Effects of repeated anaesthesia on gill and general health of Atlantic salmon, Salmo salar.

Fish are the second most widely utilized vertebrate group used for scientific procedures in the United Kingdom, but the development and application of 3Rs (the principles of replacement, reduction, and refinement) in aquaculture disease research lags behind methodologies in place for mammalian studies. With a need for individual monitoring and non-lethal sampling, the effect of repeat anaesthesia on experimental fish needs to be better understood. This study analyses the effect of repeat anaesthesia with MS-222, metomidate and AQUI-S upon the gill and general health of post-smolt Atlantic salmon Salmo salar. A single, lethal dose of anaesthetic was compared with seven anaesthetizing time points over 28 days, terminating in a lethal dose. No anaesthetic showed significant differences in accumulation in the muscle tissue, or changes in plasma glucose after repeated or single dosing. Fish repeatedly anaesthetized with MS-222 or AQUI-S exhibited upregulation of osmoregulatory genes in the gill and AQUI-S-treated individuals showed, histologically, epithelial lifting from the lamellae capillary irrespective of whether they had a single or repeated dose history. No significant changes were seen in inflammatory or stress genes in the head kidney of fish repeatedly anaesthetized with AQUI-S or metomidate, however MS-222 treatment resulted in upregulation of tnfα3. Repeated anaesthesia with MS-222 and metomidate gave a significant decrease and increase in peripheral blood neutrophils, respectively. This study concludes that no increase in cumulative stress or inflammation is induced by the repeated anaesthetization of S. salar with any of the tested anaesthetics, however gill osmotic regulation and blood parameters may be affected.

Decreased sensitivity to aspirin is associated with altered polyamine metabolism in human prostate cancer cells.

Aspirin is a well-known analgesic, anti-inflammatory and antipyretic drug and is recognised as a chemopreventative agent in cardiovascular disease and, more recently, in colorectal cancer. Although several studies indicate that aspirin is capable of reducing the risk of developing cancers, there is a lack of convincing evidence that aspirin can prevent prostate cancer in man. In this study, aspirin was shown to be an effective inhibitor of the growth of human prostate cancer cells. In order to investigate the link between polyamine catabolism and the effects of aspirin we used a "Tet off" system that induced the activity of spermidine/spermine N (1)-acetyltransferase (SSAT) in human prostate cancer cells (LNCap). Treatment with aspirin was found to decrease induced SSAT activity in these cells. A negative correlation was observed between increased polyamine catabolism via increased SSAT activity and the sensitivity to aspirin. In the presence of increased SSAT activity high amounts of N (1)-acetylspermidine and putrescine were observed. These cells were also found to grow more slowly than the non-induced cells. The results indicate that SSAT and its related polyamine metabolism may play a key role in sensitivity of cancer cells to aspirin and possibly other NSAIDs and this may have implications for the development of novel chemopreventative agents.

Gorse (Ulex europeaus) wastes with 5,6-dimethyl benzimidazole supplementation can support growth of vitamin B12 producing commensal gut microbes.

Many commensal gut microbes are recognized for their potential to synthesize vitamin B12, offering a promising avenue to address deficiencies through probiotic supplementation. While bioinformatics tools aid in predicting B12 biosynthetic potential, empirical validation remains crucial to confirm production, identify cobalamin vitamers, and establish biosynthetic yields. This study investigates vitamin B12 production in three human colonic bacterial species: Anaerobutyricum hallii DSM 3353, Roseburia faecis DSM 16840, and Anaerostipes caccae DSM 14662, along with Propionibacterium freudenreichii DSM 4902 as a positive control. These strains were selected for their potential use as probiotics, based on speculated B12 production from prior bioinformatic analyses. Cultures were grown in M2GSC, chemically defined media (CDM), and Gorse extract medium (GEM). The composition of GEM was similar to CDM, except that the carbon and nitrogen sources were replaced with the protein-depleted liquid waste obtained after subjecting Gorse to a leaf protein extraction process. B12 yields were quantified using liquid chromatography with tandem mass spectrometry. The results suggested that the three butyrate-producing strains could indeed produce B12, although the yields were notably low and were detected only in the cell lysates. Furthermore, B12 production was higher in GEM compared to M2GSC medium. The positive control, P. freudenreichii DSM 4902 produced B12 at concentrations ranging from 7 ng mL-1 to 12 ng mL-1. Univariate-scaled Principal Component Analysis (PCA) of data from previous publications investigating B12 production in P. freudenreichii revealed that B12 yields diminished when the carbon source concentration was ≤30 g L-1. In conclusion, the protein-depleted wastes from the leaf protein extraction process from Gorse can be valorised as a viable substrate for culturing B12-producing colonic gut microbes. Furthermore, this is the first report attesting to the ability of A. hallii, R. faecis, and A. caccae to produce B12. However, these microbes seem unsuitable for industrial applications owing to low B12 yields.

