Determination of Total Lipid and Lipid Classes in Marine Samples.

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being carbon- and hydrogen-rich they are also hydrophobic and can act as a solvent and absorption carrier for organic contaminants and thus can be drivers of pollutant bioaccumulation in marine ecosystems. Their hydrophobic nature facilitates their isolation from seawater or biological specimens: marine lipid analysis begins with sampling and then extraction in non-polar organic solvents, providing a convenient method for their separation from other substances in an aquatic matrix. If seawater has been sampled, the first step usually involves separation into operationally defined 'dissolved' and 'particulate' factions by filtration. Samples are collected and lipids isolated from the sample matrix typically with chloroform for truly dissolved matter and colloids, and with mixtures of chloroform and methanol for solids and biological specimens. Such extracts may contain several classes from biogenic and anthropogenic sources. At this time, total lipids and lipid classes may be determined. Total lipid can be measured by summing individually determined lipid classes which customarily have been chromatographically separated. Thin-layer chromatography (TLC) with flame ionization detection (FID) is regularly used for the quantitative analysis of lipids from marine samples. TLC-FID furnishes synoptic lipid class information and, by summing classes, a total lipid measurement. Lipid class information is especially useful when combined with measurements of individual components e.g., fatty acids and/or sterols, after their release from lipid extracts. The wide variety of lipid structures and functions means they are used broadly in ecological and biogeochemical research assessing ecosystem health and the degree of influence by anthropogenic impacts. They have been employed to measure substances of dietary value to marine fauna (e.g., aquafeeds and/or prey), and as an indicator of water quality (e.g., hydrocarbons).

Comparison of the polarization contrast of gelatinous zooplankton and a transparent single-use plastic bag-Implications for marine animals.

Plastic pollution in the ocean is an increasingly detrimental issue for marine organisms. As a form of polarized light pollution, transparent plastic debris may be more visible and pose additional threats to organisms that can detect and interpret polarized light. Plastic can mimic the visual features of common marine prey items, such as transparent gelatinous zooplankton, which may lead to more significant plastic ingestion. We measured, in situ, the polarization and radiance contrast between a transparent plastic bag and gelatinous zooplankton with an underwater video polarimeter. The plastic bag had significantly higher polarization contrast than the gelatinous zooplankton, yet both shared similar radiance contrasts. This higher polarization contrast may contribute to the observed high ingestion rates of transparent plastic by marine organisms. Further study into the connection between polarization-sensitive organisms and plastic ingestion is recommended.

Three-dimensional measurements of animal paths using handheld unconstrained GoPro cameras and VSLAM software.

I present the system PATMOS (paths and tessellated meshes from ORB_SLAM2) for measuring three-dimensional paths of animalsin situusing two handheld GoPro cameras and a small spatial reference object. Animal paths were triangulated from mobile camera positions obtained from a modified version of ORB_SLAM2, an open-source visual simultaneous localization and mapping software package. In addition to path calculation, this process provided a virtual three-dimensional surface approximation to the environment from which path to environment distances can be quantified. PATMOS can also fit a tranquil water's surface to an analytic plane if there are a sufficient number of visible objects intersecting the water's surface and can track objects over the water's surfaces with a single camera by measuring the object with its reflection. This technology was highly portable, could follow moving animals, and gave comparable spatial and temporal resolutions to fixed camera systems that use commercial cameras. An investigation of falling objects yielded a gravitational constant measurement of 978 ± 40 cm s-2. I demonstrated PATMOS's utility in terrestrial and aquatic environments by quantifying dragonfly flight characteristics and the inter-spatial distances between substrate and damselfish.

Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1.

Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral nonstructural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host species barrier. We characterized the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionarily distinct NS1 proteins counteract the IFN response using different and mutually exclusive mechanisms: while the NS1 proteins of early EIVs block general gene expression by binding to cellular polyadenylation-specific factor 30 (CPSF30), NS1 proteins from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and were rapidly selected for during EIV evolution, highlighting the importance of evolutionary processes in immune evasion mechanisms during IAV adaptation.IMPORTANCE Influenza A viruses (IAVs) infect certain avian reservoir species and occasionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efficiently infect and transmit in mammals remain unclear. H3N8 equine influenza virus (EIV) is an avian-origin virus that successfully established a new lineage in horses in the early 1960s and is currently circulating worldwide in the equine population. Here, we analyzed the molecular evolution of the virulence factor nonstructural protein 1 (NS1) and show that NS1 proteins from different time periods after EIV emergence counteract the host innate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

Genetic Analysis of Feline Panleukopenia Virus Full-length VP2 Gene in Domestic Cats Between 2006-2008 and 2012-2014, Portugal.

