Testing hypotheses on life-cycle models for Antarctic calanoid copepods, using qualitative, winter, zooplankton samples

We analysed qualitative winter zooplankton samples, collected from the ocean surface, to test portions of proposed life-cycle models for Calanus propinquus. Nine zooplankton hauls were taken in the Bransfield Strait area during June 1996. Results show that C. propinquus is present in the ocean surface in winter. However, its presence seems to be related to ice coverage below 30%

Conservation of small regulatory RNAs in Vibrio parahaemolyticus: Possible role of RNA-OUT encoded by the pathogenicity island (VPaI-7) of pandemic strains

Small regulatory RNAs (sRNAs) are molecules that play an important role in the regulation of gene expression. sRNAs in bacteria can affect important processes, such as metabolism and virulence. Previous studies showed a significant role of sRNAs in the Vibrio species, but knowledge about Vibrio parahaemolyticus is limited. Here, we examined the conservation of sRNAs between V. parahaemolyticus and other human Vibrio species, in addition to investigating the conservation between V. parahaemolyticus strains differing in pandemic origin. Our results showed that only 7% of sRNAs were conserved between V. parahaemolyticus and other species, but 88% of sRNAs were highly conserved within species. Nonetheless, two sRNAs coding to RNA-OUT, a component of the Tn10/IS10 system, were exclusively present in pandemic strains. Subsequent analysis showed that both RNA-OUT were located in pathogenicity island-7 and would interact with transposase VPA1379, according to the model of pairing of IS10-encoded antisense RNAs. According to the location of RNA-OUT/VPA1379, we also investigated if they were expressed during infection. We observed that the transcriptional level of VPA1379 was significantly increased, while RNA-OUT was decreased at three hours post-infection. We suggest that IS10 transcription increases in pandemic strains during infection, probably to favor IS10 transposition and improve their fitness when they are facing adverse conditions

Exploring the Genomic Traits of Non-toxigenic Vibrio parahaemolyticus Strains Isolated in Southern Chile

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. As reported in other countries, after the rise and fall of the pandemic strain in Chile, other post-pandemic strains have been associated with clinical cases, including strains lacking the major toxins TDH and TRH. Since the presence or absence of tdh and trh genes has been used for diagnostic purposes and as a proxy of the virulence of V. parahaemolyticus isolates, the understanding of virulence in V. parahaemolyticus strains lacking toxins is essential to detect these strains present in water and marine products to avoid possible food-borne infection. In this study, we characterized the genome of four environmental and two clinical non-toxigenic strains (tdh-, trh-, and T3SS2-). Using whole-genome sequencing, phylogenetic, and comparative genome analysis, we identified the core and pan-genome of V. parahaemolyticus of strains of southern Chile. The phylogenetic tree based on the core genome showed low genetic diversity but the analysis of the pan-genome revealed that all strains harbored genomic islands carrying diverse virulence and fitness factors or prophage-like elements that encode toxins like Zot and RTX. Interestingly, the three strains carrying Zot-like toxin have a different sequence, although the alignment showed some conserved areas with the zot sequence found in V. cholerae. In addition, we identified an unexpected diversity in the genetic architecture of the T3SS1 gene cluster and the presence of the T3SS2 gene cluster in a non-pandemic environmental strain. Our study sheds light on the diversity of V. parahaemolyticus strains from the southern Pacific which increases our current knowledge regarding the global diversity of this organism

Prolonged Activation of the Htr2b Serotonin Receptor Impairs Glucose Stimulated Insulin Secretion and Mitochondrial Function in MIN6 Cells

<div><p>Aims</p><p>Pancreatic β-cells synthesize and release serotonin (5 hydroxytryptamine, 5HT); however, the role of 5HT receptors on glucose stimulated insulin secretion (GSIS) and the mechanisms mediating this function is not fully understood. The aims of this study were to determine the expression profile of 5HT receptors in murine MIN6 β-cells and to examine the effects of pharmacological activation of 5HT receptor Htr2b on GSIS and mitochondrial function.</p><p>Materials and Methods</p><p>mRNA levels of 5HT receptors in MIN6 cells were quantified by RT qPCR. GSIS was assessed in MIN6 cells in response to global serotonergic activation with 5HT and pharmacological Htr2b activation or inhibition with BW723C86 or SB204741, respectively. In response to Htr2b activation also was evaluated the mRNA and protein levels of PGC1α and PPARy by RT-qPCR and western blotting and mitochondrial function by oxygen consumption rate (OCR) and ATP cellular content.</p><p>Results</p><p>We found that mRNA levels of most 5HT receptors were either very low or undetectable in MIN6 cells. By contrast, Htr2b mRNA was present at moderate levels in these cells. Preincubation (6 h) of MIN6 cells with 5HT or BW723C86 reduced GSIS and the effect of 5HT was prevented by SB204741. Preincubation with BW723C86 increased PGC1α and PPARy mRNA and protein levels and decreased mitochondrial respiration and ATP content in MIN6 cells.</p><p>Conclusions</p><p>Our results indicate that prolonged Htr2b activation in murine β-cells decreases glucose-stimulated insulin secretion and mitochondrial activity by mechanisms likely dependent on enhanced PGC1α/PPARy expression.</p></div

