Relationship Between Distance Run Per Week, Omega-3 Index, and Arachidonic Acid (AA)/Eicosapentaenoic Acid (EPA) Ratio: An Observational Retrospective Study in Non-elite Runners

Background: Tissue availability of polyunsaturated fatty acids (PUFA) depends on several factors, including dietary intake, physical exercise, genetic variation, and metabolic turnover. However, there is limited evidence whether running training activity per se may influence indices associated with PUFA metabolism such as Omega-3 (ω-3) index and arachidonic acid (AA; 20:4ω-6)/eicosapentaenoic acid (EPA; 20:5ω-3) ratio.Objective: To examine the association between kilometers (Km) run per week and changes in ω-3 index and AA/EPA ratio.Methods: We conducted a retrospective, observational, cohort study of 257 non-elite runners (mean age: 40.85 ± 12.17 years) who consumed no fatty acid supplements and provided a blood sample for analysis. The whole blood samples were collected by finger sticks, stored on absorbent filter paper, and then PUFA were quantified by gas chromatography (GC) and ω-3 index and AA/EPA ratio measured.Results: In a multivariate linear regression model, a gradual decrease of the ω-3 index was observed with higher weekly running distance (β = −0.033; 95% CI −0.039 to −0.026; R2 = 0.447; p < 0.0001). We also found a progressive increase of the AA/EPA ratio in subjects who ran greater weekly distances (β = 0.092; 95% CI 0.038 to 0.146; R2 = 0.320; p = 0.001). No other significant associations were observed with other variables, including years of running training and weekly training frequency (hours/week). Finally, as expected, a significant inverse correlation between ω-3 index and AA/EPA ratio (β = −2.614; 95% CI −3.407 to −1.821; R2 = 0.336; p < 0.0001) was detected.Conclusions: These findings suggest that distance running training and its weekly volume may negatively contribute to changes of the ω-3 index and AA/EPA ratio. Further studies with greater sample size will be required to replicate and extend these data

Nuclear Ras2-GTP Controls Invasive Growth in <i>Saccharomyces cerevisiae</i>

<div><p>Using an eGFP-RBD3 probe, which specifically binds Ras-GTP, we recently showed that the fluorescent probe was localized to the plasma membrane and to the nucleus in wild type cells growing exponentially on glucose medium, indicating the presence of active Ras in these cellular compartments. To investigate the nuclear function of Ras-GTP, we generated a strain where Ras2 is fused to the nuclear export signal (NES) from the HIV virus, in order to exclude this protein from the nucleus. Our results show that nuclear active Ras2 is required for invasive growth development in haploid yeast, while the expression of the NES-Ras2 protein does not cause growth defects either on fermentable or non-fermentable carbon sources and does not influence protein kinase A (PKA) activity related phenotypes analysed. Moreover, we show that the cAMP/PKA pathway controls invasive growth influencing the localization of active Ras. In particular, we show that PKA activity plays a role in the localization of active Ras and influences the ability of the cells to invade the agar: high PKA activity leads to a predominant nuclear accumulation of active Ras and induces invasive growth, while low PKA activity leads to plasma membrane localization of active Ras and to a defective invasive growth phenotype.</p></div

Nuclear active Ras2 is required for invasive growth in W303-1A-based strains.

<p>YPD exponentially growing cells of the indicated strains were spotted on YPD agar plates. After 3°C, the plates were gently washed with water from top down and pictures were taken before (Total growth) and after (Invasive growth) washing.</p

Localization of active Ras in glucose-growing <i>cyr1Δ pde2Δ yak1Δ</i> cells, before and after addition of cAMP.

<p>(A) <i>cyr1Δ pde2Δ yak1Δ</i> cells transformed with YEpeGFP-RBD3 were grown in medium containing 2% glucose at 30°C until exponential phase and then photographed with a Nikon fluorescence microscope, before and 45 min after addition of 2 mM cAMP. (B) Subcellular distribution of eGFP fluorescence.</p

Effect of expression of NES-Ras2 on growth on different carbon sources and on PKA-activity-related phenotypes.

