Photochemical internalization of bleomycin and temozolomide--in vitro studies on the glioma cell line F98.

Here we evaluate the photosensitizer meso-tetraphenyl chlorin disulphonate (TPCS2a) in survival studies of rat glioma cancer cells in combination with the novel photochemical internalization (PCI) technique. The tested anticancer drugs were bleomycin (BLM) and temozolomide (TMZ). Glioma cells were incubated with TPCS2a (0.2 µg ml(-1), 18 h, 37 °C) before BLM or TMZ stimulation (4 h) prior to red light illumination (652 nm, 50 mW cm(-2)). The cell survival after BLM (0.5 µm)-PCI (40 s light) quantified using the MTT assay was reduced to about 25% after 24 h relative to controls, and to 31% after TMZ-PCI. The supplementing quantification by clonogenic assays, using BLM (0.1 µm), indicated a long-term cytotoxic effect: the surviving fraction of clonogenic cells was reduced to 5% after light exposure (80 s) with PCI, compared to 70% in the case of PDT. In parallel, structural and morphological changes within the cells upon light treatment were examined using fluorescence microscopy techniques. The present study demonstrates that PCI of BLM is an effective method for killing F98 glioma cells, but smaller effects were observed using TMZ following the "light after" strategy. The results are the basis for further in vivo studies on our rat glioma cancer model using PDT and PCI.

Molecular ontogenesis of digestive capability and associated endocrine control in Atlantic cod (Gadus morhua) larvae.

We have profiled the expression of twelve genes, in order to provide an overview on the molecular ontogeny of digestive capability with the associated endocrine control during Atlantic cod (Gadus morhua) larval development. Enzyme activity levels for the key digestive enzyme, trypsin, was also measured. Specifically, transcripts for trypsin, amylase, lipolytic enzymes: bile salt activated lipase (BAL), phospholipase A2 (PLA2) and Acyl CoA dehydrogenase (ACADM), regulatory peptides: neuropeptide Y (NPY), orexin (OX) cholecystokinin (CCK) and cocaine and amphetamine-related transcript (CART), the somatotropic factors: growth hormone (GH), preprosomatostatin 1 (PPSS1) and thyroid hormone receptors (TRα and TRß) were analyzed using quatitative (real-time) polymerase chain reaction (qPCR). Trypsin and BAL mRNA levels peaked at approximately day 17 and 25 post-hatch, respectively, and thereafter displayed a decreasing pattern until metamorphosis. GH mRNA levels decreased moderately from 3 to 33dph, and thereafter, an increase was observed until 46dph. TRα mRNA levels showed a fluctuating pattern peaking at day 39 post-hatch. TRß mRNA levels were too low to obtain quantitative measurements. Amylase mRNA slightly increased from day 3 to 17 post-hatch, and thereafter showed a steady decrease until day 60. Interestingly, PLA2 mRNA expression showed a consistent increase throughout the study period, indicating an increasingly important role during larval development. Overall, data from this study indicate that cod larvae show differential developmental mode of expression patterns for key genes and endocrine factors that regulate digestive capability, growth and development. These data are discussed in relation to larval trypsin enzyme activity and previous reports for other teleost species.

Mid and hindgut transcriptome profiling analysis of Atlantic salmon (Salmon salar) under unpredictable chronic stress.

The intestinal epithelium is a selectively permeable barrier for nutrients, electrolytes and water, while maintaining effective protection against pathogens. Combinations of stressors throughout an animal's life, especially in agriculture and aquaculture settings, may affect the regular operativity of this organ with negative consequences for animal welfare. In the current study, we report the effects of a three-week unpredictable chronic stress (UCS) period on the intestinal morphology and transcriptome response of Atlantic salmon (Salmon salar) parr midgut and hindgut. Midgut and hindgut from both control and UCS fish were collected for histology and RNA-sequencing analysis to identify respective changes in the membrane structures and putative genes and pathways responding to UCS. Histological analysis did not show any significant effect on morphometric parameters. In the midgut, 1030 genes were differentially expressed following UCS, resulting in 279 genes which were involved in 13 metabolic pathways, including tissue repair pathways. In the hindgut, following UCS, 591 differentially expressed genes were detected with 426 downregulated and 165 upregulated. A total of 53 genes were related to three pathways. Downregulated genes include cellular senescence pathways, p53 signalling and cytokine-cytokine receptor pathways. The overall results corroborate that salmon parr were at least partly habituating to the UCS treatment. In midgut, the main upregulation was related to cell growth and repair, while in the hindgut there were indications of the activated apoptotic pathway, reduced cell repair and inhibited immune/anti-inflammatory capacity. This may be the trade-off between habituating to UCS and health resilience. This study suggests possible integrated genetic regulatory mechanisms that are tuned when farmed Atlantic salmon parr attempt to cope with UCS.