Feline panleukopenia virus (FPV) and canine parvovirus (CPV) are two closely related viruses, which cause acute gastroenteritis in carnivores, and cats may be infected by strains of both viruses. The viruses are found worldwide and may have changing host ranges and genetic variation that can be found around the world in some cases. Here, we screened a Portuguese population of cats by a conventional PCR assay for the presence of FPV/CPV viruses in faecal samples and tissues between 2006-2008 and 2012-2014. The sequence analysis of the complete VP2 gene showed that 18 of 31 animals tested were positive for FPV DNA, and no case of CPV infection was detected. The analysis of specific DNA detected three new non-synonymous substitutions in the VP2 gene that were found in single groups and were related to viruses reported elsewhere by phylogenetic analysis - some were related to Italian isolates, one was closely related to isolates from Vietnam and China, and two were related with older strains from the USA. The results of our study show that FPV circulated in the Portuguese cat population and as expected the found strains are slightly divergent from those reported previously.

Polarimetric imaging and retrieval of target polarization characteristics in underwater environment.

Polarized light fields contain more information than simple irradiance and such capabilities provide an advanced tool for underwater imaging. The concept of the beam spread function (BSF) for analysis of scalar underwater imaging was extended to a polarized BSF which considers polarization. The following studies of the polarized BSF in an underwater environment through Monte Carlo simulations and experiments led to a simplified underwater polarimetric imaging model. With the knowledge acquired in the analysis of the polarimetric imaging formation process of a manmade underwater target with known polarization properties, a method to extract the inherent optical properties of the water and to retrieve polarization characteristics of the target was explored. The proposed method for retrieval of underwater target polarization characteristics should contribute to future efforts to reveal the underlying mechanism of polarization camouflage possessed by marine animals and finally to generalize guidelines for creating engineered surfaces capable of similar polarization camouflage abilities in an underwater environment.

Open-ocean fish reveal an omnidirectional solution to camouflage in polarized environments.

Despite appearing featureless to our eyes, the open ocean is a highly variable environment for polarization-sensitive viewers. Dynamic visual backgrounds coupled with predator encounters from all possible directions make this habitat one of the most challenging for camouflage. We tested open-ocean crypsis in nature by collecting more than 1500 videopolarimetry measurements from live fish from distinct habitats under a variety of viewing conditions. Open-ocean fish species exhibited camouflage that was superior to that of both nearshore fish and mirrorlike surfaces, with significantly higher crypsis at angles associated with predator detection and pursuit. Histological measurements revealed that specific arrangements of reflective guanine platelets in the fish's skin produce angle-dependent polarization modifications for polarocrypsis in the open ocean, suggesting a mechanism for natural selection to shape reflectance properties in this complex environment.

Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia.

The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.

Polarization signaling in swordtails alters female mate preference.

Polarization of light, and visual sensitivity to it, is pervasive across aquatic and terrestrial environments. Documentation of invertebrate use of polarized light is widespread from navigation and foraging to species recognition. However, studies demonstrating that polarization body patterning serves as a communication signal (e.g., with evidence of changes in receiver behavior) are rare among invertebrate taxa and conspicuously absent among vertebrates. Here, we investigate polarization-mediated communication by northern swordtails, Xiphophorus nigrensis, using a custom-built videopolarimeter to measure polarization signals and an experimental paradigm that manipulates polarization signals without modifying their brightness or color. We conducted mate choice trials in an experimental tank that illuminates a pair of males with light passed through a polarization filter and a diffusion filter. By alternating the order of these filters between males, we presented females with live males that differed in polarization reflectance by >200% but with intensity and color differences below detection thresholds (∼5%). Combining videopolarimetry and polarization-manipulated mate choice trials, we found sexually dimorphic polarized reflectance and polarization-dependent female mate choice behavior with no polarization-dependent courtship behavior by males. Male swordtails exhibit greater within-body and body-to-background polarization contrast than females, and females preferentially associate with high-polarization-reflecting males. We also found limited support that males increase polarization contrast in social conditions over asocial conditions. Polarization cues in mate choice contexts may provide aquatic vertebrates with enhanced detection of specific display features (e.g., movements, angular information), as well as a signaling mechanism that may enhance detection by intended viewers while minimizing detection by others.

