Detrimental effects of RNAi: a cautionary note on its use in Drosophila ageing studies

RNA interference (RNAi) provides an important tool for gene function discovery. It has been widely exploited in Caenorhabditis elegans ageing research because it does not appear to have any non-specific effects on ageing-related traits in that model organism. We show here that ubiquitous, adult-onset activation of the RNAi machinery, achieved by expressing a double stranded RNA targeting GFP or lacZ for degradation, or by increasing expression of Dicer substantially reduces lifespan in Drosophila melanogaster. Induction of GFPRNAi construct also alters the response of lifespan to nutrition, exacerbating the lifespan-shortening effects of food containing a high quantity of yeast. Our study indicates that activation of the RNAi machinery may have sequence-independent side-effects on lifespan, and that caution needs to be exercised when employing ubiquitous RNAi in Drosophila ageing studies. However, we also show that RNAi restricted to certain tissues may not be detrimental to lifespan

The Ras-Erk-ETS-signaling pathway is a drug target for longevity

Summary Identifying the molecular mechanisms that underlie aging and their pharmacological manipulation are key aims for improving lifelong human health. Here, we identify a critical role for Ras-Erk-ETS signaling in aging in Drosophila. We show that inhibition of Ras is sufficient for lifespan extension downstream of reduced insulin/IGF-1 (IIS) signaling. Moreover, direct reduction of Ras or Erk activity leads to increased lifespan. We identify the E-twenty six (ETS) transcriptional repressor, Anterior open (Aop), as central to lifespan extension caused by reduced IIS or Ras attenuation. Importantly, we demonstrate that adult-onset administration of the drug trametinib, a highly specific inhibitor of Ras-Erk-ETS signaling, can extend lifespan. This discovery of the Ras-Erk-ETS pathway as a pharmacological target for animal aging, together with the high degree of evolutionary conservation of the pathway, suggests that inhibition of Ras-Erk-ETS signaling may provide an effective target for anti-aging interventions in mammals. Video Abstrac

Recruiting men from across the socioeconomic spectrum via GP registers and community outreach to a weight management feasibility randomised controlled trial

Background Men, particularly those living in disadvantaged areas, are less likely to participate in weight management programmes than women despite similar levels of excess weight. Little is known about how best to recruit men to weight management interventions. This paper describes patient and public involvement in pre-trial decisions relevant to recruitment and aims to report on recruitment to the subsequent men-only weight management feasibility trial, including the: i) acceptability and feasibility of recruitment; and ii) baseline sample characteristics by recruitment strategy. Methods Men with BMI ≥30 kg/m2 and/or waist circumference ≥ 40 in. were recruited to the feasibility trial via two strategies; community outreach (venue information stands and word of mouth) and GP letters, targeting disadvantaged areas. Recruitment activities (e.g. letters sent, researcher venue hours) were recorded systematically, and baseline characteristics questionnaire data collated. Qualitative interviews (n = 50) were conducted three months post-recruitment. Analyses and reporting followed a complementary mixed methods approach. Results 105 men were recruited within four months (community n = 60, GP letter n = 45). Community outreach took 2.3 recruiter hours per participant and GP letters had an opt-in rate of 10.2% (n = 90/879). More men were interested than could be accommodated. Most participants (60%) lived in more disadvantaged areas. Compared to community outreach, men recruited via GP letters were older (mean = 57 vs 48 years); more likely to report an obesity-related co-morbidity (87% vs 44%); and less educated (no formal qualifications, 32% vs 10%, degree educated 11% vs 41%). Recruitment strategies were acceptable, a sensitive approach and trusting relationships with recruiters valued, and the ‘catchy’ study name drew attention. Conclusions Targeted community outreach and GP letters were acceptable strategies that successfully recruited participants to a men-only weight management feasibility trial. Both strategies engaged men from disadvantaged areas, a typically underserved population. Using two recruitment strategies produced samples with different health risk profiles, which could add value to research where either primary or secondary prevention is of interest. Further work is required to examine how these strategies could be implemented and sustained in practice

Acceptability of financial incentives for breastfeeding: thematic analysis of readers' comments to UK online news reports.