Development of an enantiomer selective microsampling assay for the quantification of ketorolac suitable for paediatric pharmacokinetic studies.

BACKGROUND: Ketorolac, a potent nonsteroidal anti-inflammatory drug used for pain control in children, exists as a racemate of inactive R (+) and active S (-) enantiomers. AIM: To develop a microsampling assay for the enantioselective analysis of ketorolac in children. METHODS: Ketorolac enantiomers were extracted from 50 µl of plasma by liquid­liquid extraction and separated on a ChiralPak AD-RH. Detection was by a TSQ quantum triple quadrupole mass spectrometer with an electrospray ionisation source operating in a positive ion mode. Five children (age 13.8 (1.6) years, weight 52.7 (7.2) kg), were administered intravenous ketorolac 0.5mg/kg (maximum 10mg) and blood samples were taken at 0, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 h post administration. CL, VD and t1/2 were calculated based on non-compartmental methods. RESULTS: The standard curves for R (+) and S (-) ketorolac were linear in the range 0­2000 ng/ml. The LLOQs of the method were 0.15 ng on column and 0.31 ng on column for R (+) and S (-) ketorolac, respectively. The median (range) VD and CL of R (+) and S (-) ketorolac were 0.12 l/kg (0.07­0.17), 0.017 l/h/kg (0.12­0.29) and 0.17 (0.09­0.31) l/kg, 0.049 (0.02­0.1) l/h/kg, p = 0.043), respectively. The median (range) elimination half-life (t1/2) of the R (+) and S (-) ketorolac was 5.0 h (2.5­5.8) and 3.1 h (1.8­4.4), p = 0.043), respectively. CONCLUSION: The development of a simple, rapid and reliable ketorolac assay suitable for paediatric PK studies is reported.

Genetic Manipulation of sn-1-Diacylglycerol Lipase and CB1 Cannabinoid Receptor Gain-of-Function Uncover Neuronal 2-Linoleoyl Glycerol Signaling in Drosophila melanogaster.

Introduction: In mammals, sn-1-diacylglycerol lipases (DAGL) generate 2-arachidonoylglycerol (2-AG) that, as the major endocannabinoid, modulates synaptic neurotransmission by acting on CB1 cannabinoid receptors (CB1R). Even though the insect genome codes for inaE, which is a DAGL ortholog (dDAGL), its products and their functions remain unknown particularly because insects lack chordate-type cannabinoid receptors. Materials and Methods: Gain-of-function and loss-of-function genetic manipulations were carried out in Drosophila melanogaster, including the generation of both dDAGL-deficient and mammalian CB1R-overexpressing flies. Neuroanatomy, dietary manipulations coupled with targeted mass spectrometry determination of arachidonic acid and 2-linoleoyl glycerol (2-LG) production, behavioral assays, and signal transduction profiling for Akt and Erk kinases were employed. Findings from Drosophilae were validated by a CB1R-binding assay for 2-LG in mammalian cortical homogenates with functionality confirmed in neurons using high-throughput real-time imaging in vitro. Results: In this study, we show that dDAGL is primarily expressed in the brain and nerve cord of Drosophila during larval development and in adult with 2-LG being its chief product as defined by dietary precursor availability. Overexpression of the human CB1R in the ventral nerve cord compromised the mobility of adult Drosophilae. The causality of 2-LG signaling to CB1R-induced behavioral impairments was shown by inaE inactivation normalizing defunct motor coordination. The 2-LG-induced activation of transgenic CB1Rs affected both Akt and Erk kinase cascades by paradoxical signaling. Data from Drosophila models were substantiated by showing 2-LG-mediated displacement of [3H]CP 55,940 in mouse cortical homogenates and reduced neurite extension and growth cone collapsing responses in cultured mouse neurons. Conclusions: Overall, these results suggest that 2-LG is an endocannabinoid-like signal lipid produced by dDAGL in Drosophila.

