Sex-specific aging in flies, worms, and missing great-granddads.

Two recent studies identify how sex-specific pheromonal factors in flies and worms alter lifespan through metabolic pathways that are shared with mammals. Sex differences in human lifespans imply nonautonomous effects modulated by sex-specific gene-environment interactions that could still include pheromonal mechanisms.

Models of Replicator Proliferation Involving Differential Replicator Subunit Stability.

Several models for the origin of life involve molecules that are capable of self-replication, such as self-replicating polymers composed of RNA or DNA or amino acids. Here we consider a hypothetical replicator (AB) composed of two subunits, A and B. Programs written in Python and C programming languages were used to model AB replicator abundance as a function of cycles of replication (iterations), under specified hypothetical conditions. Two non-exclusive models describe how a reduced stability for B relative to A can have an advantage for replicator activity and/or evolution by generating free A subunits. In model 1, free A subunits associate with AB replicators to create AAB replicators with greater activity. In simulations, reduced stability of B was beneficial when the replication activity of AAB was greater than two times the replication activity of AB. In model 2, the free A subunit is inactive for some number of iterations before it re-creates the B subunit. A re-creates the B subunit with an equal chance of creating B or B', where B' is a mutant that increases AB' replicator activity relative to AB. In simulations, at moderate number of iterations (< 15), a shorter survival time for B is beneficial when the stability of B is greater than the inactive time of A. The results are consistent with the hypothesis that reduced stability for a replicator subunit can be advantageous under appropriate conditions.

Sexual dimorphism in oxidant-induced adaptive homeostasis in multiple wild-type D. melanogaster strains.

Sexual dimorphism includes the physical and reproductive differences between the sexes, including differences that are conserved across species, ranging from the common fruit fly, Drosophila melanogaster, to humans. Sex-dependent variations in adaptive homeostasis, and adaptive stress responses may offer insight into the underlying mechanisms for male and female survival differences and into differences in chronic disease incidence and severity in humans. Earlier work showed sex-specific differences in adaptive responses to oxidative stressors in hybrid laboratory strains of D. melanogaster. The present study explored whether this phenomenon is also observed in wild-type D. melanogaster strains Oregon-R (Or-R) and Canton-S (Ca-S), as well as the common mutant reference strain w[1118], in order to better understand whether such findings are descriptive of D. melanogaster in general. Flies of each strain were pretreated with non-damaging, adaptive concentrations of hydrogen peroxide (H2O2) or of different redox cycling agents (paraquat, DMNQ, or menadione). Adaptive homeostasis, and changes in the expression of the Proteasome and overall cellular proteasomal proteolytic capacity were assessed. Redox cycling agents exhibited a male-specific adaptive response, whereas H2O2 exposure provoked female-specific adaptation. These findings demonstrate that different oxidants can elicit sexually dimorphic adaptive homeostatic responses in multiple fly strains. These results (and those contained in a parallel study [1]) highlight the need to address sex as a biological variable in fundamental science, clinical research, and toxicology.

Mifepristone/RU486 acts in Drosophila melanogaster females to counteract the life span-shortening and pro-inflammatory effects of male Sex Peptide.

Males with null mutation of Sex Peptide (SP) gene were compared to wild-type males for the ability to cause physiological changes in females that could be reversed by mifepristone. Males from wild-type strains decreased median female life span by average -51%. Feeding mifepristone increased life span of these females by average +106%. In contrast, SP-null males did not decrease female life span, and mifepristone increased median life span of these females by average +14%, which was equivalent to the effect of mifepristone in virgin females (average +16%). Expression of innate immune response transgenic reporter (Drosocin-GFP) was increased in females mated to wild-type males, and this expression was reduced by mifepristone. In contrast, SP-null males did not increase Drosocin-GFP reporter expression in the female. Similarly, mating increased endogenous microbial load, and this effect was reduced or absent in females fed mifepristone and in females mated to SP-null males; no loss of intestinal barrier integrity was detected using dye-leakage assay. Reduction of microbial load by treating adult flies with doxycycline reduced the effects of both mating and mifepristone on life span. Finally, mifepristone blocked the negative effect on life span caused by transgenic expression of SP in virgin females. The data support the conclusion that the majority of the life span-shortening, immune-suppressive and pro-inflammatory effects of mating are due to male SP, and demonstrate that mifepristone acts in females to counteract these effects of male SP.

