Dermomyotome-derived endothelial cells migrate to the dorsal aorta to support hematopoietic stem cell emergence.

Development of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from ventral aortic endothelium in all vertebrate animals studied. Work in zebrafish has demonstrated that arterial and venous endothelial precursors arise from distinct subsets of lateral plate mesoderm. Here, we profile the transcriptome of the earliest detectable endothelial cells (ECs) during zebrafish embryogenesis to demonstrate that tissue-specific EC programs initiate much earlier than previously appreciated, by the end of gastrulation. Classic studies in the chick embryo showed that paraxial mesoderm generates a subset of somite-derived endothelial cells (SDECs) that incorporate into the dorsal aorta to replace HSPCs as they exit the aorta and enter circulation. We describe a conserved program in the zebrafish, where a rare population of endothelial precursors delaminates from the dermomyotome to incorporate exclusively into the developing dorsal aorta. Although SDECs lack hematopoietic potential, they act as a local niche to support the emergence of HSPCs from neighboring hemogenic endothelium. Thus, at least three subsets of ECs contribute to the developing dorsal aorta: vascular ECs, hemogenic ECs, and SDECs. Taken together, our findings indicate that the distinct spatial origins of endothelial precursors dictate different cellular potentials within the developing dorsal aorta.

Synthesis and biological evaluation of zinc chelating compounds as metallo-ß-lactamase inhibitors.

The syntheses of metallo-ß-lactamase inhibitors comprising chelating moieties, with varying zinc affinities, and peptides partly inspired from bacterial peptide sequences, have been undertaken. The zinc chelator strength was varied using the following chelators, arranged in order of ascending binding affinity: dipicolylamine (DPA, tridentate), dipicolyl-1,2,3-triazolylmethylamine (DPTA, tetradentate) dipicolyl ethylenediamine (DPED, tetradentate) and trispicolyl ethylenediamine (TPED, pentadentate). The chosen peptides were mainly based on the known sequence of the C-terminus of the bacterial peptidoglycan precursors. Biological evaluation on clinical bacterial isolates, harbouring either the NDM-1 or VIM-2 metallo-ß-lactamase, showed a clear relationship between the zinc chelator strength and restoration of meropenem activity. However, evaluation of toxicity on different cancer cell lines demonstrated a similar trend, and thus inclusion of the bacterial peptides did possess rather high toxicity towards eukaryotic cells.

Synthesis and Preclinical Evaluation of TPA-Based Zinc Chelators as Metallo-ß-lactamase Inhibitors.

The rise of antimicrobial resistance (AMR) worldwide and the increasing spread of multi-drug-resistant organisms expressing metallo-ß-lactamases (MBL) require the development of efficient and clinically available MBL inhibitors. At present, no such inhibitor is available, and research is urgently needed to advance this field. We report herein the development, synthesis, and biological evaluation of chemical compounds based on the selective zinc chelator tris-picolylamine (TPA) that can restore the bactericidal activity of Meropenem (MEM) against Pseudomonas aeruginosa and Klebsiella pneumoniae expressing carbapenemases Verona integron-encoded metallo-ß-lactamase (VIM-2) and New Delhi metallo-ß-lactamase 1 (NDM-1), respectively. These adjuvants were prepared via standard chemical methods and evaluated in biological assays for potentiation of MEM against bacteria and toxicity (IC50) against HepG2 human liver carcinoma cells. One of the best compounds, 15, lowered the minimum inhibitory concentration (MIC) of MEM by a factor of 32-256 at 50 µM within all tested MBL-expressing clinical isolates and showed no activity toward serine carbapenemase expressing isolates. Biochemical assays with purified VIM-2 and NDM-1 and 15 resulted in inhibition kinetics with kinact/ KI of 12.5 min-1 mM-1 and 0.500 min-1 mM-1, respectively. The resistance frequency of 15 at 50 µM was in the range of 10-7 to 10-9. 15 showed good tolerance in HepG2 cells with an IC50 well above 100 µM, and an in vivo study in mice showed no acute toxic effects even at a dose of 128 mg/kg.

Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus.

Infectious salmon anaemia virus (ISAV) is a piscine orthomyxovirus causing a serious disease in farmed Atlantic salmon (Salmo salar L.). The virus surface glycoprotein hemagglutinin-esterase (HE) is responsible for both viral attachment and release. Similarity to bovine and porcine torovirus hemagglutinin-esterase (BToV HE, PToV HE), bovine coronavirus HE (BCoV HE) and influenza C hemagglutinin-esterase-fusion (InfC HEF) proteins were exploited in a computational homology-based structure analysis of ISAV HE. The analysis resolved structural aspects of the protein and identified important features of relevance to ISAV HE activity. By recombinant expression and purification of secretory HE (recHE) proteins, receptor-binding and quantitative analyses of enzymatic activities displayed by ISAV HE molecules are presented for the first time. Three different recHE molecules were constructed: one representing a high virulent isolate, one a low virulent, while in the third a Ser(32) to Ala(32) amino acid substitution was introduced in the enzymatic catalytic site as inferred from the model. The three amino acid differences between the high and low virulent variants, of which two localized to the putative receptor-binding domain and one in the esterase domain, had no impact on receptor-binding or -release activities. In contrast, the Ser(32) amino acid substitution totally abolished enzymatic activity while receptor binding increased, as observed by agglutination of Atlantic salmon red blood cells. This demonstrates the essential role of a serine in the enzyme's catalytic site. In conclusion, structural analysis of ISAV HE in combination with selected recHE proteins gave insights into structure-function relationships and opens up for further studies aiming at dissecting molecular determinants of ISAV virulence.

Transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells.

The aquatic orthomyxovirus infectious salmon anemia virus (ISAV) is an important pathogen for salmonid aquaculture, however little is known about protective and pathological host responses to infection. We have investigated intracellular responses during cytopathic ISAV infection in the macrophage-like Atlantic salmon kidney (ASK) cell line by microarray analysis (1.8k SFA2.0 immunochip) and a functional assay for glutathione. Gene transcription changed rapidly and consistently with time and with minor differences between two virus isolates. While several pro-inflammatory and antiviral immune genes were induced, genes involved in cell signaling and integrity were down-regulated, suggesting isolation of infected cells from cell-to-cell interaction and responses to external signals. Differential expression of genes regulating cell cycle and apoptosis implied opposite cues from host cell and virus. This was in pace with massive down-regulation of genes involved in biosynthesis and processing of nucleotides and nucleic acids. Significant down-regulation of several genes involved in metabolism of reactive oxygen species suggested increased oxidative stress, which was confirmed by a functional assay showing reduced levels of glutathione during infection. Testing of expression data against a microarray database containing diverse experiments revealed candidate marker genes for ISAV infection. Our findings provide novel insight into cellular host responses and determinants for acute cytopathic ISAV infection.

Effects of dietary thia fatty acids on lipid composition, morphology and macrophage function of Atlantic salmon (Salmo salar L.) kidney.

High lipid levels are being used in modern salmonid diets to promote rapid growth; however there is a limiting supply of the traditional fish oils as the fish farming industry expands. One way to utilize the lipid sources better, could be to find ways to stimulate fatty acid (FA) oxidation so that Atlantic salmon use more energy for muscle growth and less for storage in perivisceral adipose tissue. We have previously shown that dietary inclusion of the thia FA tetradecylthioacetic acid (TTA) promoted hepatic beta-oxidation and reduced total body lipid levels. However, dietary TTA also had some negative effects, leading to accumulation of sulfone and sulfoxide metabolites of TTA in the kidney and increasing mortality rates, particularly at low water temperatures. Therefore we also wish to investigate the effects of TTA on kidney function at high and low temperatures, including some immune system parameters. The production of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) immunoreactive material from exogenously added arachidonic acid in isolated head kidney macrophages was affected by both diet and temperature. The phagocytic activity in these cells was reduced by DTA in the 12 degrees C group and there was significantly higher protein degradation in head kidney macrophages at 12 degrees C compared to 5 degrees C in all dietary groups. Interestingly, the incorporation of thia FAs in the kidney was higher at 5 degrees C (0.3% TTA and 0.6% DTA) than at 12 degrees C (0.1% TTA and 0.5% DTA). Additionally, there were lower levels of saturated FAs, while higher levels of polyunsaturated FAs (PUFAs) in the kidney of TTA fed fish at 5 degrees C. We also observed temperature-independent tubular dilatation and a reduction in the density of melanomacrophages of the kidney in salmon fed TTA. Nevertheless, the mRNA expression of some immune-relevant genes in head kidney tissue was not affected by TTA-inclusion in salmon diets. In conclusion, it is clear that 0.6% TTA-inclusion in the feed leads to changes in the kidney function particularly at low water temperatures.

Hepatic scavenger receptor class B, type I is stimulated by peroxisome proliferator-activated receptor gamma and hepatocyte nuclear factor 4alpha.

Excessive cellular cholesterol is transported to the liver by a pathway called 'reverse cholesterol transport.' Scavenger receptor class B, type I (SR-BI) mediates cholesterol uptake in the liver. Polyunsaturated fatty acids, known to activate peroxisome proliferator-activated receptor (PPAR), have been reported to increase hepatic cholesterol uptake. We found in the present study that PPARgamma induces expression of SR-BI in rat hepatocytes, liver endothelial cells, and Kupffer cells. In contrast, PPARalpha increased SR-BI levels only in hepatocytes and liver endothelial cells. PPARgamma/RXR binds to a response element between -459 and -472 bp in the human SR-BI promoter. Furthermore, hepatocyte nuclear factor 4alpha (HNF4alpha) was found to enhance PPARgamma-mediated SR-BI transcription. Thiazolidinedione (TZD)-activated PPARgamma/RXR increased hepatic SR-BI levels, which may lead to increased hepatic cholesterol uptake and less accumulation of lipids in peripheral tissues. The present results are in agreement with previous reports, indicating that specific PPARgamma-agonists (such as TZDs) protect against atherosclerosis.