Substitution of fish oil with camelina oil and inclusion of camelina meal in diets fed to Atlantic cod (Gadus morhua) and their effects on growth, tissue lipid classes, and fatty acids.

Developing a commercially relevant Atlantic cod aquaculture industry will require improvements in feed sustainability. Camelina oil and meal are potential replacements of fish oil and fish meal in aquaculture feeds. Camelina oil is high in 18:3ω3 (30%), with an ω3/ω6 ratio > 1. Camelina meal has a considerable crude protein level (38%), which includes significant amounts of methionine and phenylalanine. Four diets were tested; each diet was fed to triplicate tanks (3 tanks per diet) of Atlantic cod (14.4 g/fish; 70 fish per tank) for 13 wk. The diets included a fish oil/fish meal control (FO) and three diets which replaced 100% of fish oil with camelina oil: one diet contained fish meal (100CO), another solvent extracted fish meal (100COSEFM), and another had fish meal partially reduced by 15% inclusion of camelina meal (100CO15CM). Growth was measured (length and weight) and tissue samples were collected for lipid analysis (muscle, liver, brain, gut, spleen, skin, and carcass) at wk 0 (before feeding the experimental diet) and at wk 13. Cod fed camelina oil had a lower (P < 0.001) final weight than cod fed the FO diet (50.8 ± 10.3 g/fish). Cod fed 100CO15CM had a lower (P < 0.001) final weight (35.0 ± 8.0 g) than those fed 100CO (43.6 ± 8.9 g) and 100COSEFM (46.7 ± 10.7 g). Cod tissues in the 100COSEFM treatment were most impacted by dietary fatty acid profile. Multivariate statistics revealed that FO and 100COSEFM tissue fatty acid profiles were 21 to 31% different, depending on tissue type. The full replacement of fish oil with camelina oil, plus solvent extracted fish meal had an overarching effect on the entire fatty acid profile of the whole animal. Fatty acid mass balance calculations indicated that cod fed 100COSEFM elongated 13% of 18:3ω3 to 20:3ω3 and oxidized the remaining 87%, whereas cod fed fish oil showed a much lower (P < 0.001) elongation of 18:3ω3 of 1.6%. These results suggest that excess 18:3ω3 from camelina oil caused some fatty acid elongation, but little desaturation. Energy budget estimates indicated that cod fed 100COSEFM deposited the most energy throughout the trial (60 kJ/fish; P = 0.019), mostly in the liver (90%). Excess camelina lipids were not necessarily utilized for energy, which likely impacted growth. Feeding 100% camelina oil to Atlantic cod impacted growth and lipid and fatty acid composition; however, additional removal of fish oil from fish meal caused the greatest change in cod lipid composition and utilization.

Polaro-cryptic mirror of the lookdown as a biological model for open ocean camouflage.

With no object to hide behind in 3D space, the open ocean represents a challenging environment for camouflage. Conventional strategies for reflective crypsis (e.g., standard mirror) are effective against axially symmetric radiance fields associated with high solar altitudes, yet ineffective against asymmetric polarized radiance fields associated with low solar inclinations. Here we identify a biological model for polaro-crypsis. We measured the surface-reflectance Mueller matrix of live open ocean fish (lookdown, Selene vomer) and seagrass-dwelling fish (pinfish, Lagodon rhomboides) using polarization-imaging and modeling polarization camouflage for the open ocean. Lookdowns occupy the minimization basin of our polarization-contrast space, while pinfish and standard mirror measurements exhibit higher contrast values than optimal. The lookdown reflective strategy achieves significant gains in polaro-crypsis (up to 80%) in comparison with nonpolarization sensitive strategies, such as a vertical mirror. Lookdowns achieve polaro-crypsis across solar altitudes by varying reflective properties (described by 16 Mueller matrix elements m(ij)) with incident illumination. Lookdowns preserve reflected polarization aligned with principle axes (dorsal-ventral and anterior-posterior, m(22) = 0.64), while randomizing incident polarization 45° from principle axes (m(33) = -0.05). These reflectance properties allow lookdowns to reflect the uniform degree and angle of polarization associated with high-noon conditions due to alignment of the principle axes and the sun, and reflect a more complex polarization pattern at asymmetrical light fields associated with lower solar elevations. Our results suggest that polaro-cryptic strategies vary by habitat, and require context-specific depolarization and angle alteration for effective concealment in the complex open ocean environment.

