Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

Dissociation between Mature Phenotype and Impaired Transmigration in Dendritic Cells from Heparanase-Deficient Mice

To reach the lymphatics, migrating dendritic cells (DCs) need to interact with the extracellular matrix (ECM). Heparanase, a mammalian endo-β-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and ECM. The role of heparanase in the physiology of bone marrow-derived DCs was studied in mutant heparanase knock-out (Hpse-KO) mice. Immature DCs from Hpse-KO mice exhibited a more mature phenotype; however their transmigration was significantly delayed, but not completely abolished, most probably due to the observed upregulation of MMP-14 and CCR7. Despite their mature phenotype, uptake of beads was comparable and uptake of apoptotic cells was more efficient in DCs from Hpse-KO mice. Heparanase is an important enzyme for DC transmigration. Together with CCR7 and its ligands, and probably MMP-14, heparanase controls DC trafficking

Effect of aliskiren on post-discharge outcomes among diabetic and non-diabetic patients hospitalized for heart failure: insights from the ASTRONAUT trial

Aims The objective of the Aliskiren Trial on Acute Heart Failure Outcomes (ASTRONAUT) was to determine whether aliskiren, a direct renin inhibitor, would improve post-discharge outcomes in patients with hospitalization for heart failure (HHF) with reduced ejection fraction. Pre-specified subgroup analyses suggested potential heterogeneity in post-discharge outcomes with aliskiren in patients with and without baseline diabetes mellitus (DM). Methods and results ASTRONAUT included 953 patients without DM (aliskiren 489; placebo 464) and 662 patients with DM (aliskiren 319; placebo 343) (as reported by study investigators). Study endpoints included the first occurrence of cardiovascular death or HHF within 6 and 12 months, all-cause death within 6 and 12 months, and change from baseline in N-terminal pro-B-type natriuretic peptide (NT-proBNP) at 1, 6, and 12 months. Data regarding risk of hyperkalaemia, renal impairment, and hypotension, and changes in additional serum biomarkers were collected. The effect of aliskiren on cardiovascular death or HHF within 6 months (primary endpoint) did not significantly differ by baseline DM status (P = 0.08 for interaction), but reached statistical significance at 12 months (non-DM: HR: 0.80, 95% CI: 0.64-0.99; DM: HR: 1.16, 95% CI: 0.91-1.47; P = 0.03 for interaction). Risk of 12-month all-cause death with aliskiren significantly differed by the presence of baseline DM (non-DM: HR: 0.69, 95% CI: 0.50-0.94; DM: HR: 1.64, 95% CI: 1.15-2.33; P < 0.01 for interaction). Among non-diabetics, aliskiren significantly reduced NT-proBNP through 6 months and plasma troponin I and aldosterone through 12 months, as compared to placebo. Among diabetic patients, aliskiren reduced plasma troponin I and aldosterone relative to placebo through 1 month only. There was a trend towards differing risk of post-baseline potassium ≥6 mmol/L with aliskiren by underlying DM status (non-DM: HR: 1.17, 95% CI: 0.71-1.93; DM: HR: 2.39, 95% CI: 1.30-4.42; P = 0.07 for interaction). Conclusion This pre-specified subgroup analysis from the ASTRONAUT trial generates the hypothesis that the addition of aliskiren to standard HHF therapy in non-diabetic patients is generally well-tolerated and improves post-discharge outcomes and biomarker profiles. In contrast, diabetic patients receiving aliskiren appear to have worse post-discharge outcomes. Future prospective investigations are needed to confirm potential benefits of renin inhibition in a large cohort of HHF patients without D

Histological appearance and neutrophil infiltration of distal colon sections.

<p>(<b>A</b>) H&E appearance. (<b>A-I</b>) DSS uptake leads to severe epithelial damage (black arrowed) while (<b>A-II</b>) apoptotic cell treatment maintain integrity of treated mice (<b>A-III</b>) histological score of distal colon sections of DSS-treated Balb/c mice Results from 3 independent experiments (*<i>p</i><0.05, t-test). (<b>B</b>) Apoptotic cell treatment inhibits neutrophil accumulation in inflamed colon. Mouse colon tissue sections were stained by immunohistochemistry assay using a rabbit monoclonal antibody against mouse myeloperoxidase (MPO). After immunostaining, slides were counterstained by hematoxylin. Images show the MPO stain followed by HRP-anti rabbit secondary antibody. All images are x200. <b>(B-I)</b> Staining control. Untreated colon stained with HRP-anti rabbit secondary antibody only, without anti-MPO. <b>(B-II)</b> Normal colon control. MPO-stained neutrophils in untreated colon. <b>(B-III)</b> DSS treatment. MPO-stained neutrophils in 3% DSS treated colon (3% DSS+PBS). <b>(B-IV)</b> Apoptotic cell & DSS treatment. MPO-stained neutrophils in 3% DSS-treated colon with apoptotic cell infusion (3% DSS+ApoCell).</p

IL-1β inhibition by apoptotic cells pre and post NF-κB triggering by LPS. (A-B).

