Equisetum arvense standardized dried extract hinders age-related osteosarcopenia.

Age-associated osteosarcopenia is an unresolved syndrome characterized by the concomitant loss of bone (osteopenia) and skeletal muscle (sarcopenia) tissues increasing falls, immobility, morbidity, and mortality. Unbalanced resorption of bone in the remodeling process and excessive protein breakdown, especially fast type II myosin heavy chain (MyHC-II) isoform and myofiber metabolic shift, are the leading causes of bone and muscle deterioration in the elderly, respectively. Equisetum arvense (EQ) is a plant traditionally recommended for many pathological conditions due to its anti-inflammatory properties. Thus, considering that a chronic low-grade inflammatory state predisposes to both osteoporosis and sarcopenia, we tested a standardized hydroalcoholic extract of EQ in in vitro models of muscle atrophy [C2C12 myotubes treated with proinflammatory cytokines (TNFα/IFNγ), excess glucocorticoids (dexamethasone), or the osteokine, receptor activator of nuclear factor kappa-B ligand (RANKL)] and osteoclastogenesis (RAW 264.7 cells treated with RANKL). We found that EQ counteracted myotube atrophy, blunting the activity of several pathways depending on the applied stimulus, and reduced osteoclast formation and activity. By in silico target fishing, IKKB-dependent nuclear factor kappa-B (NF-κB) inhibition emerges as a potential common mechanism underlying EQ's anti-atrophic effects. Consumption of EQ (500 mg/kg/day) by pre-geriatric C57BL/6 mice for 3 months translated into: i) maintenance of muscle mass and performance; ii) restrained myofiber oxidative shift; iii) slowed down age-related modifications in osteoporotic bone, significantly preserving trabecular connectivity density; iv) reduced muscle- and spleen-related inflammation. EQ can preserve muscle functionality and bone remodeling during aging, potentially valuable as a natural treatment for osteosarcopenia.

KYMASIN UP Natural Product Inhibits Osteoclastogenesis and Improves Osteoblast Activity by Modulating Src and p38 MAPK.

The imbalance in osteoblast (OB)-dependent bone formation in favor of osteoclast (OC)-dependent bone resorption is the main cause of loss of tissue mineral mass during bone remodeling leading to osteoporosis conditions. Thus, the suppression of OC activity together with the improvement in the OB activity has been proposed as an effective therapy for maintaining bone mass during aging. We tested the new dietary product, KYMASIN UP containing standardized Withania somnifera, Silybum marianum and Trigonella foenum-graecum herbal extracts or the single extracts in in vitro models mimicking osteoclastogenesis (i.e., RAW 264.7 cells treated with RANKL, receptor activator of nuclear factor kappa-Β ligand) and OB differentiation (i.e., C2C12 myoblasts treated with BMP2, bone morphogenetic protein 2). We found that the dietary product reduces RANKL-dependent TRAP (tartrate-resistant acid phosphatase)-positive cells (i.e., OCs) formation and TRAP activity, and down-regulates osteoclastogenic markers by reducing Src (non-receptor tyrosine kinase) and p38 MAPK (mitogen-activated protein kinase) activation. Withania somnifera appears as the main extract responsible for the anti-osteoclastogenic effect of the product. Moreover, KYMASIN UP maintains a physiological release of the soluble decoy receptor for RANKL, OPG (osteoprotegerin), in osteoporotic conditions and increases calcium mineralization in C2C12-derived OBs. Interestingly, KYMASIN UP induces differentiation in human primary OB-like cells derived from osteoporotic subjects. Based on our results, KYMASIN UP or Withania somnifera-based dietary supplements might be suggested to reverse the age-related functional decline of bone tissue by re-balancing the activity of OBs and OCs, thus improving the quality of life in the elderly and reducing social and health-care costs.

Responses to Developmental Temperature Fluctuation in Life History Traits of Five Drosophila Species (Diptera: Drosophilidae) from Different Thermal Niches.