Particle and Photon Detection: Counting and Energy Measurement.

Fundamental limits for photon counting and photon energy measurement are reviewed for CCD and CMOS imagers. The challenges to extend photon counting into the visible/nIR wavelengths and achieve energy measurement in the UV with specific read noise requirements are discussed. Pixel flicker and random telegraph noise sources are highlighted along with various methods used in reducing their contribution on the sensor's read noise floor. Practical requirements for quantum efficiency, charge collection efficiency, and charge transfer efficiency that interfere with photon counting performance are discussed. Lastly we will review current efforts in reducing flicker noise head-on, in hopes to drive read noise substantially below 1 carrier rms.

Natural variation of gene models in Drosophila melanogaster.

BACKGROUND: Variation within splicing regulatory sequences often leads to differences in gene models among individuals within a species. Two alleles of the same gene may express transcripts with different exon/intron structures and consequently produce functionally different proteins. Matching genomic and transcriptomic data allows us to identify putative regulatory variants associated with changes in splicing patterns. RESULTS: Here we analyzed natural variation of splicing patterns in the transcriptomes of 81 natural strains of Drosophila melanogaster with known genotypes. We identified dozens of genotype-specific splicing patterns associated with putative cis-splicing quantitative trait loci (sQTL). The majority of changes can be explained by mutations in splice sites. Allelic-imbalance in splicing patterns confirmed that the majority are regulated mainly by cis-genetic effects. Remarkably, allele-specific splicing changes often lead to qualitative changes in gene models, yielding many isoforms not previously annotated. The observed alterations are typically outside protein-coding regions or affect only very short protein segments. CONCLUSIONS: Overall, the sets of gene models appear to be flexible within D. melanogaster populations. The observed variation in splicing patterns are predicted to have limited effects on the encoded protein sequences. To our knowledge, this is the first sQTL mapping study in Drosophila.

Mitochondrial maintenance failure in aging and role of sexual dimorphism.

Gene expression changes during aging are partly conserved across species, and suggest that oxidative stress, inflammation and proteotoxicity result from mitochondrial malfunction and abnormal mitochondrial-nuclear signaling. Mitochondrial maintenance failure may result from trade-offs between mitochondrial turnover versus growth and reproduction, sexual antagonistic pleiotropy and genetic conflicts resulting from uni-parental mitochondrial transmission, as well as mitochondrial and nuclear mutations and loss of epigenetic regulation. Aging phenotypes and interventions are often sex-specific, indicating that both male and female sexual differentiation promote mitochondrial failure and aging. Studies in mammals and invertebrates implicate autophagy, apoptosis, AKT, PARP, p53 and FOXO in mediating sex-specific differences in stress resistance and aging. The data support a model where the genes Sxl in Drosophila, sdc-2 in Caenorhabditis elegans, and Xist in mammals regulate mitochondrial maintenance across generations and in aging. Several interventions that increase life span cause a mitochondrial unfolded protein response (UPRmt), and UPRmt is also observed during normal aging, indicating hormesis. The UPRmt may increase life span by stimulating mitochondrial turnover through autophagy, and/or by inhibiting the production of hormones and toxic metabolites. The data suggest that metazoan life span interventions may act through a common hormesis mechanism involving liver UPRmt, mitochondrial maintenance and sexual differentiation.

Selectively advantageous instability in biotic and pre-biotic systems and implications for evolution and aging.

Rules of biology typically involve conservation of resources. For example, common patterns such as hexagons and logarithmic spirals require minimal materials, and scaling laws involve conservation of energy. Here a relationship with the opposite theme is discussed, which is the selectively advantageous instability (SAI) of one or more components of a replicating system, such as the cell. By increasing the complexity of the system, SAI can have benefits in addition to the generation of energy or the mobilization of building blocks. SAI involves a potential cost to the replicating system for the materials and/or energy required to create the unstable component, and in some cases, the energy required for its active degradation. SAI is well-studied in cells. Short-lived transcription and signaling factors enable a rapid response to a changing environment, and turnover is critical for replacement of damaged macromolecules. The minimal gene set for a viable cell includes proteases and a nuclease, suggesting SAI is essential for life. SAI promotes genetic diversity in several ways. Toxin/antitoxin systems promote maintenance of genes, and SAI of mitochondria facilitates uniparental transmission. By creating two distinct states, subject to different selective pressures, SAI can maintain genetic diversity. SAI of components of synthetic replicators favors replicator cycling, promoting emergence of replicators with increased complexity. Both classical and recent computer modeling of replicators reveals SAI. SAI may be involved at additional levels of biological organization. In summary, SAI promotes replicator genetic diversity and reproductive fitness, and may promote aging through loss of resources and maintenance of deleterious alleles.

