Moderate dietary restriction delays the onset of age-associated sarcopenia in Caenorhabditis elegans due to reduced myosin UNC-54 degradation.

Sarcopenia, a gradual decrease in skeletal muscle mass and strength, is a major component of frailty in the elderly, with age, (lack of) exercise and diet found to be the major risk factors. The nematode Caenorhabditis elegans is an important model of sarcopenia. Although many studies describe loss of muscle function in ageing C. elegans, surprisingly few report on the loss of muscle mass. Here, in order to quantify loss of muscle mass under various dietary restriction (DR) conditions, we used an internal GFP standard to determine levels of the major body wall muscle myosin (UNC-54) in transgenic unc-54::gfp worms over their lifespan. Myosin density linearly increased during the first week of adulthood and there was no significant effect of DR. In contrast, an exponential decrease in myosin density was seen during the second week of adulthood, with reduced rates of myosin loss for mild and medium DR compared to control. UNC-54 turnover rates, previously determined using pulse-labelling methods, correspond well with the t1/2 value found here for UNC-54-GFP using fluorescence (control t1/2 = 12.0 days), independently validating our approach. These data indicate that sarcopenia is delayed in worms under mild and medium DR due to a reduced rate of myosin UNC-54 degradation, thereby maintaining protein homeostasis.

Supplementation of porcine in vitro maturation medium with FGF2, LIF, and IGF1 enhances cytoplasmic maturation in prepubertal gilts oocytes and improves embryo quality.

In porcine in vitro production (IVP) systems, the use of oocytes derived from prepubertal gilts, whilst being commercially attractive, remains challenging due to their poor developmental competence following in vitro maturation (IVM). Follicular fluid contains important growth factors and plays a key role during oocyte maturation; therefore, it is a common supplementation for porcine IVM medium. However, follicular fluid contains many poorly characterized components, is batch variable, and its use raises biosecurity concerns. In an effort to design a defined IVM system, growth factors such as cytokines have been previously tested. These include leukaemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1), the combination of which is termed 'FLI'. Here, using abattoir-derived oocytes in a well established porcine IVP system, we compared follicular fluid and FLI supplementation during both IVM and embryo culture to test the hypothesis that FLI can substitute for follicular fluid without compromising oocyte nuclear and cytoplasmic maturation. We demonstrate that in oocytes derived from prepubertal gilts, FLI supplementation enhances oocyte meiotic maturation and has a positive effect on the quality and developmental competence of embryos. Moreover, for the first time, we studied the effects of follicular fluid and FLI combined showing no synergistic effects.

Synthesis of Gold Functionalised Nanoparticles with the Eranthis hyemalis Lectin and Preliminary Toxicological Studies on Caenorhabditis elegans.

The lectin found in the tubers of the Winter Aconite (Eranthis hyemalis) plant (EHL) is a Type II Ribosome Inactivating Protein (RIP). Type II RIPs have shown anti-cancer properties and have great potential as therapeutic agents. Similarly, colloidal gold nanoparticles are successfully used in biomedical applications as they can be functionalised with ligands with high affinity and specificity for target cells to create therapeutic and imaging agents. Here we present the synthesis and characterization of gold nanoparticles conjugated with EHL and the results of a set of initial assays to establish whether the biological effect of EHL is altered by the conjugation. Gold nanoparticles functionalised with EHL (AuNPs@EHL) were successfully synthesised by bioconjugation with citrate gold nanoparticles (AuNPs@Citrate). The conjugates were analysed by UV-Vis spectroscopy, Dynamic Light Scattering (DLS), Zeta Potential analysis, and Transmission Electron Microscopy (TEM). Results indicate that an optimal functionalisation was achieved with the addition of 100 µL of EHL (concentration 1090 ± 40 µg/mL) over 5 mL of AuNPs (concentration [Au°] = 0.8 mM). Biological assays on the effect of AuNPs@EHL were undertaken on Caenorhabditis elegans, a free-living nematode commonly used for toxicological studies, that has previously been shown to be strongly affected by EHL. Citrate gold nanoparticles did not have any obvious effect on the nematodes. For first larval stage (L1) nematodes, AuNPs@EHL showed a lower biological effect than EHL. For L4 stage, pre-adult nematodes, both EHL alone and AuNPs@EHL delayed the onset of reproduction and reduced fecundity. These assays indicate that EHL can be conjugated to gold nanoparticles and retain elements of biocidal activity.

Winter Aconite (Eranthis hyemalis) Lectin as a cytotoxic effector in the lifecycle of Caenorhabditis elegans.

The lectin found in the tubers of the Winter Aconite (Eranthis hyemalis) plant is an N-acetyl-D-galactosamine specific Type II Ribosome Inactivating Protein (RIP); Type II RIPs have shown anti-cancer properties, and hence have potential as therapeutic agents. Here we present a modified protocol for the extraction and purification of the E. hyemalis lectin (EHL) using affinity chromatography. De novo amino acid sequencing of EHL confirms its classification as a Type II Ribosome Inactivating Protein. The biocidal properties of EHL have been investigated against the nematode Caenorhabditis elegans. Arrested first stage larvae treated with EHL have shown some direct mortality, with surviving larvae subsequently showing a range of phenotypes including food avoidance, reduced fecundity, developmental delay and constitutive dauer larvae formation. Both inappropriate dauer larvae development and failure to locate to bacterial food source are consistent with the disruption of chemosensory function and the ablation of amphid neurons. Further investigation indicates that mutations that disrupt normal amphid formation can block the EHL-induced dauer larvae formation. In combination, these phenotypes indicate that EHL is cytotoxic and suggest a cell specific activity against the amphid neurons of C. elegans.