A rapid and reliable detection procedure of Atlantic trout introgression at the diagnostic lactate dehydrogenase chain-1 gene

The Italian-native Mediterranean brown trout (Salmo ghigii) is a seriously threatened freshwater fish, especially by anthropogenic hybridisation with the domestic strains of Atlantic origin that have been repeatedly released into the wild for angling. A PCR-restriction fragment length polymorphism (RFLP) assay of the diagnostic lactate dehydrogenase chain-1 (LDH-C1) gene sequences has been routinely applied to distinguish exotic from native brown trout lineages and detect Atlantic introgression signals in the Mediterranean wild populations. Here, we used dermal swab DNA obtained from 28 wild trout to improve laboratory procedures to genetically characterise trout samples at the LDH-C1gene through (1) a capillary electrophoresis analysis of the RFLP fragments and (2) the optimisation of a diagnostic single nucleotide polymorphism analysable through mini-sequencing approaches. The developed methods were fully consistent with those obtained through the traditional approach, but their analytical process is almost entirely automated and digitalised, thus improving result readability and accuracy in the detection of alien introgressed traces in wild Mediterranean brown trout populations

Fatty acids rather than hormones restore in vitro angiogenesis in human male and female endothelial cells cultured in charcoal-stripped serum.

Charcoal-stripped serum (CSS) is a well-accepted method to model effects of sex hormones in cell cultures. We have recently shown that human endothelial cells (ECs) fail to growth and to undergo in vitro angiogenesis when cultured in CSS. However, the mechanism(s) underlying the CSS-induced impairment of in vitro EC properties are still unknown. In addition, whether there is any sexual dimorphism in the CSS-induced EC phenotype remains to be determined. Here, by independently studying human male and female ECs, we found that CSS inhibited both male and female EC growth and in vitro angiogenesis, with a more pronounced effect on male EC sprouting. Reconstitution of CSS with 17-β estradiol, dihydrotestosterone, or the lipophilic thyroid hormone did not restore EC functions in both sexes. On the contrary, supplementation with palmitic acid or the acetyl-CoA precursor acetate significantly rescued the CSS-induced inhibition of growth and sprouting in both male and female ECs. We can conclude that the loss of metabolic precursors (e.g., fatty acids) rather than of hormones is involved in the impairment of in vitro proliferative and angiogenic properties of male and female ECs cultured with CSS

Effect of CSS on eNOS expression in F- and M-ECs.

<p>(<b>A</b>) Total eNOS protein was evaluated by immunoblotting in F- and M-EC lysates prepared after 48 h of incubation in SM (solid bars) or in CSS-M (open bars). β-actin was used as a loading control. A representative blot and the densitometric analysis of eNOS protein expression normalized to β-actin are shown. **p<0.01 <i>vs</i> SM-cultured M-ECs, <i>t</i> test, n = 13–12 for F- and M-ECs, respectively. (<b>B</b>) F-EC lysates were prepared after 48 h of incubation in SM (solid bar), CSS-M (open bar), or CSS-M + E2 (10 nM, spotted bar). β-actin was used as a loading control. A representative blot and the densitometric analysis of eNOS protein expression normalized to β-actin are shown. n = 4.</p

CSS drastically impairs <i>in vitro</i> angiogenesis.

<p>(<b>A</b>) Representative images of spheroids from F-ECs (upper panels) or M-ECs (lower panels) embedded in collagen gels in the presence of SM (left panels) or CSS-M (right panels). Photographs were taken 24 h later. Quantification of the cumulative length (<b>B</b>), the average length (<b>C</b>), and the number of sprouts (<b>D</b>) emerging from F- or M-EC spheroids incubated in the presence of SM (solid bars) or CSS-M (open bars). *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> SM; ^§p<0.05<i>vs</i> F-ECs cultured in CSS-M, n = 7, <i>t</i> test. F- and M-ECs are orange and blue, respectively.</p

The inhibitory effects of CSS are independent of the presence of sex and thyroid hormones.

<p>ECs were incubated in Standard Medium (SM, solid bars), CSS-medium (CSS-M, open bars), CSS-M + E2 (1 nM, spotted bars), CSS-M + DHT (10 nM, diagonal bars), or CSS-M + T3 (10 nM, squared bars). MTT absorbance (<b>A, C, E</b>) or cell number (<b>B, D, F</b>) were measured after 48h of incubation. Cell number was expressed as percent of control, <i>i</i>.<i>e</i>. cells cultured in SM, set at 100%. In (<b>A</b>), (<b>C</b>), and (<b>E</b>), *p<0.05, **p<0.01 (CSS-M <i>vs</i> SM), ns (CSS-M <i>vs</i> CSS-M + E2, DHT or T3), n = 3, <i>t</i> test. In (<b>B</b>), (<b>D</b>), and (<b>F</b>), *p<0.05, **p<0.01, ***p<0.001 (CSS-M <i>vs</i> SM), ns (CSS-M <i>vs</i> CSS-M + E2, DHT or T3), n = 3, <i>t</i> test. (<b>G</b>) CSS-M was replaced with CSS-M (open bars) or SM (diagonal bars), and MTT was measured after further 48h of incubation. Solid bars: SM replaced with SM. ***p<0.001 <i>vs</i> SM, °°°p<0.001 <i>vs</i> CSS-M, n = 3, <i>t</i> test. F- and M-ECs are orange and blue, respectively.</p

Palmitic acid and acetate rescue the CSS-induced inhibition of cell number.