Developmental effects in fish embryos exposed to oil dispersions - The impact of crude oil micro-droplets.

During accidental crude oil spills and permitted discharges of produced water into the marine environment, a large fraction of naturally occurring oil components will be contained in micron-sized oil droplets. Toxicity is assumed to be associated with the dissolved fraction of oil components, however the potential contribution of oil droplets to toxicity is currently not well known. In the present work we wanted to evaluate the contribution of oil droplets to effects on normal development of Atlantic cod (Gadus morhua) through exposing embryos for 96 h to un-filtered (dispersions containing droplets) and filtered (water soluble fractions) dispersions in a flow-through system at dispersion concentrations ranging from 0.14 to 4.34 mg oil/L. After exposure, the embryos were kept in clean seawater until hatch when survival, development and morphology were assessed. The experiment was performed at two different stages of embryonic development to cover two potentially sensitive stages (gastrulation and organogenesis). Exposure of cod embryos to crude oil dispersions caused acute and delayed toxicity, including manifestation of morphological deformations in hatched larvae. Oil droplets appear to contribute to some of the observed effects including mortality, larvae condition (standard length, body surface, and yolk sac size), spinal deformations as well as alterations in craniofacial and jaw development. The timing of exposure may be essential for the development of effects as higher acute mortality was observed when embryos were exposed from the start of gastrulation (Experiment 1) than when exposed during organogenesis (Experiment 2). Even though low mortality was observed when exposed during organogenesis, concentration-dependent mortality was observed during recovery.

An oil containing EPA and DHA from transgenic Camelina sativa to replace marine fish oil in feeds for Atlantic salmon (Salmo salar L.): Effects on intestinal transcriptome, histology, tissue fatty acid profiles and plasma biochemistry.

New de novo sources of omega 3 (n-3) long chain polyunsaturated fatty acids (LC-PUFA) are required as alternatives to fish oil in aquafeeds in order to maintain adequate levels of the beneficial fatty acids, eicosapentaenoic and docosahexaenoic (EPA and DHA, respectively). The present study investigated the use of an EPA+DHA oil derived from transgenic Camelina sativa in Atlantic salmon (Salmo salar) feeds containing low levels of fishmeal (35%) and fish oil (10%), reflecting current commercial formulations, to determine the impacts on tissue fatty acid profile, intestinal transcriptome, and health of farmed salmon. Post-smolt Atlantic salmon were fed for 12-weeks with one of three experimental diets containing either a blend of fish oil/rapeseed oil (FO), wild-type camelina oil (WCO) or transgenic camelina oil (DCO) as added lipid source. The DCO diet did not affect any of the fish performance or health parameters studied. Analyses of the mid and hindgut transcriptomes showed only mild effects on metabolism. Flesh of fish fed the DCO diet accumulated almost double the amount of n-3 LC-PUFA than fish fed the FO or WCO diets, indicating that these oils from transgenic oilseeds offer the opportunity to increase the n-3 LC-PUFA in farmed fish to levels comparable to those found a decade ago.

Allometric growth and development of organs in ballan wrasse (Labrus bergylta Ascanius, 1767) larvae in relation to different live prey diets and growth rates.