Temperature has profound effects on biochemical processes as suggested by the extensive variation in performance of organisms across temperatures. Nonetheless, the use of fluctuating temperature (FT) regimes in laboratory experiments compared to constant temperature (CT) regimes is still mainly applied in studies of model organisms. We investigated how two amplitudes of developmental temperature fluctuation (22.5/27.5 °C and 20/30 °C, 12/12 h) affected several fitness-related traits in five Drosophila species with markedly different thermal resistance. Egg-to-adult viability did not change much with temperature except in the cold-adapted D. immigrans. Developmental time increased with FT among all species compared to the same mean CT. The impact of FT on wing size was quite diverse among species. Whereas wing size decreased quasi-linearly with CT in all species, there were large qualitative differences with FT. Changes in wing aspect ratio due to FT were large compared to the other traits and presumably a consequence of thermal stress. These results demonstrate that species of the same genus but with different thermal resistance can show substantial differences in responses to fluctuating developmental temperatures not predictable by constant developmental temperatures. Testing multiple traits facilitated the interpretation of responses to FT in a broader context.

Daily increasing or decreasing photoperiod affects stress resistance and life history traits in four Drosophila species.

Photoperiod is one of the most reliable seasonal cues that organisms can use to prepare for upcoming environmental changes. Evidence suggests that exposure to different photoperiod can activate plastic responses in stress resistance traits, while there is limited evidence on the plastic response induced by daily progressive cumulative changes in photoperiod. In this study, we assayed the effect of within generation daily uni-directional and cumulative changes in photoperiod on stress resistance and life history traits in four Drosophila species. We predicted that daily increasing photoperiod, mimicking upcoming summer conditions, should lead to an increase in heat resistance and establish trade-offs with other fitness related traits. On the other hand, we predicted that daily decreasing photoperiod should reflect upcoming winter conditions leading to an increase in cold resistance. We found that within genreation changes in photoperiod had a significant effect on life history and stress resistance traits in the four Drosophila species. The observed response was different across species, with D. melanogaster showing five out of six studied traits affected, while in D. mercatorum only one trait was significantly affected. The exposure to changing photoperiod led to an increased upper thermal resistance in D. melanogaster and D. mercatorum and a decreased lower thermal resistance in D. melanogaster and D. simulans, as well as a decreased starvation and desiccation resistance in D. virilis. The developmental time was shorter when flies were exposed to the two photoperiod regimes compared to constant day length control in D. melanogaster and D. simulans. A limited effect was observed on egg-to-adult-viability and desiccation resistance. The results of this study show that daily change in photoperiod induced a plastic response in different traits of drosophilids, suggesting that this environmental parameter needs to be carefully considered in evolutionary studies.

Identification of Withania somnifera-Silybum marianum-Trigonella foenum-graecum Formulation as a Nutritional Supplement to Contrast Muscle Atrophy and Sarcopenia.

Background: Muscle atrophy, i.e., the loss of skeletal muscle mass and function, is an unresolved problem associated with aging (sarcopenia) and several pathological conditions. The imbalance between myofibrillary protein breakdown (especially the adult isoforms of myosin heavy chain, MyHC) and synthesis, and the reduction of muscle regenerative potential are main causes of muscle atrophy. Methods: Starting from one-hundred dried hydroalcoholic extracts of medical plants, we identified those able to contrast the reduction of C2C12 myotube diameter in well-characterized in vitro models mimicking muscle atrophy associated to inflammatory states, glucocorticoid treatment or nutrient deprivation. Based on their ability to rescue type II MyHC (MyHC-II) expression in atrophying conditions, six extracts with different phytochemical profiles were selected, mixed in groups of three, and tested on atrophic myotubes. The molecular mechanism underpinning the effects of the most efficacious formulation, and its efficacy on myotubes obtained from muscle biopsies of young and sarcopenic subjects were also investigated. Results: We identified WST (Withania somnifera, Silybum marianum, Trigonella foenum-graecum) formulation as extremely efficacious in protecting C2C12 myotubes against MyHC-II degradation by stimulating Akt (protein kinase B)-dependent protein synthesis and p38 MAPK (p38 mitogen-activated protein kinase)/myogenin-dependent myoblast differentiation. WST sustains trophism in C2C12 and young myotubes, and rescues the size, developmental MyHC expression and myoblast fusion in sarcopenic myotubes. Conclusion: WST strongly counteracts muscle atrophy associated to different conditions in vitro. The future validation in vivo of our results might lead to the use of WST as a food supplement to sustain muscle mass in diffuse atrophying conditions, and to reverse the age-related functional decline of human muscles, thus improving people quality of life and reducing social and health-care costs.

