Cellular response to stress in control and c.709-1G>A PGRN carriers lymphoblasts.

<p>Lymphoblasts from control and c.709-1G>A PGRN carriers were incubated in serum-free RPMI medium for 72 h (SW) or with 10% FBS in the presence of H₂O₂ or 2 deoxy Ribose (2dRib) for 24 h. The cells were then counted by Trypan blue dye exclusion or by the MTT methods. Results are expressed as % of the number of cells at day 0, and are the mean±SE of four independent experiments. Statistical difference: *p<0.05 from lymphoblasts from control individuals.</p

Serum deprivation-induced apoptosis is accompanied by changes in caspase activation.

<p>A: Influence of the pan-caspase z-VAD-fmk inhibitor on survival of lymphoblasts derived from control, asymptomatic and FTLD patients following serum deprivation. Cells were seeded at an initial density of 1×10⁶/ml and incubated in serum-free RPMI medium for 72 h in the absence or in the presence of 50 µM z-VAD-fmk for 72 h. Results shown are the mean±SE of different experiments carried out with cell lines from four control subjects, asymptomatic or FTLD patients, carrying the PGRN c.709-1G>A mutation, respectively. *p<0.05 significantly different from control cells. B: Caspase activation in serum-deprived lymphoblasts from control and c.709-1G>A carriers. Cells were incubated as above and then labeled with the FLICA reagent, following the manufacture’s recommendation to detect its binding to active caspases 3 and 7. A representative flow cytometric analysis of the frequency distribution of cells according their green fluorescence is showing. Below it is shown the percentage of cells with active caspases 3 and 7 (mean±SE) of 3 observations carried out in different cell lines from control or c.709-1G>A PGRN mutation carriers individuals. *p<0.05 significantly different from control cells.</p

Effects of exogenous progranulin in the serum deprivation-induced cell death.

<p>Lymphoblasts from control or c.709-1G>A carriers individuals were incubated in serum-free RPMI medium in the absence or in the presence of recombinant PGRN (100 ng/ml), alone or in combination with 10 µM SB or 1 µM PD332991. Cell survival was determined after 72 hours of serum deprivation. Data shown are the mean±SE of four determinations carried out with different cell lines. *p<0.05 significantly different from control cells. **p<0.05 significantly different from untreated cells. ⁺p<0.05 significantly different from cells treated with PGRN alone. Below it is shown representative immunoblots showing the effects of these drugs, alone or in combination of exogenous progranulin on CDK6 and pRb proteins levels.</p

Demographic characteristics of the subjects enrolled in the study.

<p>Control: individuals without sign of neurological degeneration. Key: c.709-1G>A, progranulin mutation; FTLD, frontotemporal lobar degeneration.</p

CDK6 mRNA, protein levels and pRb phosphorylation in lymphoblasts from control and c.709-1G>A carriers individuals.

<p>Immortalized lymphocytes from control and c.709-1G>A carriers, FTLD patients or asymptomatic individuals were seeded at an initial density of 1×10⁶/ml and incubated in serum-free RPMI medium. 48 hours later cells were harvested to isolate RNA and to prepare cell lysates. A: CDK6 mRNA expression levels were analyzed by quantitative RT-PCR (left panel), and the CDK6 protein content was analyzed by WB (right panel). The inmunoblot shows two different cell extracts from control, asymptomatic and FTLD patients is shown. The data represent the mean±SE for six observations in different cell lines. *p<0.05 significantly different from control cells. B: Representative inmunoblots showing pRb and p130 phosphorylation status in two different control, asymptomatic or FTLD individuals is shown. pp =  the hyperphosphorylated form of the pRb or the p130 protein. Below it is shown the densitometric analysis of the hyperphosphorylated form of pRb and p130. Data represent the mean±SE for six independent observations in different cell lines. C: Representative immunoblots showing the cellular content of Cyclin D1, D2 and D3, and the CDK inhibitors p16 y p18 in two different control, asymptomatic or FTLD individuals.</p

Effects of sodium butyrate and PD332991 on CDK6 mRNA and protein levels and in the survival of control and c.709-1G>A carriers lymphoblasts.