Lipid class and fatty acid content of the leptocephalus larva of tropical eels.

The leptocephalus larva of eels distinguishes the elopomorph fishes from all other bony fishes. The leptocephalus is long lived and increases in size primarily through the synthesis and deposition of glycosaminoglycans. Energy stored during the larval stage, in the form of glycosaminoglycan and lipids, is required to fuel migration, metamorphosis and metabolism of the subsequent glass eel stage. Despite the importance of energy storage by leptocephali for survival and recruitment, their diet, condition and lipid content and composition is essentially unknown. To gain further insight into energy storage and condition of leptocephali, we determined the lipid class and fatty acid concentration of larvae collected on a cross-shelf transect off Broome, northwestern Australia. The total lipid concentration of two families and four sub-families of leptocephali ranged from 2.7 to 7.0 mg g wet weight(-1), at the low end of the few published values. Phospholipid and triacylglycerol made up ca. 63 % of the total lipid pool. The triacylglycerol:sterol ratio, an index of nutritional condition, ranged from 0.9 to 3.7, indicating that the leptocephali were in good condition. The predominant fatty acids were 16:0 (23 mol%), 22:6n-3 (docosahexaenoic acid, DHA, 16 mol%), 18:0 (8.2 mol%), 20:5n-3 (eicosapentaenoic acid, EPA, 6.7 mol%), 18:1n-9 (6.4 mol%) and 16:1n-7 (6.3 mol%). The DHA:EPA ratio ranged from 2.4 to 2.9, sufficient for normal growth and development of fish larvae generally. The leptocephali had proportions of bacterial markers >4.4 %, consistent with the possibility that they consume appendicularian houses or other marine snow that is bacteria rich.

Anticipating the species jump: surveillance for emerging viral threats.

Zoonotic disease surveillance is typically triggered after animal pathogens have already infected humans. Are there ways to identify high-risk viruses before they emerge in humans? If so, then how and where can identifications be made and by what methods? These were the fundamental questions driving a workshop to examine the future of predictive surveillance for viruses that might jump from animals to infect humans. Virologists, ecologists and computational biologists from academia, federal government and non-governmental organizations discussed opportunities as well as obstacles to the prediction of species jumps using genetic and ecological data from viruses and their hosts, vectors and reservoirs. This workshop marked an important first step towards envisioning both scientific and organizational frameworks for this future capability. Canine parvoviruses as well as seasonal H3N2 and pandemic H1N1 influenza viruses are discussed as exemplars that suggest what to look for in anticipating species jumps. To answer the question of where to look, prospects for discovering emerging viruses among wildlife, bats, rodents, arthropod vectors and occupationally exposed humans are discussed. Finally, opportunities and obstacles are identified and accompanied by suggestions for how to look for species jumps. Taken together, these findings constitute the beginnings of a conceptual framework for achieving a virus surveillance capability that could predict future species jumps.

Gastroparesis and functional dyspepsia: excerpts from the AGA/ANMS meeting.

BACKGROUND: Despite the relatively high prevalence of gastroparesis and functional dyspepsia, the aetiology and pathophysiology of these disorders remain incompletely understood. Similarly, the diagnostic and treatment options for these two disorders are relatively limited despite recent advances in our understanding of both disorders. PURPOSE: This manuscript reviews the advances in the understanding of the epidemiology, pathophysiology, diagnosis, and treatment of gastroparesis and functional dyspepsia as discussed at a recent conference sponsored by the American Gastroenterological Association (AGA) and the American Neurogastroenterology and Motility Society (ANMS). Particular focus is placed on discussing unmet needs and areas for future research.

Treatment of gastroparesis: a multidisciplinary clinical review.

This clinical review on the treatment of patients with gastroparesis is a consensus document developed by the American Motility Society Task Force on Gastroparesis. It is a multidisciplinary effort with input from gastroenterologists and other specialists who are involved in the care of patients with gastroparesis. To provide practical guidelines for treatment, this document covers results of published research studies in the literature and areas developed by consensus agreement where clinical research trials remain lacking in the field of gastroparesis.

