A new method for isolation of interstitial fluid from human solid tumors applied to proteomic analysis of ovarian carcinoma tissue

Major efforts have been invested in the identification of cancer biomarkers in plasma, but the extraordinary dynamic range in protein composition, and the dilution of disease specific proteins make discovery in plasma challenging. Focus is shifting towards using proximal fluids for biomarker discovery, but methods to verify the isolated sample's origin are missing. We therefore aimed to develop a technique to search for potential candidate proteins in the proximal proteome, i.e. in the tumor interstitial fluid, since the biomarkers are likely to be excreted or derive from the tumor microenvironment. Since tumor interstitial fluid is not readily accessible, we applied a centrifugation method developed in experimental animals and asked whether interstitial fluid from human tissue could be isolated, using ovarian carcinoma as a model. Exposure of extirpated tissue to 106 g enabled tumor fluid isolation. The fluid was verified as interstitial by an isolated fluid:plasma ratio not significantly different from 1.0 for both creatinine and Na(+), two substances predominantly present in interstitial fluid. The isolated fluid had a colloid osmotic pressure 79% of that in plasma, suggesting that there was some sieving of proteins at the capillary wall. Using a proteomic approach we detected 769 proteins in the isolated interstitial fluid, sixfold higher than in patient plasma. We conclude that the isolated fluid represents undiluted interstitial fluid and thus a subproteome with high concentration of locally secreted proteins that may be detected in plasma for diagnostic, therapeutic and prognostic monitoring by targeted methods.     

Notes on feeding ecology of macrourid fishes from the mid-Atlantic Ridge, North Atlantic

At least 16 macrourid fishes (Gadiformes, Macrouridae) inhabit the mid-Atlantic Ridge of the North Atlantic. To investigate the roles of macrourids in the food-web and compare feeding patterns of the most frequent co-occurring species, diet information on five of the species were described and compared. Pelagic and benthopelagic copepods were the most numerous prey but did not contribute much on a weight basis. Cephalopods were by far the most important prey of the small grenadiers, while shrimps and fish became increasingly significant with increasing size. Previous studies from other areas have also found pelagic prey to be important, but in contrast to this study, cephalopods were more important on the mid-Atlantic Ridge than in continental margin locations.     

Candida utilis and Chlorella vulgaris Counteract Intestinal Inflammation in Atlantic Salmon (Salmo salar L.)

Intestinal inflammation, caused by impaired intestinal homeostasis, is a serious condition in both animals and humans. The use of conventional extracted soybean meal (SBM) in diets for Atlantic salmon and several other fish species is known to induce enteropathy in the distal intestine, a condition often referred to as SBM induced enteropathy (SBMIE). In the present study, we investigated the potential of different microbial ingredients to alleviate SBMIE in Atlantic salmon, as a model of feed-induced inflammation. The dietary treatments consisted of a negative control based on fish meal (FM), a positive control based on 20% SBM, and four experimental diets combining 20% SBM with either one of the three yeasts Candida utilis (CU), Kluyveromyces marxianus (KM), Saccharomyces cerevisiae (SC) or the microalgae Chlorella vulgaris (CV). Histopathological examination of the distal intestine showed that all fish fed the SC or SBM diets developed characteristic signs of SBMIE, while those fed the FM, CV or CU diets showed a healthy intestine. Fish fed the KM diet showed intermediate signs of SBMIE. Corroborating results were obtained when measuring the relative length of PCNA positive cells in the crypts of the distal intestine. Gene set enrichment analysis revealed decreased expression of amino acid, fat and drug metabolism pathways as well as increased expression of the pathways for NOD-like receptor signalling and chemokine signalling in both the SC and SBM groups while CV and CU were similar to FM and KM was intermediate. Gene expression of antimicrobial peptides was reduced in the groups showing SBMIE. The characterisation of microbial communities using PCR-DGGE showed a relative increased abundance of Firmicutes bacteria in fish fed the SC or SBM diets. Overall, our results show that both CU and CV were highly effective to counteract SBMIE, while KM had less effect and SC had no functional effects.     

Purification of protein synthesis initiation factor eIF-3 from rat liver microsomes by affinity chromatography on rRNA-cellulose

An efficient four-step procedure is described for preparing highly purified polypeptide chain initiation factor eIF-3 from rat liver microsomal saltwash. The method involves fractionation with ammonium sulfate between 25–40% saturation (0°C) followed by affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation. eIF-3 is eluted from the affinity column at a KCl concentration of 0.18 M. The purification is 10-times and the recovery of activity better than 85%. In the sucrose gradients, eIF-3 sediments as a 15 S particle indicating a total mass of 650 000 Da. The purified eIF-3 is highly active in stimulating globin synthesis in a fractionated translation system. Factor eIF-3 contains eight subunits with molecular weights ranging from 40 000 to 110 000. Seven of the subunits are present in one copy per eIF-3, whereas the factor contains two copies of one subunit. The isoelectric points of the factor subunits range from 5.5 to 7.3 with most of the polypeptides being acidic.     