Small fish larvae grow allometrically, but little is known about how this growth pattern may be affected by different growth rates and early diet quality. The present study investigates how different growth rates, caused by start-feeding with copepods or rotifers the first 30 days post-hatch (dph), affect allometric growth and development of nine major organs in ballan wrasse (Labrus bergylta) larvae up to experimental end at 60 dph. Feeding with cultivated copepod nauplii led to both increased larval somatic growth and faster development and growth of organ systems than feeding with rotifers. Of the organs studied, the digestive and respiratory organs increased the most in size between 4 and 8 dph, having a daily specific growth rate (SGR) between 30 and 40% in larvae fed copepods compared with 20% or less for rotifer-fed larvae. Muscle growth was prioritised from flexion stage and onwards, with a daily SGR close to 30% between 21 and 33 dph regardless of treatment. All larvae demonstrated a positive linear correlation between larval standard length (SL) and increase in total tissue volume, and no difference in allometric growth pattern was found between the larval treatments. A change from positive allometric to isometric growth was observed at a SL close to 6.0 mm, a sign associated with the start of metamorphosis. This was also where the larvae reached postflexion stage, and was accompanied by a change in growth pattern for most of the major organ systems. The first sign of a developing hepatopancreas was, however, first observed in the largest larva (17.4 mm SL, 55 dph), indicating that the metamorphosis in ballan wrasse is a gradual process lasting from 6.0 to at least 15-17 mm SL.

Skeletal muscle growth dynamics and the influence of first-feeding diet in Atlantic cod larvae (Gadus morhua L.).

Dynamics between hypertrophy (increase in cell size) and hyperplasia (increase in cell numbers) of white and red muscle in relation to body size [standard length (SL)], and the influence of the first-feeding diets on muscle growth were investigated in Atlantic cod larvae (Gadus morhua). Cod larvae were fed copepod nauplii or rotifers of different nutritional qualities from 4 to 29 days post hatching (dph), Artemia nauplii from 20 to 40 dph and a formulated diet from 36 to 60 dph. The short period of feeding with cultivated copepod nauplii had a positive effect on both muscle hyperplasia and hypertrophy after the copepod/rotifer phase (19 dph), and a positive long term effect on muscle hypertrophy (60 dph). The different nutritional qualities of rotifers did not significantly affect muscle growth. We suggest here a model of the dynamics between hyperplasia and hypertrophy of red and white muscle fibre cells in relation to cod SL (4 to 30 mm), where the different red and white muscle growth phases clearly coincided with different metamorphosis stages in cod larvae. These shifts could be included as biomarkers for the different stages of development during metamorphosis. The main dietary muscle effect was that hypertrophic growth of red muscle fibres was stronger in cod larvae that were fed copepods than in larvae that were fed rotifers, both in relation to larval age and size. Red muscle fibres are directly involved in larval locomotory performance, but may also play an important role in the larval myogenesis. This can have a long term effect on growth potential and fish performance.

Differential spatio-temporal expression and functional diversification of the myogenic regulatory factors MyoD1 and MyoD2 in Atlantic halibut (Hippoglossus hippoglossus).

Development of the vertebrate skeletal muscle is orchestrated by the myogenic regulatory factors MyoD, Myf5, myogenin and MRF4, which likely arose from the duplications of a single ancestral gene early in vertebrate evolution. We have isolated two myod genes from Atlantic halibut and examined their differential expression during embryogenesis using quantitative PCR and in situ hybridization to address their functional roles in this asymmetrically organized flatfish. myod1 was initially maternally expressed, while myod2 mRNA was first detectable during gastrulation. The myod1 mRNA levels predominated throughout somitogenesis, and both slow and fast muscle precursor cells displayed the bilateral symmetric myod1 signal during the formation of the symmetric somite pairs. In contrast, myod2 was left-right asymmetrically expressed in the fast muscle precursors. The random expression of myod2 was not associated with the right-sided eye migration and the development of thicker fast skeletal muscle on the eyed side than on the blind side. The nucleotide substitution analysis indicated that the teleost MyoDs essentially have evolved under purifying selection, but a subset of amino acid sites under strong positive selection were identified in the MyoD2 branch. Altogether, halibut MyoD1 seems to have retained the central role of MyoD in driving skeletal myogenesis, whereas the function of MyoD2 is uncertain in this flatfish species.

Molecular cloning of the Atlantic salmon activin receptor IIB cDNA - Localization of the receptor and myostatin in vivo and in vitro in muscle cells.