<p>Lymphoblasts were incubated as in the legend of Fig. 6 in the absence or in the presence of 10 µM SB (A, B and C) or 1 µM PD332991 (E, F and G) for 48 h. CDK6 mRNA analysis was performed by quantitative RT-PCR, protein levels were assessed by WB. Cell survival was determined by trypan blue exclusion under inverted phase-contrast microscopy. Values shown are the mean±SE for four independent observations carried out in different cell lines. *p<0.05 significantly different from control cells. **p<0.05 significantly different from untreated cells.</p

Enhanced release of cytochrome c to the cytosol in serum-deprived lymphoblasts bearing the c.709-1G>A PGRN mutation.

<p>A: Lymphoblasts from control and c.709-1G>A carriers were serum deprived for 72 h. Cell lysates were fractionated to isolate cytoplasmic and crude mitochondria. The presence of cytochrome c in cytosolic and mitochondrial fractions was assessed by WB analysis using the ApoTrack antibody cocktail, which demonstrates the purity of the fractions and loading. A representative blot of three independent experiments is shown. B: Cytochorme c detection in cytosolic extracts from control and PGRN deficient lymphoblasts. A representative immunoblot showing cytosolic cytochorme c in two different individuals for each condition is shown (left panel). Densitometric analysis is presented in the right panel. The data represent the mean±SE of the cytosolic cytochrome c for four observations in different cell lines. *p<0.05 significantly different from control cells.</p

Influence of PGRN haploinsufficiency on cell response to serum stimulation or withdrawal.

<p>A: Immortalized lymphocytes from control and c.709-1G>A carriers, FTLD patients or asymptomatic individuals were seeded at an initial density of 1×10⁶/ml and incubated in RPMI medium with decreasing concentrations of FBS or in the absence of serum for 72 h. Cell viability was determined by trypan blue exclusion under inverted phase-contrast microscopy. Data are the mean±SE for at least four independent experiments carried out with cell lines from different individuals *p<0.01 significantly different from control cells. B: Proliferative response of control and PGRN deficient cells in the presence or in the absence of serum. Lymphoblasts (5000 cells/well) were seeded in 96-well plates in the presence of 10% FBS or serum replacement (SR). After 24 h, cells were pulsed with 10 µM BrdU for 4 h. DNA synthesis was assessed by BrdU incorporation method according to the manufacturer’s instructions. Proliferation was expressed as absorbance of stimulated minus that of nonstimulated cultures. Each bar represents the mean±SE of three independent experiments performed in triplicate. C: Effect of PGRN deficiency on the serum deprivation-induced cell death. Lymphoblasts from control and c.709-1G>A carriers, FTLD patients or asymptomatic individuals were seeded as above and incubated in serum-free RPMI medium for 72 h. Cells were harvested every day thereafter and cell viability was determined by trypan blue exclusion under inverted phase-contrast microscopy. Data shown are the mean±SE of all cell lines used in this study (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037057#pone-0037057-t002" target="_blank">Table 2</a>). * and ⁺p<0.05 differences significantly different between control and asymptomatic or FTLD patients respectively.</p

The relationship between coping style and psychological distress in people with head and neck cancer: a systematic review

Objective Individuals diagnosed with head and neck cancer (HNC) are at an elevated risk of experiencing psychological distress and a reduced quality of life. The aim of this review was to systematically examine and assess the quality of empirical evidence on the associations between coping mechanisms and psychological distress among people with HNC. Methods CINAHL, MEDLINE, PsycINFO, EMBASE and Web of Science were accessed to conduct this review. Studies were included if they used reliable and valid measures to investigate the relationship between coping style and psychological distress. Study quality was assessed and rated according to pre-set criteria, and showed variability in relation to selection methods. Results 12 studies (nine cross-sectional and three prospective designs) involving 1281 patients were reviewed. There was considerable heterogeneity in study samples and coping measures. Moderate to large associations between disengagement coping mechanisms (e.g. avoidance) and psychological distress were observed. Engagement coping strategies (e.g. direct action) were not consistently associated with psychological distress across studies. Conclusions Several studies observed a significant relationship between coping styles aimed at disengaging and distancing from cancer and increased psychological distress. To understand directionality of these associations and further develop an understanding of temporal features of the relationship between coping styles and distress, longitudinal designs could be used in future research

Additional file 5: of Mutations in LRRK2 impair NF-κB pathway in iPSC-derived neurons

Uncropped blots related to Fig. 6