Differences in morphine and cocaine reinforcement under fixed and progressive ratio schedules; effects of extinction, reacquisition and schedule design.

Opiate reinforcement was evaluated under a progressive ratio (PR) schedule often used for psychostimulant self-administration (termed 'PR 3-4' because the third response requirement was four lever presses) and three additional schedules that were modified to provide successively lower levels of difficulty by decreasing the steepness of response requirement progression (termed 'PR 9-4', 'PR 14-4', and 'PR 26-4' because a response requirement of four was reached with step numbers of 9, 14 and 26, respectively). With the exception of the PR 3-4 schedule, all of the schedules supported morphine self-administration, and morphine self-administration during initial exposure and reacquisition did not differ by more than 10%. In contrast to morphine, cocaine was self-administered under the PR 3-4 schedule, with responding clearly exceeding levels during extinction. The PR 9-4 schedule was most suitable for morphine self-administration because it provided an intermediate level of difficulty, which supported levels of self-administration that exceeded values obtained under extinction but were less than those observed under FR-1. Under the PR 9-4 schedule, the number of self-administered injections of morphine was 61.5% of the number of injections obtained under a simple FR-1 schedule. This compares with a value of 21.0% for cocaine self-administration under the PR 3-4 schedule compared to an FR-1 schedule. These results show important differences in self-administration behavior supported by morphine and cocaine, which are consistent with a lower reinforcing efficacy for opiates relative to psychostimulants.

Flow cytometric characterization of perfused human bone marrow cultures: identification of the major cell lineages and correlation with the CFU-GM assay.

BACKGROUND: Prolific cultures of human bone marrow mononuclear cells (BM MNCs) were recently developed that include a full spectrum of hematopoietic and accessory cells, with the presence of autofluorescent cells indicating adequate cell expansion. However, phenotypic and functional clonogenic characterizations of the autofluorescent cells and the various other subpopulations present in these cultures have not been carried out. METHODS: Cells from a continuously perfused bioreactor inoculated with BM MNCs and cultured for 12 days in serum-containing medium with PIXY321, erythropoietin, and with or without FLT3-L were evaluated by using flow cytometry. RESULTS: Two antibodies, CD71 and CD13, allowed the separation of the autofluorescent cells into two distinct populations. The CD71+CD13++ autofluorescent population contained the colony-forming unit (CFU) fibroblast, and the CD71++CD13++ autofluorescent population contained macrophage/dendritic like cells. The CFU-granulocyte/macrophage (CFU-GM) could not be thoroughly evaluated with CD71 and CD13. However, the number of CD13+/++Lin- cells correlated with the number of CFU-GM (r = 0.83), with approximately 1 CFU-GM for every 30 CD13+/++Lin- cells. CONCLUSIONS: The data showed that CD71 and CD13 antibodies separate the autofluorescent cells into two populations but do not separate hematopoietic cells into specific phenotypic populations. The data also showed that the number of CD13+/++Lin- cells correlated with the number of CFU-GM. These data present the initial step toward detailed phenotypic analysis of ex vivo expanded human BM MNC cultures.

Sex differences in hepatic lipids of Toxaphene-exposed juvenile yellowtail flounder (Pleuronectes ferrugineus Storer).

Histochemical and biochemical effects of Toxaphene on liver were investigated in laboratory-bred female and male juvenile yellowtail flounder (Pleuronectes ferrugineus). Fish were fed uncontaminated food, or food contaminated with hexane (the solvent for Toxaphene) or with one of two concentrations of Toxaphene (0.02 or 0.2 microg/g fish/day) for 2 weeks. Males were more advanced in sexual maturity than females, although all were from the same year-class (0(+)). Liver tissue examined histochemically (Sudan black B, oil red O) revealed that Toxaphene affected storage of total and neutral lipids according to sex and dose. The sexes differed in the amount of total and neutral lipids. Neutral lipid droplets were considerably larger in the liver of females. Lipids were extracted and analyzed using the latroscan TLC/FID system. Triacylglycerols comprised the majority of lipids. Animals exposed to the lowest concentration of Toxaphene stored low amounts of total and neutral lipids and high amounts of polar lipids, while animals exposed to a 10 times higher concentration showed the reverse. Sterols were highest in animals exposed to the highest dose. Thus Toxaphene can alter the lipid composition in the liver of yellowtail flounder, which may have consequences for physiological processes involving the liver, such as lipid metabolism and reproduction.