<p>Cell number was measured in F-ECs (<b>A</b>, orange bars) and M-ECs (<b>B</b>, blue bars) after 48h of incubation in SM (solid bars), CSS-M + vehicle (ethanol/BSA, cross-hitched bars), CSS-M + palmitic acid (250 μM, vertical bars), CSS-M (open bars), or CSS-M + acetate (20 mM, horizontal bars). In (<b>A</b>) and (<b>B</b>), **p<0.01 (CSS-M <i>vs</i> SM), *p<0.05 (CSS-M <i>vs</i> CSS-M + palmitic or CSS-M + acetate), n = 3–4 for F- and M-ECs, respectively, <i>t</i> test.</p

CSS reduces cell number in human F- and M-ECs.

<p>MTT absorbance (<b>A</b>), cell number (<b>B</b>), and ATP luminescence (<b>C</b>) were measured after 48h of incubation in Standard Medium (SM, solid bars) or CSS-medium (CSS-M, open bars). Mean values were compared by Student’s <i>t</i> test. In (<b>A</b>), *p<0.05, n = 14–11 for F- and M-ECs, respectively; in (<b>B</b>), ***p<0.001, n = 3; in (<b>C</b>), *p<0.05, **p<0.01, n = 11–10 for F- and M-ECs, respectively. In (<b>D</b>), ATP luminescence was normalized to the corresponding protein levels. F- and M-ECs are orange and blue, respectively.</p

Lack of endocrine response in F- and M-ECs cultured in FA-reconstituted CSS-medium.

<p>E2 (1 nM) or DHT (10 nM) were added to CSS-M in the absence (open bars) or in the presence (solid bars) of palmitic acid (250 μM), and cell number was measured after 48 of incubation in F-ECs (<b>A</b>, orange bars) and M-ECs (<b>B</b>, blue bars). In (<b>A</b>), ***p<0.001; in (<b>B</b>), **p<0.01, n = 3, Two-way ANOVA. The presence of E2 or DHT did not significantly affect the results. (<b>C</b>) E2 (10 nM) was added to CSS-M in the absence (open bars) or in the presence (solid bars) of palmitic acid (250 μM), and the cumulative length of sprouts emerging from F- and M-ECs was measured after 24 h of incubation. ***p<0.001, n = 3, Two-way ANOVA. The presence of E2 did not significantly affect the results. F- and M-ECs are orange and blue, respectively.</p

Palmitic acid and acetate rescue the CSS-induced inhibition of <i>in vitro</i> angiogenesis.

<p>(<b>A</b>) Representative images of spheroids from F-ECs incubated for 24 h in SM, CSS-M, CSS-M + palmitic acid, or CSS-M + acetate. The cumulative (<b>B</b>, <b>D</b>) and the average (<b>C, E</b>) length of sprouts emerging from F-ECs (<b>B</b>, <b>C</b>) and M-ECs (<b>D</b>, <b>E</b>) were measured after 24h of incubation. Treatments and bars are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189528#pone.0189528.g005" target="_blank">Fig 5</a>: SM, solid bars; CSS + vehicle (ethanol/BSA, cross-hitched bars), CSS + palmitic acid (250 μM, vertical bars); CSS-M, open bars; CSS-M + acetate (20 mM, horizontal bars). Data are expressed as percent of control, <i>i</i>.<i>e</i>. the cumulative and the average length of sprouts from cells cultured in SM, set at 100%. In (<b>B</b>) and (<b>D</b>), ***p<0.001 (CSS-M + vehicle <i>vs</i> SM), *p<0.05, **p<0.01 (CSS-M + vehicle <i>vs</i> CSS-M + palmitic acid), n = 3; °°°p<0.001 (CSS-M <i>vs</i> SM), °p<0.05 (CSS-M <i>vs</i> CSS-M + acetate), n = 4, <i>t</i> test. In (<b>C</b>) and (<b>E</b>), **p<0.01 (CSS-M + vehicle <i>vs</i> SM), *p<0.05, ***p<0.001 (CSS-M + vehicle <i>vs</i> CSS-M + palmitic acid), n = 3; °p<0.05, °°°p<0.001 (CSS-M <i>vs</i> SM), °°p<0.01, °°°p<0.001 (CSS-M <i>vs</i> CSS-M + acetate), n = 4, <i>t</i> test. F- and M-ECs are orange and blue, respectively.</p

The Modulation of Cholesterol Metabolism Is Involved in the Antiviral Effect of Nitazoxanide

We previously investigated the role of Nitazoxanide (NTZ), a thiazolide endowed with antiviral and antiparasitic activity, in HIV-1 infection. NTZ treatment in primary isolated PBMCs was able to reduce HIV-1 infection in vitro by inducing the expression of a number of type-I interferon-stimulated genes. Among them, NTZ was able to induce cholesterol-25-hydroxylase (CH25H), which is involved in cholesterol metabolism. In the present study, we wanted to deepen our knowledge about the antiviral mechanism of action of NTZ. Indeed, by inducing CH25H, which catalyzes the formation of 25-hydroxycholesterol from cholesterol, NTZ treatment repressed cholesterol biosynthetic pathways and promoted cholesterol mobilization and efflux from the cell. Such effects were even more pronounced upon stimulation with FLU antigens in combination. It is already well known how lipid metabolism and virus replication are tightly interconnected; thus, it is not surprising that the antiviral immune response employs genes related to cholesterol metabolism. Indeed, NTZ was able to modulate cholesterol metabolism in vitro and, by doing so, enhance the antiviral response. These results give us the chance to speculate about the suitability of NTZ as adjuvant for induction of specific natural immunity. Moreover, the putative application of NTZ to alimentary-related diseases should be investigated