Main fatty acid changes in NB100 cell membranes after fatty acids supplementation.

<p>(<b>A</b>) In the left panel, the main fatty acid variations in NB100 cell membranes after 150 µM PA supplementation are reported. In the right panel, fluctuations of the corresponding fatty acid families are evidenced. (<b>B</b>) In the left panel, the main fatty acid variations in NB100 cell membranes after 150 µM PA +50 µM OA +50 µM AA supplementation are reported. The right panel evidences the fatty acid family changes after this supplementation. (<b>C</b>) In the left panel, the main fatty acid variations and the corresponding fatty acid families are evidenced in NB100 cell membranes after 50 µM PA supplementation. In the right panel, the same fatty acid variations are reported for cells incubated with 150 µM PA for 8 hours and 24 hours. Data are obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone-0055537-t001" target="_blank">Tables 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone-0055537-t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone-0055537-t003" target="_blank">3</a>. Values are means ± SD. Statistical significances are as reported in the notes to the tables.</p

Membrane phospholipid fatty acids of NB100 cells treated for the indicated times with 50 µM and 150 µM PA and compared to controls grown in the same conditions without PA supplementation at each time intervals.

<p> <b>The values are reported as % rel of the total fatty acid peak areas detected in the GC analysis. They are mean values ± SD of the n repetitions of the same experiment.</b></p>a<p>FAME are obtained from total lipid extraction, derivatization, and GC analysis.</p>b<p>The identification of the peaks have been performed by authentic samples and the identified peaks accounted for >98% of the total peaks.</p>c<p>Values higher than untreated control (***P = 0.0001).</p>d<p>Values lower than untreated control (***P = 0.0001).</p>e<p>Values higher than untreated control (**P<0.001).</p>f<p>Values lower than untreated control (**P<0.001).</p>g<p>Values higher than untreated control (*P<0.01).</p>h<p>Values lower than untreated control (*P<0.01).</p>*<p>Evaluated with standard compounds (mono-trans arachidonic acid isomers) obtained following references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone.0055537-Ferreri1" target="_blank">[12]</a>.</p>#<p>This value includes EPA and DHA.</p

Membrane phospholipid fatty acids of NB100 cells treated for the indicated times with 150 µM PA, 50 µM OA, 50 µM AA and compared to controls grown in the same conditions without fatty acid supplementation.

<p> <b>Controls are the mean of cells cultured from 0 to 3 hours in the absence of fatty acid supplementation. The values are reported as % rel of the total fatty acid peak areas detected in the GC analysis. They are mean values ± SD of the n repetitions of the same experiment.</b></p>a<p>FAME are obtained from total lipid extraction, derivatization, and GC analysis.</p>b<p>The identification of the peaks have been performed by authentic samples and the identified peaks accounted for >98% of the total peaks.</p>c<p>Values higher than untreated control (*P = 0.01).</p>d<p>Values lower than untreated control (*P = 0.01).</p>e<p>Values higher than untreated control (*P<0.04).</p>f<p>Values lower than untreated control (*P<0.04).</p>*<p>Evaluated with standard compounds (mono-trans arachidonic acid isomers) obtained following references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone.0055537-Ferreri1" target="_blank">[12]</a>.</p>#<p>This value includes EPA and DHA.</p

Membrane phospholipid fatty acids of NB100 cells treated for the indicated times with 150 µM PA and compared to controls grown in the same conditions without PA supplementation.

<p> <b>Controls are the mean of cells cultured from 0 to 3 hours in the absence of PA. The values are reported as % rel of the total fatty acid peak areas detected in the GC analysis. They are mean values ± SD of the n repetitions of the same experiment.</b></p>a<p>FAME are obtained from total lipid extraction, derivatization, and GC analysis.</p>b<p>The identification of the peaks have been performed by authentic samples and the identified peaks accounted for >98% of the total peaks.</p>c<p>Values higher than untreated control (***P = 0.0001).</p>d<p>Values lower than untreated control (***P = 0.0001).</p>e<p>Values higher than untreated control (**P<0.001).</p>f<p>Values lower than untreated control (**P<0.001).</p>g<p>Values higher than untreated control (*P<0.01).</p>h<p>Values lower than untreated control (*P<0.01).</p>*<p>Evaluated with standard compounds (mono-trans arachidonic acid isomers) obtained following references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone.0055537-Ferreri1" target="_blank">[12]</a>.</p>#<p>This value includes EPA and DHA.</p

Evaluation of apoptosis in NB100 cells supplemented with fatty acids.

<p>(<b>A</b>) Caspase activation in NB100 cells exposed to PA 150 µM (▪) or PA 150 µM+OA 50 µM+AA 50 µM (•). Caspase-2, -8, -9 and -3/7 activation was determined at 3, 8, 16, 24 hours as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#s2" target="_blank">Materials and Methods</a>. Caspase activity is expressed as percentage of control values obtained form cultures grown in the absence of FA supplementation. Mean results ± SD are reported. (<b>B</b>) Cell viability was evaluated at 24 hours on NB100 cells pretreated with 30 µM of the irreversible tetrapeptide pan-caspase inhibitor Z-VAD-fmk, added to the culture 3 hours before the 150 µM PA supplementation.</p

Effect of PA on NB100 cell line and cell morphology.