Prolonged activation of Htr2b reduces GSIS in MIN6 cells.

<p>(A) MIN6 cells were treated with vehicle or increasing concentrations (10 nM– 10 μM) of the selective Htr2b agonist BW723C86 for 6 hours and then subjected to GSIS procedure. (B) MIN6 cells were treated with vehicle, 50 μM 5HT or co-treated with 5HT (50 μM) plus a selective Htr2b antagonist SB204741 (100 nM) for 6 hours and then subjected to GSIS procedure. The GSIS assays were carry out with KRH buffer 0 (white bars) or 20 mM glucose (black bars). Concentration of insulin accumulated over 1 hour was measured by ELISA. The bars represent mean ± SEM of three independent experiments in triplicate. Number over the bars represents the stimulation index (SI). ** p<0.01, *** p<0.001 and ns, non-significant. One-way ANOVA analysis.</p

5HT receptors mRNA levels in MIN6 cells.

<p>Total RNA was extracted from MIN6 cells and 5HT receptors mRNA levels assessed by RT-qPCR. The mRNA levels was expressed as fold-change to Insulin receptor (Insr) (2^{−ΔCt 5HT receptors}/2^{−ΔCt Insr}). Bars correspond to mean ± SEM of three independent experiments. Numbers over the bars correspond to mean Ct values.</p

Prolonged Htr2b activation increases PGC1α and PPARy levels in MIN6 cells and the effect of PGC1α1 overexpression on GSIS.

<p>(A) MIN6 cells were treated for 6 hours with the Htr2b selective agonist BW723C86 (10 μM) and then the mRNA levels of PGC1α and PPARy were quantified by RT-qPCR. Relative mRNA levels are expressed as fold-change of 2^−ΔCt in treated vs. control cells (2^{−ΔCt treated}/2^{−ΔCt control}). Numbers over bars correspond to mean Ct values. (B) MIN6 cells were treated for 24 hours with BW723C86 (10 μM) and then, PGC1α and Gapdh protein expression was evaluated by immunoblot. (C and D) MIN6 cells were transfected with a pcDNA3.1-PGC1α1 or an empty vector (mock) for 48 hours. (C) Protein levels of PGC1α1 and (D) insulin secretion were measured by immunoblot and GSIS procedure, respectively. Graph bars represent means ± SEM of three independent experiments in triplicate. ** p<0.01 and *** p<0.001 in one-way ANOVA analysis (A and D) or Student’s t test (B and C).</p

Proposed model for the inhibitory effect of prolonged Htr2b activation on GSIS in MIN6 cells.

<p>MIN6 cells constitute a functional microserotoninergic system, with the ability to synthesis 5HT from its precursors (tryptophan or 5HTP), storage, degrade and release this monoamine. The extracellular 5HT may decrease GSIS through the activation of the Htr2b receptor, the most abundantly 5HT receptor expressed in MIN6 cells. Prolonged Htr2b activation is accompanied with calcium release from endoplasmic reticulum and its inhibitory effects on GSIS is likely the result of enhanced PGC1α/PPARy expression and mitochondrial dysfunction with decreased glucose-stimulated ATP synthesis.</p

5HTP increases extracellular 5HT levels in MIN6 cells and 5HT reduces GSIS.

<p>(A) MIN6 cells were incubated with 5HT precursor 5HTP (500 μM) (black bars) or vehicle (white bars) for 6 hours and then 5HT concentration was quantify in the conditioned medium by HPLC. (B) MIN6 cells were incubated with 5HT (50 μM) or vehicle during 6 hours in DMEM without FBS and then subjected to GSIS procedure with 0 (white bars) or 20 mM glucose (black bars) for 1 hour. Insulin concentration in the KRH buffer was quantified by ELISA. Graph bars represent mean ± SEM of measurements of three independent experiments in triplicate. The symbol *** denotes p<0.001 in Student’s t (A) tests and in one-way ANOVA (B).</p