Pronounced Plastic and Evolutionary Responses to Unpredictable Thermal Fluctuations in Drosophila simulans.

Organisms are exposed to temperatures that vary, for example on diurnal and seasonal time scales. Thus, the ability to behaviorally and/or physiologically respond to variation in temperatures is a fundamental requirement for long-term persistence. Studies on thermal biology in ectotherms are typically performed under constant laboratory conditions, which differ markedly from the variation in temperature across time and space in nature. Here, we investigate evolutionary adaptation and environmentally induced plastic responses of Drosophila simulans to no fluctuations (constant), predictable fluctuations or unpredictable fluctuations in temperature. We whole-genome sequenced populations exposed to 20 generations of experimental evolution under the three thermal regimes and examined the proteome after short-term exposure to the same three regimes. We find that unpredictable fluctuations cause the strongest response at both genome and proteome levels. The loci showing evolutionary responses were generally unique to each thermal regime, but a minor overlap suggests either common laboratory adaptation or that some loci were involved in the adaptation to multiple thermal regimes. The evolutionary response, i.e., loci under selection, did not coincide with induced responses of the proteome. Thus, genes under selection in fluctuating thermal environments are distinct from genes important for the adaptive plastic response observed within a generation. This information is key to obtain a better understanding and prediction of the effects of future increases in both mean and variability of temperatures.

How much starvation, desiccation and oxygen depletion can Drosophila melanogaster tolerate before its upper thermal limits are affected?

Heat tolerance is commonly assessed as the critical thermal maximum (CTmax) using the dynamic method exposing organisms to a gradually increasing (ramping) temperature until organisms fall into a coma. The CTmax estimate is dependent on the ramping rate, with decreased rates leading to longer treatments and ultimately lower CTmax estimates. There is a current discussion surrounding the physiological dynamics of the effect of the time of exposure by temperature interaction on these estimates. Besides temperature the time of exposure to limited food (starvation), desiccation, and reduced levels of oxygen or increased levels of CO2 may, in interaction with ramping rate, act as confounding factors when assessing upper thermal limits using the dynamic method. Here we test the role of the different potentially confounding factors for assaying thermal tolerance using a ramping assay under four different ramping rates, varying from 0.01 °C/min to 0.2 °C/min. We find that CTmax values are higher at faster ramping rates and that oxygen or CO2 concentration does not show any negative effect on the CTmax values obtained within the experimental pre-treatment period (32 h). Both water (up to 6 h) and food (up to 42 h) deprivation prior to assay showed a negative correlation with thermal tolerance of the flies. For both traits, we found a significant interaction with ramping rate, most likely due to prolonged assays at lower rates. However, as little water was lost during the ramping assay and as food deprivation only modestly affected CTmax values, results were very robust to the conditions experienced during the assay (even at slow rates) and mainly affected by the conditions experienced prior to performing the assay. Thus, for the most commonly applied experimental conditions CTmax estimates are unlikely to be biased or confounded by ramping rate, starvation, desiccation or deteriorating atmospheric composition.