In mammals, the activin receptor type IIB (ActRIIB) binds with high affinity several members of the transforming growth factor-beta (TGF-beta) superfamily, including the negative muscle regulator myostatin (MSTN). In this study, an actRIIB cDNA of 1443 bp was isolated by reverse transcription (RT)-PCR from the liver of Atlantic salmon (Salmo salar) encoding almost the complete receptor. The deduced salmon ActRIIB of 481 amino acids (aa) contained the conserved catalytic domain of serine/threonine protein kinases, and showed the highest sequence identity (83-87%) to the zebrafish, chicken and goldfish ActRIIB. Salmon actRIIB mRNA was identified by RT-PCR in all the examined tissues of juvenile fish that was confirmed by in situ hybridization. In comparison, the salmon MSTN signal was less widespread, and co-expression of the receptor and this putative ligand was only demonstrated in skeletal muscle. Consistently, both ActRIIB and MSTN were immunocytologically identified in salmon myoblasts and differentiated myotubes in culture.

Somite formation and expression of MyoD, myogenin and myosin in Atlantic halibut (Hippoglossus hippoglossus L.) embryos incubated at different temperatures: transient asymmetric expression of MyoD.

Genes encoding the myogenic regulating factors MyoD and myogenin and the structural muscle proteins myosin light chain 2 (MyLC2) and myosin heavy chain (MyHC) were isolated from juvenile Atlantic halibut (Hippoglossus hippoglossus L.). The impact of temperature on their temporal and spatial expression during somitogenesis were examined by incubating halibut embryos at 4, 6 and 8 degrees C, and regularly sampling for whole-mount in situ hybridisation and reverse transcription (RT)-PCR. There were no significant effects of temperature on the onset of somitogenesis or number of somites at hatching. The rate of somite formation increased with increasing temperature, and the expression of MyoD, myogenin and MyHC followed the cranial-to-caudal somite formation. Hence, no significant effect of temperature on the spatial and temporal expression of the genes studied was found in relation to somite stage. MyoD, which has subsequently been shown to encode the MyoD2 isoform, displayed a novel bilaterally asymmetric expression pattern only in white muscle precursor cells during early halibut somitogenesis. The expression of myogenin resembled that previously described for other fish species, and preceded the MyHC expression by approximately five somites. Two MyLC2 cDNA sequences were for the first time described for a flatfish, probably representing embryonic (MyLC2a) and larval/juvenile (MyLC2b) isoforms. Factors regulating muscle determination, differentiation and development have so far mostly been studied in vertebrates with external bilateral symmetry. The findings of the present study suggest that more such investigations of flatfish species could provide valuable information on how muscle-regulating mechanisms work in species with different anatomical, physiological and ecological traits.

Molecular characterization of a PDZ-LIM protein in Atlantic salmon (Salmo salar): a fish ortholog of the alpha-actinin-associated LIM-protein (ALP).

A protein containing both PDZ and LIM protein-protein interaction motifs has for the first time been identified in a lower vertebrate species. A full-length cDNA encoding the ortholog of the alpha-actinin-associated LIM protein (ALP) was isolated from white skeletal muscle of Atlantic salmon (Salmo salar). Whereas ALP is expressed as two muscle specific isoforms in mammals and chicken as the result of alternative splicing, a single ALP transcript was found in both muscle and non-muscular tissues of Atlantic salmon. On the other hand, Western blot analysis revealed several immunoreactive ALP variants in salmon muscle tissues, including a 45 kDa protein in white and red skeletal muscle and a 37-40 kDa protein in heart and smooth muscle. Salmon ALP and alpha-actinin showed similar striated patterns in serial longitudinal sections of white and red skeletal muscle and heart muscle. Expression of ALP was initiated at the 45-somite stage of the salmon embryogenesis contemporary with the first appearance of alpha-actinin transcripts. The similarities in both the spatial and temporal expression patterns of salmon ALP and alpha-actinin strongly indicate that the two proteins are associated as in higher vertebrates, and that the assumed involvement of ALP in the organization and/or maintenance of the Z-lines in striated muscle has been conserved during vertebrate evolution. However, in contrast to the restricted expression of ALP in higher vertebrates, the ubiquitous expression of salmon ALP suggest that this factor is involved in the assembly of additional multi-protein complexes in fish.