Conditional Inhibition of Eip75B Eliminates the Effects of Mating and Mifepristone on Lifespan in Female Drosophila.

Mating in female Drosophila melanogaster causes midgut hypertrophy and reduced lifespan, and these effects are blocked by the drug mifepristone. Eip75B is a transcription factor previously reported to have pleiotropic effects on Drosophila lifespan. Because Eip75B null mutations are lethal, conditional systems and/or partial knock-down are needed to study Eip75B effects in adults. Previous studies showed that Eip75B is required for adult midgut cell proliferation in response to mating. To test the possible role of Eip75B in mediating the lifespan effects of mating and mifepristone, a tripartite FLP-recombinase-based conditional system was employed that provides controls for genetic background. Expression of a Hsp70-FLP transgene was induced in third instar larvae by a brief heat pulse. The FLP recombinase catalyzed the recombination and activation of an Actin5C-GAL4 transgene. The GAL4 transcription factor in turn activated expression of a UAS-Eip75B-RNAi transgene. Inhibition of Eip75B activity was confirmed by loss of midgut hypertrophy upon mating, and the lifespan effects of both mating and mifepristone were eliminated. In addition, the negative effects of mifepristone on egg production were eliminated. The data indicate that Eip75B mediates the effects of mating and mifepristone on female midgut hypertrophy, egg production, and lifespan.

A conserved role for the 20S proteasome and Nrf2 transcription factor in oxidative stress adaptation in mammals, Caenorhabditis elegans and Drosophila melanogaster.

In mammalian cells, hydrogen peroxide (H(2)O(2))-induced adaptation to oxidative stress is strongly dependent on an Nrf2 transcription factor-mediated increase in the 20S proteasome. Here, we report that both Caenorhabditis elegans nematode worms and Drosophila melanogaster fruit flies are also capable of adapting to oxidative stress with H(2)O(2) pre-treatment. As in mammalian cells, this adaptive response in worms and flies involves an increase in proteolytic activity and increased expression of the 20S proteasome, but not of the 26S proteasome. We also found that the increase in 20S proteasome expression in both worms and flies, as in mammalian cells, is important for the adaptive response, and that it is mediated by the SKN-1 and CNC-C orthologs of the mammalian Nrf2 transcription factor, respectively. These studies demonstrate that stress mechanisms operative in cell culture also apply in disparate intact organisms across a wide biological diversity.

Markers and mechanisms of death in Drosophila.

Parameters correlated with age and mortality in Drosophila melanogaster include decreased negative geotaxis and centrophobism behaviors, decreased climbing and walking speed, and darkened pigments in oenocytes and eye. Cessation of egg laying predicts death within approximately 5 days. Endogenous green fluorescence in eye and body increases hours prior to death. Many flies exhibit erratic movement hours before death, often leading to falls. Loss of intestinal barrier integrity (IBI) is assayed by feeding blue dye ("Smurf" phenotype), and Smurf flies typically die within 0-48 h. Some studies report most flies exhibit Smurf, whereas multiple groups report most flies die without exhibiting Smurf. Transgenic reporters containing heat shock gene promoters and innate immune response gene promoters progressively increase expression with age, and partly predict remaining life span. Innate immune reporters increase with age in every fly, prior to any Smurf phenotype, in presence or absence of antibiotics. Many flies die on their side or supine (on their back) position. The data suggest three mechanisms for death of Drosophila. One is loss of IBI, as revealed by Smurf assay. The second is nervous system malfunction, leading to erratic behavior, locomotor malfunction, and falls. The aged fly is often unable to right itself after a fall to a side-ways or supine position, leading to inability to access the food and subsequent dehydration/starvation. Finally, some flies die upright without Smurf phenotype, suggesting a possible third mechanism. The frequency of these mechanisms varies between strains and culture conditions, which may affect efficacy of life span interventions.