Constitutive up-regulation of Turandot genes rather than changes in acclimation ability is associated with the evolutionary adaptation to temperature fluctuations in Drosophila simulans.

Most research on thermal adaptation of ectotherms is based on experiments performed at constant temperatures. However, for short-lived insects daily fluctuations of temperature could be an important environmental parameter involved in evolutionary adaptation to thermal heterogeneity. In this study we investigated the mechanisms underlying evolutionary adaptation to daily fluctuating temperatures. We studied replicated selection lines of Drosophila simulans evolved in a constant or a daily fluctuating thermal regime. Previous studies of these lines have shown clear acclimation benefits to heat tolerance induced by the fluctuating regime. First, we tested the existence of an evolved circadian controlled adjustment of heat resistance in selected flies. This was done by investigating the daily variation in time to heat knockdown in flies from both selection regimes when exposed to either a constant or a daily fluctuating thermal regime for a single generation. While daily variation in heat resistance was found, the results suggest that there was neither an evolved adaptive circadian controlled adjustment of heat resistance nor a continuous acclimation response induced by fluctuating temperatures in these lines. Second, in order to reveal functional candidates for adaptation to the fluctuating thermal regime, we investigated the global transcriptomic response to a high temperature exposure in flies from both regimes. We found that flies selected both in constant and fluctuating thermal regimes responded similarly to increasing temperature. However, we found that evolutionary adaptation to the fluctuating thermal regime led to transcriptional enrichment of the GO terms eggshell chorion assembly and cellular response to heat. The latter category was constituted by a constitutive up-regulation of four Turandot genes and not heat shock protein genes, suggesting that Turandot genes could play a prominent role for adaptation to daily fluctuating thermal conditions.

Genetic rescue of an endangered domestic animal through outcrossing with closely related breeds: A case study of the Norwegian Lundehund.

Genetic rescue, outcrossing with individuals from a related population, is used to augment genetic diversity in populations threatened by severe inbreeding and extinction. The endangered Norwegian Lundehund dog underwent at least two severe bottlenecks in the 1940s and 1960s that each left only five inbred dogs, and the approximately 1500 dogs remaining world-wide today appear to descend from only two individuals. The Lundehund has a high prevalence of a gastrointestinal disease, to which all remaining dogs may be predisposed. Outcrossing is currently performed with three Nordic Spitz breeds: Norwegian Buhund, Icelandic Sheepdog, and Norrbottenspets. Examination of single nucleotide polymorphism (SNP) genotypes based on 165K loci in 48 dogs from the four breeds revealed substantially lower genetic diversity for the Lundehund (HE 0.035) than for other breeds (HE 0.209-0.284). Analyses of genetic structure with > 15K linkage disequilibrium-pruned SNPs showed four distinct genetic clusters. Pairwise FST values between Lundehund and the candidate breeds were highest for Icelandic Sheepdog, followed by Buhund and Norrbottenspets. We assessed the presence of outlier loci among candidate breeds and examined flanking genome regions (1 megabase) for genes under possible selection to identify potential adaptive differences among breeds; outliers were observed in flanking regions of genes associated with key functions including the immune system, metabolism, cognition and physical development. We suggest crossbreeding with multiple breeds as the best strategy to increase genetic diversity for the Lundehund and to reduce the incidence of health problems. For this project, the three candidate breeds were first selected based on phenotypes and then subjected to genetic investigation. Because phenotypes are often paramount for domestic breed owners, such a strategy could provide a helpful approach for genetic rescue and restoration of other domestic populations at risk, by ensuring the involvement of owners, breeders and managers at the start of the project.

Environmental heterogeneity does not affect levels of phenotypic plasticity in natural populations of three Drosophila species.