BACKGROUND: Whilst it is recommended that babies are breastfed exclusively for the first six months, many mothers do not maintain breastfeeding for this length of time. Previous research confirms that women and midwives value financial incentives for breastfeeding, but limited research has explored the wider acceptability of these interventions to the general public. This paper examines opinion towards financial incentives for breastfeeding using reader responses to UK on-line media coverage of a study undertaken in this area. METHODS: This study used netnography to undertake a thematic analysis of 3,373 reader comments posted in response to thirteen articles, published in November 2013, which reported findings from a feasibility study of financial incentives for breastfeeding. All articles were published on one of six UK news websites that achieved a monthly audience of at least five million viewers across laptop and desktop computers and mobile devices during April-May 2013. RESULTS: Nine analytical themes were identified, with a majority view that financial incentives for breastfeeding are unacceptable. These themes cover a range of opinions: from negligent parents unable to take responsibility for their own actions; through to psychologically vulnerable members of society who should be protected from coercion and manipulation; to capable and responsible women who can, and should be allowed to, make their own decisions. Many views focused on the immediate costs of the intervention, concluding that this was something that was currently unaffordable to fund (e.g. by the NHS). Others contrasted the value of the incentive against other 'costs' of breastfeeding. There was some consideration of the issue of cost-effectiveness and cost-saving, where the potential future benefit from initial investment was identified. Many commenters identified that financial incentives do not address the many structural and cultural barriers to breastfeeding. CONCLUSIONS: Overall, those commenting on the on-line UK news articles viewed financial incentives for breastfeeding as unacceptable and that alternative, structural, interventions were likely to be more effective. Further consideration of how best to conduct internet-based qualitative research to elicit opinion towards public health issues is required

Ubiquitous adult-induced expression of dsRNA targeting <i>lacZ</i> for RNAi, or of <i>Dicer2</i>, shortens lifespan in <i>Drosophila</i> females.

<p>A Mated <i>ActGS>lacZRNAi</i> female flies of the indicated genotype were fed 1 SYA food containing RU486 (red lines) or not (black lines) from day two of adulthood. Log-rank test detected significant differences between the induced and uninduced conditions (p = 0.004, n =  ∼120 per condition). B Same as in A but for <i>ActGS>Dcr2</i>. Log-rank test detected significant differences between the induced and uninduced conditions (p = 0.0006, n =  ∼150 per condition).</p

Cell-nonautonomous effects of dFOXO/DAF-16 in aging.

Drosophila melanogaster and Caenorhabditis elegans each carry a single representative of the Forkhead box O (FoxO) family of transcription factors, dFOXO and DAF-16, respectively. Both are required for lifespan extension by reduced insulin/Igf signaling, and their activation in key tissues can extend lifespan. Aging of these tissues may limit lifespan. Alternatively, FoxOs may promote longevity cell nonautonomously by signaling to themselves (FoxO to FoxO) or other factors (FoxO to other) in distal tissues. Here, we show that activation of dFOXO and DAF-16 in the gut/fat body does not require dfoxo/daf-16 elsewhere to extend lifespan. Rather, in Drosophila, activation of dFOXO in the gut/fat body or in neuroendocrine cells acts on other organs to promote healthy aging by signaling to other, as-yet-unidentified factors. Whereas FoxO-to-FoxO signaling appears to be required for metabolic homeostasis, our results pinpoint FoxO-to-other signaling as an important mechanism through which localized FoxO activity ameliorates aging

Ubiquitous adult-induced expression of dsRNA targeting <i>GFP</i> for RNAi shortens lifespan in <i>Drosophila</i> females.