<p>IL-1β measured by ELISA (<b>Upper panel</b>) and western blot (<b>Lower panel</b>). IL-1β and caspase-1 were measured in supernatant (SN) and/or intracellular in cell lysate (CL). B6 pMΦ cells were incubated either in the presence of apoptotic cells for 2h followed by LPS priming for 1h (sixth from left, dark bar), or first primed with LPS (to promote NF-κB signaling) for 1h and then incubated with apoptotic cells for 2h (seventh from left, white bar, separated by dashed line). pMΦ were then incubated with various inflammasome inducers. <b>A</b>: nigericin 2.5μM; <b>B</b>: calcium pyrophosphate dihydrate 200μg/mL (CPPD). An anti-mouse actin served as a loading control. Shown are data for A-B, as means ± SEM of 3 independent experiments done in duplicates and WB representative data of three experiments (*<i>p</i><0.001, one way ANOVA).</p

Lysosomal damage and K+ efflux in pMΦ.

<p><b>(A)</b> Flow-cytometer analysis of B6 pMΦ treated for 2h with apoptotic cells and/or 24h with DSS were stained with fluorochrome acridine orange (AO). Loss of fluorescence, which correlates with reduced numbers of lysosomes, was analyzed by flow-cytometer, excluding dead cells base on FSC/SSC parameters. Shown are means ± SEM of 4 independent experiments (*<i>p</i><0.05, **<i>p</i><0.03, one way ANOVA). <b>(B)</b> confocal microscopy of LPS primed pMΦ incubated (or not; left) for 2h with apoptotic cells (middle) or DAPI-stained apoptotic cells (right), stained with 1μg/mL acridine orange for 15 min and then incubated for 2.5 h with 3% DSS. Representative data from four experiments. <b>(C)</b> Apoptotic cell treatment inhibits nigericin-induced IL-1β secretion. B6 pMΦ cells were treated with nigericin at the indicated concentrations in the presence of LPS priming, with or without apoptotic cell treatment (*<i>p</i><0.01, unpaired t-test).</p

Reactive oxygen species (ROS) in pMΦ.

<p><b>(A)</b> Upper Panel: <b>I</b>. pMΦ as seen in bright field. <b>II</b>. pMΦ seen by fluorescent microscope following incubation with an ROS-sensitive dye (1μM). <b>III</b>. pMΦ following 3% DSS treatment. Generation of ROS is seen. <b>IV</b>. pMΦ following 3% DSS and apoptotic cell treatment. Inhibition of ROS generation is seen. Original magnification: All panels x100. pMΦ extracted from mice were seeded overnight onto eight-chamber slides at a density of 1x10⁵ cells/chamber. After washing, pMΦ were treated for 2h with apoptotic cells followed by treatment with 3% DSS for 30 min. Negative control samples were treated with media only. ROS generation was determined by fluorescence microscopy using a fluorescein fluorescent probe for 30 min with a green filter. The experiments were repeated 3 times, independently; one representative experiment is shown. <b>(B)</b> Flow-cytometer analysis of pMΦ stained with ROS-sensitive dye. ROS generation was determined by flow-cytometer using a fluorescence probe as above, excluding dead cells base on FSC/SSC parameters. Shown are means ± SEM of 3 experiments done in triplicates (*<i>p</i><0.05, one way ANOVA).</p

Apoptotic cell treatment inhibits NF-κB in DSS-induced colitis.

<p>Mouse colon tissue sections were stained by immunohistochemistry assay using an antibody against mouse phospho-NF-κB (pNF-κB) p65. After immunostaining, slides were counterstained by hematoxylin. Images show pNF-κB p65 staining. All images are x200. <b>(I)</b> Untreated colon stained with HRP-anti rabbit secondary antibody only, without anti-NF-κB. <b>(II)</b> pNF-κB p65 staining in untreated colon. <b>(III)</b> Large pNF-κB p65 positive stain (black arrow) in 3% DSS-treated colon (3% DSS+PBS). <b>(IV)</b> Fewer pNF-κB p65 positive stain (black arrow) in 3% DSS treated colon with apoptotic cell infusion (3% DSS+ApoCell).</p

Apoptotic cell treatment protects mice from DSS-induced colitis.

<p>Balb/c mice were offered distilled water (filled circles), or distilled water with 3% DSS orally ad libitum with treatment of PBS (filled squares) or apoptotic cell (filled triangles). (<b>A</b>) Mean weight of indicated animal number per group (*<i>p</i><0.05, **<i>p</i><0.001, t-test). (<b>B</b>) IBD Clinical Score. Numbers inside boxes indicate the mean score of each parameter with error bar (*<i>p</i><0.001, t-test). Data is presented as mean ± SEM of 3 independent experiments. Weight change, hematochezia and stool consistency were monitored daily. (<b>C</b>) Macroscopic changes of colon and spleen in DSS-treated mice. Photographs of the dissected large intestines and spleens of four mice treated with 3% DSS without- (DSS+PBS) or with apoptotic cell treatment (DSS+ApoCell). <b>(D)</b> IL-1β cytokine level in colonic homogenate from DSS-treated mice. Levels of IL-1β were analyzed by ELISA. Data is presented as mean ± SEM, 3 mice per group (*<i>p</i><0.001, one way ANOVA). <b>(E)</b> IL-1β mRNA levels in colonic homogenate from DSS-treated mice. mRNA was measured by RT-PCR and normalized to untreated colons. Data is presented as mean ± SEM, 4–5 mice per group (*<i>p</i><0.02, t-test)</p