L tanks (seven fish/tank) connected to a brackish water (5‐7%v) recirculating culture system with biofiltration. Weight gain ranged from 195 to 236% of initial weight (60 g) and was significantly greater (P < 0.1) for fish fed diets containing 25% carbohydrate as dextrin or as 70% amylose and significantly lower in fish fed diets in which carbohydrate was composed of 30% amylose, 100% amylopectin, or glucose. Feed efficiency ranged from 0.52 to 0.61 and was higher in fish fed the diet containing the highest concentration of amylose and lower in fish fed the diet containing glucose. Hepatosomatic index was highest (2.71) in fish fed the diet containing glucose and lowest (1.401.45) in fish fed diets containing high‐amylose cornstarch. Intraperitoneal fat ratio was distinctly lower in fish fed diets containing some amylose as compared to those fed diets without amylose. Liver lipid was significantly lower (4.8%) in fish fed the diet containing glucose and almost twice as high (7.3‐8.9%) in fish fed the diets containing any starch. Glycogen content of the liver decreased from approximately 12% in fish fed the diet containing glucose to 5% in fish fed the diets containing amylose. Muscle proximate composition and ratio were unaffected by the dietary treatments. Fasting levels (15 h) of blood glucose in fish reared for 10 wk on the diet containing glucose were significantly elevated (5.5 mmol/L) when compared to fasting levels of those that had been reared on diets containing starch (3.4‐1.1 mmol/L). Fish fed the diet containing glucose exhibited maximum blood concentrations (14.6 mmoVL) 4 h postprandial then rapidly declined to nearly fasting levels within 8 h postprandial. In contrast, maximum plasma glucose concentrations in fish fed diets containing starch were roughly half (6.8‐8.1 mmol/L) those of fish fed the diet containing glucose. Blood glucose in fish fed diets containing dextrin or predominantly amylopectin starch remained elevated longer than that of fish fed diets containing glucose or predominantly amylose starch. Glycemic response appeared to decrease with increasing dietary amylose content. These data suggest that feeding diets in which a greater portion of the starch is amylose may be a useful strategy for improving carbohydrate use in sunshine bass.     

Massive Hepatic Necrosis and Nodular Regeneration in Largemouth Bass Fed Diets High in Available Carbohydrate

Largemouth bass Micropterus salmoides are piscivorous fish raised on farms then sold live in Asian fish markets on the east and west coasts of the United States. In the winter of 1998, a major producer of feed‐trained bass suffered a significant increase in fish mortality both during shipping and while the fish were still in ponds. No bacterial, viral, or significant parasitic pathogens were found at necropsy. Livers of affected fish were pale and translucent with 3–10 mm pink nodules on their surface and deeper in the parenchyma. Histological examination of these livers showed that the translucent regions of the liver contained few hepatocytes and were composed of tissue consistent with a chronic inflammatory response. Also present were eosinophils, islands of pancreatic and biliary cells, and granulomas that did not stain positively for mycobacteria. The pink nodules were areas of multifocal regeneration of normal hepatocytes. Sequential studies of bass in ponds revealed that the bass were progressively accumulating glycogen in their hepatocytes to an extent sufficient to explain the massive necrosis of that organ. In order to determine the effect of diets varying in available carbohydrate on fish growth, survival, and liver glycogen content, a 12‐wk feeding trial was conducted in aquaria with juvenile largemouth bass. Nitrogen‐free extract values indicated that an extruded trout diet, a steelhead trout diet, and a diet designed to contain 45% protein and 25% fat, contained 35, 27, and 21% carbohydrate, respectively. Weight gain was lowest in fish fed the extruded trout diet, while liver glycogen was significantly higher in fish fed diets with >27% carbohydrate than in fish fed the diet with 21% carbohydrate. The farmer switched to a diet similar to the 45–25 diet used in our trial. Subsequently, 16‐mo‐old fish examined in October 2000 had no hepatic nodules or necrosis, were significantly larger and less variable in size than previous crops, and the farmer experienced no significant losses in ponds or during shipping.