Adaptation of natural populations to variable environmental conditions may occur by changes in trait means and/or in the levels of plasticity. Theory predicts that environmental heterogeneity favors plasticity of adaptive traits. Here we investigated the performance in several traits of three sympatric Drosophila species freshly collected in two environments that differ in the heterogeneity of environmental conditions. Differences in trait means within species were found in several traits, indicating that populations differed in their evolutionary response to the environmental conditions of their origin. Different species showed distinct adaptation with a very different role of plasticity across species for coping with environmental changes. However, geographically distinct populations of the same species generally displayed the same levels of plasticity as induced by fluctuating thermal regimes. This indicates a weak and trait-specific effect of environmental heterogeneity on plasticity. Furthermore, similar levels of plasticity were found in a laboratory-adapted population of Drosophila melanogaster with a common geographic origin but adapted to the laboratory conditions for more than 100 generations. Thus, this study does not confirm theoretical predictions on the degree of adaptive plasticity among populations in relation to environmental heterogeneity but shows a very distinct role of species-specific plasticity.

Phospholipid fatty acid composition linking larval-density to lifespan of adult Drosophila melanogaster.

Pre-adult density-associated alterations in the composition of storage lipids may affect the cell membrane fatty acid profile (mainly phospholipids), membrane integrity, and cell function. The present study evaluated the impact of pre-adult density conditions, sex, and the selection regime on the composition of phospholipid fatty acids and lifespan of Drosophila melanogaster. The phospholipid profile of adult flies developed under larval crowding contained a higher proportion of polyunsaturated fatty acids, lower proportion of monounsaturated fatty acids, and greater risk of peroxidation. There was also a negative correlation between the peroxidation index (PI) and longevity. The longevity-selected females showed a lower PI compared with control lines under both densities. The present results indicate that pre-adult density may play a significant role in the lifespan of adult flies by altering the composition of phospholipids and shaping cell membrane bilayers with different susceptibilities to peroxidation.

The Role of Storage Lipids in the Relation between Fecundity, Locomotor Activity, and Lifespan of Drosophila melanogaster Longevity-Selected and Control Lines.

The contribution of insect fat body to multiple processes, such as development, metamorphosis, activity, and reproduction results in trade-offs between life history traits. In the present study, age-induced modulation of storage lipid composition in Drosophila melanogaster longevity-selected (L) and non-selected control (C) lines was studied and the correlation between total body fat mass and lifespan assessed. The trade-offs between fecundity, locomotor activity, and lifespan were re-evaluated from a lipid-related metabolic perspective. Fewer storage lipids in the L lines compared to the C lines supports the impact of body fat mass on extended lifespan. The higher rate of fecundity and locomotor activity in the L lines may increase the lipid metabolism and enhance the lipolysis of storage lipids, reducing fat reserves. The correlation between neutral lipid fatty acids and fecundity, as well as locomotor activity, varied across age groups and between the L and C lines. The fatty acids that correlated with egg production were different from the fatty acids that correlated with locomotor activity. The present study suggests that fecundity and locomotor activity may positively affect the lifespan of D. melanogaster through the inhibition of fat accumulation.

Inbreeding affects locomotor activity in Drosophila melanogaster at different ages.

The ability to move is essential for many behavioural traits closely related to fitness. Here we studied the effect of inbreeding on locomotor activity (LA) of Drosophila melanogaster at different ages under both dark and light regimes. We expected to find a decreased LA in inbred lines compared to control lines. We also predicted an increased differentiation between lines due to inbreeding. LA was higher in the dark compared to the light regime for both inbred and outbred control lines. As expected, inbreeding increased phenotypic variance in LA, with some inbred lines showing higher and some lower LA than control lines. Moreover, age per se did not affect LA neither in control nor in inbred lines, while we found a strong line by age interaction between inbred lines. Interestingly, inbreeding changed the daily activity pattern of the flies: these patterns were consistent across all control lines but were lost in some inbred lines. The departure in the daily pattern of LA in inbred lines may contribute to the inbreeding depression observed in inbred natural populations.