Migration patterns of a potamodromous piscivore,asp (Leuciscus aspius), in a river–lake system

Potamodromous fishes require safe migration routes between spawning, feeding and wintering habitats to complete their life cycle. As knowledge on asp migrations is restricted, this work investigated the movements of adult asp tagged with acoustic transmitters for 3 years in the large Peipsi-Emajõgi-Võrtsjärv lake–river system, Estonia, which is free of migration barriers. Asp showed complex migration patterns, moving between and within different waterbodies (lakes, river, tributaries) in all seasons, but with a tendency to repeat habitat use patterns between years. Lakes were mainly used for feeding during spring and summer (after spawning 65% of the fish migrated to Lake Peipsi), and more so by large fish. The majority (80–96%) of the fish spent the winter in the rivers, mostly close to their subsequent spawning area. Spawning areas were in swift-flowing waters in tributaries and the main river. The results indicate that asp may benefit from an extensive and diverse complex of habitats, and any migration barrier during any season may restrict the natural habitat use by asp. Maintenance and restoration of habitat heterogeneity and connectivity is critical to protect behaviourally diverse fish populations and increase resilience in rivers negatively impacted by various human activities.     

Site fidelity of Atlantic cod Gadus morhua L. as deduced from telemetry and stable isotope studies

A study was performed to test the hypothesis that Atlantic cod Gadus morhua captured within fjords do not migrate across the sill into outer fjord segments or coastal waters. An 18 month long telemetry experiment, corroborated by recapture patterns, showed that 40% of the tagged fish crossed the sill. The majority of the fish that crossed the sill, however, returned after short excursions of only a few days. Many fish remained stationary at or near the release location, and migrations up the fjord into brackish water appeared more frequent than migration out of the fjord. The stable isotope analyses showed significant differences between the inner and the outer fjord segments, and this was seen for both adults and juveniles. This suggests that the Atlantic cod in the two areas have different diets and probably utilize different foraging ranges. Thus, both the isotope studies and the telemetry experiment suggest limited cross-sill migration and rather high site fidelity. Marketing restrictions only apply to fish caught in the fjord proper, not the comparatively clean outer fjord system beyond the sill. Based on this study of migratory tendencies, it is concluded that contamination levels in Atlantic cod in both the Frierfjord proper and the outer fjords probably result from local exposure and appear little affected by inter-fjord migration.     

Female turnover rate differs between two Northern Goshawk Accipiter gentilis nesting areas,as revealed by DNA analysis of moulted feathers

The turnover rate of breeding Northern Goshawk Accipiter gentilis females in two study areas in southern Norway, which are assumed to experience different levels of illegal human persecution, was estimated using DNA extracted from moulted feathers, along with other characteristics. Female turnover rate over a 4‐year period for each nesting territory during 1990–2015 was significantly higher in Aust‐Agder County (31.9%; 23 territories), where hunters have long shown antipathy to Goshawks, than in Telemark County (17.3%; 25 territories). Turnover rate was not related to territory quality. The proportion of successful nesting attempts was lower in Aust‐Agder (70–75%) than in Telemark (82–84%), but for newly built nests, which are less likely to be known by hunters, the difference was not significant (77.4 vs 79.6%). The proportion of juvenile female Goshawks among recruits and in the total breeding population also did not differ between the two areas. Our findings support the notion that Goshawk persecution was higher in Aust‐Agder than in Telemark. We found that the use of phenotypic characteristics for individual recognition was successful in 96.2% of 130 comparisons of female Goshawks from two consecutive nesting attempts, when assessed using genetic markers.     

Responses of Microbial Communities in Arctic Sea Ice After Contamination by Crude Petroleum Oil

Microbial communities associated with Arctic fjord ice polluted with petroleum oils were investigated in this study. A winter field experiment was conducted in the Van Mijen Fjord (Svalbard) from February to June 2004, in which the ice was contaminated with a North Sea paraffinic oil. Holes were drilled in the ice and oil samples frozen into the ice at the start of the experiment. Samples, including cores of both oil-contaminated and clean ice, were collected from the field site 33, 74, and 112 days after oil application. The sampled cores were separated into three sections and processed for microbiological and chemical analyses. In the oil-contaminated cores, enumerations of total prokaryotic cells by fluorescence microscopy and colony-forming units (CFU) counts of heterotrophic prokaryotes both showed stimulation of microbial growth, while concentrations of oil-degrading prokaryotes remained at similar levels in contaminated and clean ice. Analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments by denaturing gradient gel electrophoresis (DGGE) revealed that bacterial communities in oil-contaminated ice generated fewer bands than communities in clean ice, although banding patterns changed both in contaminated and clean ice during the experimental period. Microbial communities in unpolluted ice and in cores contaminated with the paraffinic oil were examined by cloning and sequence analysis. In the contaminated cores, the communities became predominated by Gammaproteobacteria related to the genera Colwellia, Marinomonas, and Glaciecola, while clean ice included more heterogeneous populations. Chemical analysis of the oil-contaminated ice cores with determinations of n-C17/Pristane and naphthalene/phenanthrene ratios indicated slow oil biodegradation in the ice, primarily in the deeper parts of the ice with low hydrocarbon concentrations.     

DNA evidence that Marshallagia marshalli Ransom, 1907 and M. occidentalis Ransom, 1907 (Nematoda: Ostertagiinae) from Svalbard reindeer are conspecific

The gastro-intestinal parasitic nematodes of ruminants Marshallagia marshalli and M. occidentalis are morphs of a single species according to indirect evidence. In this study, their taxonomic status and molecular identification were assessed more directly in isolates from the abomasal nematode community of Svalbard reindeer using genetic data. DNA sequences of the first and second internal transcribed spacers of nuclear ribosomal RNA genes were obtained from individual nematodes by the polymerase chain reaction (PCR). Both taxa contained virtually identical sequences of each ITS and shared most of the polymorphisms detected. A PCR assay based on ITS-2 sequences previously developed to identify M. marshalli and Ostertagia gruehneri, the second common species in this community, gave identical results for M. marshalli andM. occidentalis. Genetic data thus confirmed that M. marshalli andM. occidentalis are conspecific.