<p>Mated female flies of the indicated genotype were fed 1 SYA food containing RU486 (red lines) or not (black lines) from day two of adulthood. <b>A</b> and <b>B</b> Log-rank test detected significant differences between the induced and uninduced conditions (p<0.0001, n =  ∼100 per condition). <b>C</b>, <b>D</b> and <b>E</b> Log-rank test did not detected significant differences between the induced and uninduced conditions (p>0.05, n =  ∼100 per condition).</p

Interplay of dFOXO and Two ETS-Family Transcription Factors Determines Lifespan in <i>Drosophila melanogaster</i>

<div><p>Forkhead box O (FoxO) transcription factors (TFs) are key drivers of complex transcriptional programmes that determine animal lifespan. FoxOs regulate a number of other TFs, but how these TFs in turn might mediate the anti-ageing programmes orchestrated by FoxOs <i>in vivo</i> is unclear. Here, we identify an E-twenty six (ETS)-family transcriptional repressor, <i>Anterior open</i> (<i>Aop</i>), as regulated by the single <i>Drosophila melanogaster</i> FoxO (dFOXO) in the adult gut. AOP, the functional orthologue of the human Etv6/Tel protein, binds numerous genomic sites also occupied by dFOXO and counteracts the activity of an ETS activator, <i>Pointed</i> (<i>Pnt</i>), to prevent the lifespan-shortening effects of co-activation of dFOXO and PNT. This detrimental synergistic effect of dFOXO and PNT appears to stem from a mis-regulation of lipid metabolism. At the same time, AOP activity in another fly organ, the fat body, has further beneficial roles, regulating genes in common with <i>dfoxo</i>, such as the secreted, non-sensory, odorant binding protein (<i>Obp99b</i>), and robustly extending lifespan. Our study reveals a complex interplay between evolutionarily conserved ETS factors and dFOXO, the functional significance of which may extend well beyond animal lifespan.</p></div

<i>Aop</i> extends lifespan.

<p><b>A</b> Survival of <i>S₁106>Aop^ACT</i> female flies in the presence or absence or RU486. Log-rank test detected significant differences (p = 2×10⁻¹⁰; total dead/censored: − RU486 145/1, + RU486 129/3; median/maximum lifespan: − RU486: 74/87, + RU486 84/97). <b>B</b> Median lifespan extension achieved by RU486 feeding in <i>S₁106>Aop^ACT</i> (6 experiments) or <i>TIGS>Aop^ACT</i> (3 experiments) females. Log-rank test detected significant extension (p<0.05) of lifespan in 6 out of 6 <i>S₁106>Aop^ACT</i> and in 1 out of 3 <i>TIGS>Aop^ACT</i> trials. In one <i>TIGS>Aop^ACT</i> trial lifespan was shortened. <b>C</b> Haemolymph glucose and trehalose in <i>S₁106>Aop^ACT</i> females fed or not RU486, where RU486 had a significant effect in each case (t-test, p = 0.01 and 0.02 respectively; n = 8 where 10 measurements were made and the highest and lowest measurement removed from each group). <b>D</b> Survival of <i>S₁106>Aop^ACT</i> or dfoxoΔ/Δ <i>S₁106>Aop^ACT</i> female flies in the presence or absence or RU486. Log-rank test detected significant differences for both: <i>S₁106>Aop^ACT</i> (p<10⁻⁴; total dead/censored: − RU486 139/3, + RU486 136/7; median/maximum lifespan: − RU486: 77/87, + RU486 86/94) and dfoxoΔ/Δ <i>S₁106>Aop^ACT</i> (p<10⁻⁴; total dead/censored: − RU486 138/0, + RU486 136/4; median/maximum lifespan: − RU486: 56/81, + RU486 72/90). CHP analysis revealed significant effects of RU486 (p<10⁻¹⁵) and <i>dfoxo</i> (p<10⁻¹⁵) but no significant difference in the response to RU486 between the two lines (p = 0.2).</p