Dhr96[1] mutation and maternal tudor[1] mutation increase life span and reduce the beneficial effects of mifepristone in mated female Drosophila.

Mating and receipt of male Sex Peptide hormone cause increased egg laying, increased midgut size and decreased life span in female Drosophila. Feeding mated females with the synthetic steroid mifepristone decreases egg production, reduces midgut size, and increases life span. Here, several gene mutations were assayed to investigate possible mechanisms for mifepristone action. Drosophila Dhr96 is a hormone receptor, and a key positive regulator of midgut lipid uptake and metabolism. Dhr96[1] null mutation increased female life span, and reduced the effects of mifepristone on life span, suggesting that Dhr96[1] mutation and mifepristone may act in part through the same mechanism. Consistent with this idea, lipidomics analysis revealed that mating increases whole-body levels of triglycerides and fatty-acids in triglycerides, and these changes are reversed by mifepristone. Maternal tudor[1] mutation results in females that lack the germ-line and produce no eggs. Maternal tudor[1] mutation increased mated female life span, and reduced but did not eliminate the effects of mating and mifepristone on life span. This indicates that decreased egg production may be related to the life span benefits of mifepristone, but is not essential. Mifepristone increases life span in w[1118] mutant mated females, but did not increase life span in w[1118] mutant virgin females. Mifepristone decreased egg production in w[1118] mutant virgin females, indicating that decreased egg production is not sufficient for mifepristone to increase life span. Mifepristone increases life span in virgin females of some, but not all, white[+] and mini-white[+] strains. Backcrossing of mini-white[+] transgenes into the w[1118] background was not sufficient to confer a life span response to mifepristone in virgin females. Taken together, the data support the hypothesis that mechanisms for mifepristone life span increase involve reduced lipid uptake and/or metabolism, and suggest that mifepristone may increase life span in mated females and virgin females through partly different mechanisms.

Increased internal and external bacterial load during Drosophila aging without life-span trade-off.

The role of microbial load during aging of the adult fruit fly Drosophila melanogaster is incompletely understood. Here we show dramatic increases in aerobic and anaerobic bacterial load during aging, both inside the body and on the surface. Scanning electron microscopy and cell staining analyses of the surface of aged flies detected structures resembling abundant small bacteria and bacterial biofilms. Bacteria cultured from laboratory flies included aerobic species Acetobacter aceti, Acetobacter tropicalis, and Acetobacter pasteurianus and anaerobic species Lactobacillus plantarum and Lactobacillus sp. MR-2; Lactobacillus homohiochii, Lactobacillus fructivorans, and Lactobacillus brevis were identified by DNA sequencing. Reducing bacterial load and antimicrobial peptide gene expression by axenic culture or antibiotics had no effect on life span. We conclude that Drosophila can tolerate a significant bacterial load and mount a large innate immune response without a detectable trade-off with life span; furthermore, microbes do not seem to limit life span under optimized laboratory conditions.

A screen of small molecule and genetic modulators of life span in female Drosophila identifies etomoxir, RH5849 and unanticipated temperature effects.

Mifepristone increases life span in female Drosophila melanogaster, and its molecular target(s) remain unclear. Here small molecule and genetic interventions were tested for ability to mimic mifepristone, or to decrease life span in a way that can be rescued by mifepristone. Etomoxir inhibits lipid metabolism, and significantly increased life span in virgin and mated females, but not males, at 50 µM concentration. Pioglitazone is reported to activate both mammalian PPARγ and its Drosophila homolog Eip75B. Pioglitazone produced minor and inconsistent benefits for female Drosophila life span, and only at the lowest concentrations tested. Ecdysone is a Drosophila steroid hormone reported to regulate responses to mating, and RH5849 is a potent mimic of ecdysone. RH5849 reduced virgin female life span, and this was partly rescued by mifepristone. Mifepristone did not compete with RH5849 for activation of an ecdysone receptor (EcR)-responsive transgenic reporter, indicating that the relevant target for mifepristone is not EcR. The conditional GAL4/GAL80ts system was used in attempt to test the effect of an Eip75B RNAi construct on female life span. However, the 29°C temperature used for induction reduced or eliminated mating-induced midgut hypertrophy, the negative life span effects of mating, and the positive life span effects of mifepristone. Even when applied after mating was complete, a shift to 29°C temperature reduced mating-induced midgut hypertrophy by half, and the life span effects of mating by 4.8-fold. Taken together, these results identify promising small molecules for further analysis, and inform the design of experiments involving the GAL4/GAL80ts system.