<p>(A) Cells were gown in YPD medium at 30°C until exponential phase. Then cells were harvested by centrifugation, washed three times with sterile water and resuspended in sterile water at 10⁷ cells/ml. 5 µl from the concentrated suspension and from 10–fold dilutions were spotted on agar plates containing the indicated carbon sources. Pictures were taken after 48 h at 30°C. (B) Heat-shock resistance in exponentially growing cells. Cells were incubated synthetic complete medium containing 2% glucose to exponential phase, diluted in fresh medium to a concentration of 1.25×10⁶ cells/ml and then exposed to heat shock at 51°C for 0, 1, 2 and 3 min. Approximately 10⁴ cells were spotted on YPD agar and incubated at 30°C for 24 hours. (C) Oxidative and osmotic stress resistance in exponentially growing cells. Cells were incubated in YPD medium until exponential phase. Then cells were harvested by centrifugation, washed three times with sterile water and resuspended in sterile water at 10⁷ cells/ml. 5 µl from the concentrated suspension and from 10–fold dilutions were spotted on glucose agar plates containing respectively 2 mM H₂O₂, 6 mM H₂O₂ and 0.5 M NaCl. After 48 hours at 30°C pictures were taken.</p

Effect of PKA activity on the localization of active Ras.

<p>(A) W303-1A, <i>cyr1</i>Δ <i>pde2</i>Δ<i>yak1</i>Δ and <i>bcy1</i>Δ cells transformed with YEpeGFP-RBD3. Cells were grown in medium containing 2% glucose at 30°C until exponential phase and then photographed with a Nikon fluorescence microscope. (B) Subcellular distribution of eGFP fluorescence.</p

Effect of expression of NES-Ras2 on proteins level, growth rate, localization of Ras-GTP and invasive growth.

<p>(A) Expression of the Ras2 and NES-Ras2 proteins using a SDS-PAGE and western blotting analysis. (B) The Tlys86-NES-RAS2 strain (Δ) grew in minimal medium containing 2% glucose at a rate comparable to that of the Tlys86 wild type strain (▴). (C) Tlys86 and Tlys86-NES-RAS2 cells transformed with YEpeGFP-RBD3. Cells were grown in medium containing 2% glucose at 30°C until exponential phase and then photographed with a Nikon fluorescence microscope. (D) YPD exponentially growing cells of the indicated strains were spotted on YPD agar plates. After 3 days at 30°C, the plates were washed under the running water and pictures were taken before (Total growth) and after (Invasive growth) washing.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Peptide-Nanoparticle Ligation Mediated by <i>Cutinase</i> Fusion for the Development of Cancer Cell-Targeted Nanoconjugates

The relationship between the positioning of ligands on the surface of nanoparticles and the structural features of nanoconjugates has been underestimated for a long time, albeit of primary importance to promote specific biological recognition at the nanoscale. In particular, it has been formerly observed that a proper molecular orientation can play a crucial role, first optimizing ligand immobilization onto the nanoparticles and, second, improving the targeting efficiency of the nanoconjugates. In this work, we present a novel strategy to afford peptide-oriented ligation using genetically modified <i>cutinase</i> fusion proteins, which combines the presence of a site-directed “capture” module based on an enzymatic unit and a “targeting” moiety consisting of the ligand terminal end of a genetically encoded polypeptide chain. As an example, the oriented presentation of U11 peptide, a sequence specific for the recognition of urokinase plasminogen activator receptor (uPAR), was achieved by enzyme-mediated conjugation with an irreversible inhibitor of cutinase, an alkylphosphonate <i>p</i>-nitrophenol ester linker, covalently bound to the surface of iron oxide nanoparticles. The targeting efficiency of the resulting protein–nanoparticle conjugates was assessed using uPAR-positive breast cancer cells exploiting confocal laser scanning microscopy and quantitative fluorescence analysis of confocal images. Ultrastructural analysis of transmission electron micrographs provided evidence of a receptor-mediated pathway of endocytosis. Our results showed that, despite the small average number of targeting peptides presented on the nanoparticles, our ligand-oriented nanoconjugates proved to be very effective in selectively binding to uPAR and in promoting the uptake in uPAR-positive cancer cells