Placental function and homeostasis is very much dependent upon trophoblast mitochondria. Mitochondria are responsible for a myriad of functions including energy production, endocrine functions, nutrient and oxygen transfer as well as protecting the placenta from oxidative insult and cell stress. The materno-fetal interface is comprised of 2 related cell lineages, cytotrophoblast cells which fuse to form the overlaying syncytium. Mitochondrial morphology and function in these 2 lineages is different and, in this study, we describe methods for mitochondrial isolation, cryopreservation and preliminary biochemical characterisation of these discrete forms of mitochondria. Villous tissue was collected from normal term placentae following vaginal delivery (n=7). Differential centrifugation was used to isolate 2 fractions, larger mitochondria predominantly from the cytotrophoblast cells and smaller mitochondria from the syncytiotrophoblast. Freshly isolated mitochondria were assessed for Complex I and Complex II respiration and maximal respiratory capacity and these were compared to equivalent isolates that had been frozen, stored at-80 o C and revived. Other important functional parameters of mitochondrial activity were also assessed post cryopreservation including membrane potential, ATP production, progesterone biosynthesis and anti-oxidant expression. The methods described resulted in 2 mitochondrial populations which match the types of mitochondria observed in situ with election microscopy. Both forms of mitochondria were metabolically active and recovered well from cryopreservation. There were important functional differences between cyto-mitochondria and syncytio-mitochondria including decreased respiration, decreased oxidative phosphorylation, decreased anti-oxidant expression but increased expression of the enzyme CYP11A1 and increased progesterone production. This study highlights the need to consider different types of trophoblast mitochondria in placental studies and provides methods to facilitate this.

The aim of this study was to identify markers of training stress and characteristics of middle-distance runners related to the incidence of overreaching following overload training. Twenty-four highly-trained runners (n=16 male; VO2peak=73.3(4.3) mL·kg·min-1; n=8 female, VO2peak=63.2(3.4) mL·kg·min-1) completed 3 weeks of normal training (NormTr), 3 weeks of high-volume training (HVTr; a 10, 20 and 30% increase in training volume each successive week from NormTr), and a 1-week taper (TapTr; 55% exponential reduction in training volume from HVTr week 3). Before, and immediately after each training period, an incremental treadmill-running test was performed, while resting metabolic rate (RMR), subjective fatigue responses and various resting blood biomarkers were assessed. Muscle fiber typology of the gastrocnemius was estimated by quantification of muscle carnosine using proton magnetic resonance spectroscopy and expressed as a z-score relative to a non-athlete control group. Twelve runners were classified as functionally overreached (FOR) following HVTr (decreased running TTE), whereas the other twelve were classified as acutely fatigued (AF; no decrease in running TTE). The FOR group did not demonstrate systematic alterations in RMR, resting blood biomarkers or submaximal exercise responses compared to the AF group. Gastrocnemius carnosine z-score was significantly higher in FOR (-0.44 ± 0.57) compared to AF (-1.25 ± 0.49, p = 0.004, d = 1.53) and was also associated with changes in running TTE from pre- to post-HVTr (r=-0.55, p=0.005) and pre-HVTr to post-TapTr (r=-0.64, p=0.008). Muscle fiber typology is related to the incidence of overreaching and performance super-compensation following increased training volume and a taper.

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This clinical trial (ACTRN12619001296123) investigated the impact of silymarin (Legalon®) on circulating bilirubin concentration, lipid status, systemic inflammation, and antioxidant status. The study design was a randomized, placebo‐controlled, single‐blind crossover trial of healthy men (18‐65 years), conducted at Griffith University, Gold Coast, Australia. Participants were recruited from Griffith University and were randomized to silymarin (140 mg silymarin capsules thrice daily) or placebo (3 capsules containing mannitol taken daily) for 14 days followed by a ≥4‐week washout and crossover to the other arm. The main outcomes were whether silymarin treatment would increase serum bilirubin concentration by >0.29 mg/dL, change serum lipid status (cholesterol and triglycerides), inflammation (c‐reactive protein), and antioxidant capacity (ferric reducing ability of plasma) compared with baseline. Silymarin consumption (n = 17) did not affect serum concentrations of unconjugated bilirubin (0.73 versus 0.67 mg/dL, P = .79), cholesterol (185 versus 189 mg/dL, P = .19), triglycerides (94.2 versus 92.3 mg/dL, P = .79), c‐reactive protein (0.17 versus 0.09 mg/dL, P = .23), or antioxidant status (6.61 versus 6.67 mg Fe2+/dL, P = .40). These findings challenge previous reports and manufacturer claims of hyperbilirubinemia following silymarin treatment and are critical to guiding researchers toward an effective means to mildly elevate bilirubin, which evidence suggests could protect from cardiovascular disease.

Background Bilirubin, a by-product of haem catabolism, possesses potent endogenous antioxidant and platelet inhibitory properties. These properties may be useful in inhibiting inappropriate platelet activation and ROS production; for example, during storage for transfusion. Given the hydrophobicity of unconjugated bilirubin (UCB), we investigated the acute platelet inhibitory and ROS scavenging ability of a water-soluble bilirubin analogue, bilirubin ditaurate (BRT) on ex vivo platelet function to ascertain its potential suitability for inclusion during platelet storage. Methods The inhibitory potential of BRT (10–100 μM) was assessed using agonist induced platelet aggregation, dense granule exocytosis and flow cytometric analysis of P-selectin and GPIIb/IIIa expression. ROS production was investigated by analysis of H 2 DCFDA fluorescence following agonist simulation while mitochondrial ROS production investigated using MitoSOX™ Red. Platelet mitochondrial membrane potential and viability was assessed using TMRE and Zombie Green™ respectively. Results Our data shows ≤35 μM BRT significantly inhibits both dense and alpha granule exocytosis as measured by ATP release and P-selectin surface expression, respectively. Significant inhibition of GPIIb/IIIa expression was also reported upon ≤35 μM BRT exposure. Furthermore, platelet exposure to ≤10 μM BRT significantly reduces platelet mitochondrial ROS production. Despite the inhibitory effect of BRT, platelet viability, mitochondrial membrane potential and agonist induced aggregation were not perturbed. Conclusions These data indicate, for the first time, that BRT, a water-soluble bilirubin analogue, inhibits platelet activation and reduces platelet ROS production ex vivo and may, therefore, may be of use in preserving platelet function during storage.