Mifepristone Increases Life Span in Female Drosophila Without Detectable Antibacterial Activity.

Mifepristone dramatically increases the life span of mated female Drosophila while reducing the expression of innate immune response genes. Previous results indicated that mifepristone also reduced the load of aero-tolerant bacteria in mated females. Experiments were conducted to further investigate the possible role of bacteria in mifepristone life span effects. Life span was assayed in flies grown from sterilized eggs on autoclaved media and in normally cultured controls in two independent assays. Sterilization increased mated female life span (+8.3% and +57%, respectively), and the effect of mifepristone was additive (+53% and +93%, respectively). High-throughput sequencing of 16S sequences revealed that sterilization reduced the abundance of multiple species and the classes Bacteroidia, Bacilli, Actinobacteria, and Cytophagia. By contrast, mifepristone caused no decreases and instead increased the abundance of three species. Five aero-tolerant bacterial species were cultured from extracts of mated female flies, including both Gram-positive and Gram-negative species (Acetobacter sicerae, Enterococcus faecalis, Lactobacillus plantarum, Serratia rubidea, and Paenibacillus glucanolyticus). There was no detectable effect of mifepristone on the growth of these bacteria in vitro, indicating that mifepristone does not have a direct antibiotic effect. To test if antibiotics could mimic the effects of mifepristone in vivo, mated female flies were treated throughout adult life span with high concentrations of the individual antibiotics doxycycline, ampicillin, kanamycin, and streptomycin, in replicate experiments. No significant effect on life span was observed for ampicillin, kanamycin, or streptomycin, and an inconsistent benefit was observed for doxycycline. Finally, supplementation of media with Enterococcus faecalis did not alter adult female life span in the presence or absence of mifepristone. Taken together, the results indicate the life span benefits of mifepristone are not due to an antibiotic effect.

The murmur of old broken heartstrings.

Although it has long been hypothesized that gene expression might become more variable or noisy during aging, direct evidence has been scarce so far. A new study reports detection, by PCR analysis of individual cells, of increased cell-to-cell variability in gene expression in aging mouse heart; moreover, a similar variability can be generated in cultured cells using oxidative stress (Bahar et al., 2006).

In vivo assay and modelling of protein and mitochondrial turnover during aging.

To maintain homoeostasis, cells must degrade damaged or misfolded proteins and synthesize functional replacements. Maintaining a balance between these processes, known as protein turnover, is necessary for stress response and cellular adaptation to a changing environment. Damaged mitochondria must also be removed and replaced. Changes in protein and mitochondrial turnover are associated with aging and neurodegenerative disease, making it important to understand how these processes occur and are regulated in cells. To achieve this, reliable assays of turnover must be developed. Several methods exist, including pulse-labelling with radioactive or stable isotopes and strategies making use of fluorescent proteins, each with their own advantages and limitations. Both cell culture and live animals have been used for these studies, in systems ranging from yeast to mammals. In vivo assays are especially useful for connecting turnover to aging and disease. With its short life cycle, suitability for fluorescent imaging, and availability of genetic tools, Drosophila melanogaster is particularly well suited for this kind of analysis.

Incorporating antagonistic pleiotropy into models for molecular replicators.

In modern cells, chromosomal genes composed of DNA encode multi-subunit protein/RNA complexes that catalyze the replication of the chromosome and cell. One prevailing theory for the origin of life posits an early stage involving self-replicating macromolecules called replicators, which can be considered genes capable of self-replication. One prevailing theory for the genetics of aging in humans and other organisms is antagonistic pleiotropy, which posits that a gene can be beneficial in one context, and detrimental in another context. We previously reported that the conceptual simplicity of molecular replicators facilitates the generation of two simple models involving antagonistic pleiotropy. Here a third model is proposed, and each of the three models is presented with improved definition of the time variable. Computer simulations were used to calculate the proliferation of a hypothetical two-subunit replicator (AB), when one of the two subunits (B) exhibits antagonistic pleiotropy, leading to an advantage for B to be unstable. In model 1, instability of B yields free A subunits, which in turn stimulate the activity of other AB replicators. In model 2, B is lost and sometimes replaced by a more active mutant form, B'. In model 3, B becomes damaged and loses activity, and its instability allows it to be replaced by a new B. For each model, conditions were identified where instability of B was detrimental, and where instability of B was beneficial. The results are consistent with the hypothesis that antagonistic pleiotropy can promote molecular instability and system complexity, and provide further support for a model linking aging and evolution.