Proinflammatory markers in prediction of posttraumatic psychological symptoms: a prospective cohort study.

INTRODUCTION: Posttraumatic psychopathology (PTP) describes the spectrum of conditions that can complicate the recovery from commonly occurring musculoskeletal trauma. There is a clear association with the activation of the hypothalamic-pituitary-adrenal axis (HPAA), and we wished to examine the predictive value of proinflammatory markers of the HPAA and of the GABA, which acts as an inhibitory regulator. METHODS: Levels of proinflammatory markers and GABA were measured in 84 patients who had suffered musculoskeletal injuries requiring hospitalisation. PTP was assessed by the use of the General Health Questionnaire (GHQ) at presentation and again at two- and six-month reviews. RESULTS: Significant psychological disturbance was noted in 39% of patients at two months and falling back to 18% by six months. There was no correlation between any of the markers tested at presentation and PTP at follow-up. DISCUSSION: The HPAA response to trauma and the development of PTP are extremely complex. It is unlikely that a simple blood assay will provide significant predictive information, while incident specific information and patient perception are of more practical use.

Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signalling.

Local environmental cues are indispensable for axonal growth and guidance during brain circuit formation. Here, we combine genetic and pharmacological tools, as well as systems neuroanatomy in human fetuses and mouse models, to study the role of endocannabinoid and Slit/Robo signalling in axonal growth. We show that excess 2-arachidonoylglycerol, an endocannabinoid affecting directional axonal growth, triggers corpus callosum enlargement due to the errant CB1 cannabinoid receptor-containing corticofugal axon spreading. This phenotype mechanistically relies on the premature differentiation and end-feet proliferation of CB2R-expressing oligodendrocytes. We further show the dependence of both axonal Robo1 positioning and oligodendroglial Slit2 production on cell-type-specific cannabinoid receptor activation. Accordingly, Robo1 and/or Slit2 manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can configure focal Slit2/Robo1 signalling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic deficits and prenatal drug exposure.

Generation of hepatocytes expressing functional cytochromes P450 from a pancreatic progenitor cell line in vitro.

The proliferating AR42J-B13 pancreatic cell line is known to respond to glucocorticoid treatment by producing foci of cells that express the liver-specific albumin gene. We demonstrate that this cell line also expresses liver-specific or liver-enriched functional cytochrome P450 proteins when stimulated to trans-differentiate into hepatocytes by glucocorticoid. These data suggest that this cell line has an unusual ability to trans-differentiate into functional hepatocytes and that it could be possible to generate a limitless supply of functional hepatocyte-like cells in vitro.

Mechanism of action of the antifibrogenic compound gliotoxin in rat liver cells.

Gliotoxin has been shown to promote a reversal of liver fibrosis in an animal model of the disease although its mechanism of action in the liver is poorly defined. The effects of gliotoxin on activated hepatic stellate cells (HSCs) and hepatocytes have therefore been examined. Addition of gliotoxin (1.5 microM) to culture-activated HSCs resulted in its rapid accumulation, resulting in increased levels of glutathione and apoptosis without any evidence of oxidative stress. In contrast, although hepatocytes also rapidly sequestered gliotoxin, cell death only occurred at high (50-microM) concentrations of gliotoxin and by necrosis. At high concentrations, gliotoxin was metabolized by hepatocytes to a reduced (dithiol) metabolite and glutathione was rapidly oxidized. Fluorescent dye loading experiments showed that gliotoxin caused oxidative stress in hepatocytes. Antioxidants--but not thiol redox active compounds--inhibited both oxidative stress and necrosis in hepatocytes. In contrast, HSC apoptosis was not affected by antioxidants but was potently abrogated by thiol redox active compounds. The adenine nucleotide transporter (ANT) is implicated in mitochondrial-dependent apoptosis. HSCs expressed predominantly nonliver ANT isoform 1, and gliotoxin treatment resulted in a thiol redox-dependent alteration in ANT mobility in HSC extracts, but not hepatocyte extracts. In conclusion, these data suggest that gliotoxin stimulates the apoptosis of HSCs through a specific thiol redox-dependent interaction with the ANT. Further understanding of this mechanism of cell death will aid in finding therapeutics that specifically stimulate HSC apoptosis in the liver, a promising approach to antifibrotic therapy.