<p>(<b>A</b>) Effect of FA supplementation on NB100 cell viability. Cell viability was determined by MTS assay. Values are means ± SD of four determinations. Left graph: cells were incubated in complete medium supplemented with 50 µM PA (□), 150 µM PA (▪), 150 µM PA +50 µM OA (♦), 150 µM PA +50 µM AA (▴), 150 µM PA +50 µM OA +50 µM AA (○). Right graph: cells were incubated in complete medium supplemented with PA at various concentrations for 1 hour (•) or for 2 hours (○), and then incubated in complete medium for 48 hours after wash. (<b>B</b>) NB100 cells morphology assessed by phase contrast microscopy. Control cultures grown in the absence of FA supplementation are shown in comparison with cells treated for 24 hours with 150 µM PA or 150 µM PA +50 µM OA +50 µM AA. Magnification 200×. (<b>C</b>) Nuclei of NB100 cells stained with DAPI and assessed by fluorescence microscopy (×600 magnification objective). Cells were incubated in complete medium supplemented with 150 µM PA (4, 8, 16, 24 hours) or 150 µM PA +50 µM OA +50 µM AA (24 hours). Control cultures grown in the absence of FA supplementation (24 hours) are also shown.</p

cPLA2 activation under PA supplementation.

<p>(<b>A</b>) Western blot analyses of the effects of PA treatment on cPLA₂ protein activation in its phosphorylated form in NB100 cells. Cells were examined after treatment for 15–180 min with 150 µM PA. Cell lysates were resolved by SDS/PAGE. Proteins were blotted and detected with monoclonal antibodies against cPLA₂ or phospho-cPLA₂. Representative gels are shown. (<b>B</b>) The bar graph represents the band intensity values obtained by the Image J analysis, expressed as percentage of the corresponding control (Time 0) (white bars: total cPLA₂; black bars: phospho-cPLA₂). AA quantity in membranes as % of control (grey bars) is obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055537#pone-0055537-t001" target="_blank">Table 1</a>. Results are mean ± SD of at least three independent experiments. Phospho-cPLA₂ is significantly higher than controls at 15, 30 and 60 min (p<0.0001). AA is significantly higher at 30 min and significantly lower at 180 min (p = 0.001).</p

DataSheet1_Applying the estimand and target trial frameworks to external control analyses using observational data: a case study in the solid tumor setting.DOCX

Introduction: In causal inference, the correct formulation of the scientific question of interest is a crucial step. The purpose of this study was to apply causal inference principles to external control analysis using observational data and illustrate the process to define the estimand attributes.Methods: This study compared long-term survival outcomes of a pooled set of three previously reported randomized phase 3 trials studying patients with metastatic non-small cell lung cancer receiving front-line chemotherapy and similar patients treated with front-line chemotherapy as part of routine clinical care. Causal inference frameworks were applied to define the estimand aligned with the research question and select the estimator to estimate the estimand of interest.Results: The estimand attributes of the ideal trial were defined using the estimand framework. The target trial framework was used to address specific issues in defining the estimand attributes using observational data from a nationwide electronic health record-derived de-identified database. The two frameworks combined allow to clearly define the estimand and the aligned estimator while accounting for key baseline confounders, index date, and receipt of subsequent therapies. The hazard ratio estimate (point estimate with 95% confidence interval) comparing the randomized clinical trial pooled control arm with the external control was close to 1, which is indicative of similar survival between the two arms.Discussion: The proposed combined framework provides clarity on the causal contrast of interest and the estimator to adopt, and thus facilitates design and interpretation of the analyses.</p

Influence of Serotonin Transporter Gene Polymorphisms and Adverse Life Events on Depressive Symptoms in the Elderly: A Population-Based Study

<div><p>Background</p><p>Depression is common in the elderly. The role of genetic and environmental factors in modulating depressive symptoms is not clear.</p><p>Methods</p><p>We evaluated the influence of serotonin transporter gene polymorphisms and recent adverse life events on depressive symptoms in an elderly Italian population. We used data from “InveCe.Ab”, a population-based study of 1321 subjects aged 70–74 years. We used the 15-item Geriatric Depression Scale (GDS) to assess depressive symptoms–a GDS score ≥5 points (GDS≥5) indicated the presence of clinically relevant symptoms–and performed 5-HTTLPR and rs25531 genotyping to obtain the triallelic polymorphism of the serotonin transporter. We used the Geriatric Adverse Life Events Scale to measure adverse life events, and logistic regression models to evaluate the role of genotype and recent adverse life events in depressive symptoms, controlling for potential confounders and independent predictors.</p><p>Results</p><p>Two hundred subjects (15.76%) had a GDS≥5. The 5-HTTLPR triallelic polymorphism was significantly associated with GDS≥5. Only S′S′ carriers showed an increased risk of depressive symptoms (OR_adj = 1.81, <i>p</i> = .022); one extra adverse life event increased this risk by 14% (<i>p</i> = .061) independently of genotype. Other factors significantly related to GDS≥5 were: female gender (OR_adj = 2.49, <i>p</i> < .001), age (OR_adj = 1.19, <i>p</i> = .007), a history of depression (OR_adj = 4.73, <i>p</i> < .001), and comorbidity (OR_adj = 1.23, <i>p</i> = .001). One extra adverse life event increased the risk of depressive symptoms by 57% (<i>p</i> = .005) only in the L′L′ carriers, while antidepressant intake was directly related to GDS≥5 in the L′S′ carriers (OR_adj = 2.46, <i>p</i> = .036) and borderline significant in the S′S′ carriers (OR_adj = 2.41, <i>p</i> = .081).</p><p>Discussion</p><p>The S′S′ genotype and recent exposure to adverse life events were independently associated with depressive symptoms. The S′S′ genotype, compared with the environment, exerted a predominant effect on depressive symptoms, suggesting that it reduces the efficacy of antidepressant therapy. We conclude that genetics may be an important risk factor for depressive symptoms in late adulthood.</p></div

Overview of the baseline characteristics of the cohort.

<p>The summary statistics (mean and standard deviation) or the frequency distribution of each variable are shown in the overall population and by the presence/absence of clinically relevant depressive symptoms (GDS≥5 vs GDS<5).The statistical tests and p-values relative to analyses by GDS categories are also reported.</p