Mifepristone Increases Life Span of Virgin Female Drosophila on Regular and High-fat Diet Without Reducing Food Intake.

Background: The synthetic steroid mifepristone is reported to have anti-obesity and anti-diabetic effects in mammals on normal and high-fat diets (HFD). We previously reported that mifepristone blocks the negative effect on life span caused by mating in female Drosophila melanogaster. Methods: Here we asked if mifepristone could protect virgin females from the life span-shortening effect of HFD. Mifepristone was assayed for effects on life span in virgin females, in repeated assays, on regular media and on media supplemented with coconut oil (HFD). The excrement quantification (EX-Q) assay was used to measure food intake of the flies after 12 days mifepristone treatment. In addition, experiments were conducted to compare the effects of mifepristone in virgin and mated females, and to identify candidate mifepristone targets and mechanisms. Results: Mifepristone increased life span of virgin females on regular media, as well as on media supplemented with either 2.5 or 5% coconut oil. Food intake was not reduced in any assay, and was significantly increased by mifepristone in half of the assays. To ask if mifepristone might rescue virgin females from all life span-shortening stresses, the oxidative stressor paraquat was tested, and mifepristone produced little to no rescue. Analysis of extant metabolomics and transcriptomics data suggested similarities between effects of mifepristone in virgin and mated females, including reduced tryptophan breakdown and similarities to dietary restriction. Bioinformatics analysis identified candidate mifepristone targets, including transcription factors Paired and Extra-extra. In addition to shortening life span, mating also causes midgut hypertrophy and activation of the lipid metabolism regulatory factor SREBP. Mifepristone blocked the increase in midgut size caused by mating, but did not detectably affect midgut size in virgins. Finally, mating increased activity of a SREBP reporter in abdominal tissues, as expected, but reporter activity was not detectably reduced by mifepristone in either mated or virgin females. Conclusion: Mifepristone increases life span of virgin females on regular and HFD without reducing food intake. Metabolomics and transcriptomics analyses suggest some similar effects of mifepristone between virgin and mated females, however reduced midgut size was observed only in mated females. The results are discussed regarding possible mifepristone mechanisms and targets.

Metabolic Signatures of Life Span Regulated by Mating, Sex Peptide, and Mifepristone/RU486 in Female Drosophila melanogaster.

Mating and transfer of male sex peptide (SP), or transgenic expression of SP, causes inflammation and decreased life span in female Drosophila. Mifepristone rescues these effects, yielding dramatic increases in life span. Here targeted metabolomics data were integrated with further analysis of extant transcriptomic data. Each of 7 genes positively correlated with life span were expressed in the brain or eye and involved regulation of gene expression and signaling. Genes negatively correlated with life span were preferentially expressed in midgut and involved protein degradation, amino acid metabolism, and immune response. Across all conditions, life span was positively correlated with muscle breakdown product 1/3-methylhistidine and purine breakdown product urate, and negatively correlated with tryptophan breakdown product kynurenic acid, suggesting a SP-induced shift from somatic maintenance/turnover pathways to the costly production of energy and lipids from dietary amino acids. Some limited overlap was observed between genes regulated by mifepristone and genes known to be regulated by ecdysone; however, mifepristone was unable to compete with ecdysone for activation of an ecdysone-responsive transgenic reporter. In contrast, genes regulated by mifepristone were highly enriched for genes regulated by juvenile hormone (JH), and mifepristone rescued the negative effect of JH analog methoprene on life span in adult virgin females. The data indicate that mifepristone increases life span and decreases inflammation in mated females by antagonizing JH signaling downstream of male SP. Finally, mifepristone increased life span of mated, but not unmated, Caenorhabditis elegans, in 2 of 3 trials, suggesting possible evolutionary conservation of mifepristone mechanisms.