Sex differences in leucocyte telomere length in a free-living mammal.

Mounting evidence suggests that average telomere length reflects previous stress and predicts subsequent survival across vertebrate species. In humans, leucocyte telomere length (LTL) is consistently shorter during adulthood in males than in females, although the causes of this sex difference and its generality to other mammals remain unknown. Here, we measured LTL in a cross-sectional sample of free-living Soay sheep and found shorter telomeres in males than in females in later adulthood (>3 years of age), but not in early life. This observation was not related to sex differences in growth or parasite burden, but we did find evidence for reduced LTL associated with increased horn growth in early life in males. Variation in LTL was independent of variation in the proportions of different leucocyte cell types, which are known to differ in telomere length. Our results provide the first evidence of sex differences in LTL from a wild mammal, but longitudinal studies are now required to determine whether telomere attrition rates or selective disappearance are responsible for these observed differences.

Proteome turnover in the green alga Ostreococcus tauri by time course 15N metabolic labeling mass spectrometry.

Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters; however, recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast, and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with (15)N. We applied it in a study of the unicellular pico-alga Ostreococcus tauri and observed high relative turnover in chloroplast-encoded ATPases (0.42-0.58% h(-1)), core photosystem II proteins (0.34-0.51% h(-1)), and RbcL (0.47% h(-1)), while nuclear-encoded RbcS2 is more stable (0.23% h(-1)). Mitochondrial targeted ATPases (0.14-0.16% h(-1)), photosystem antennae (0.09-0.14% h(-1)), and histones (0.07-0.1% h(-1)) were comparatively stable. The calculation of degradation and synthesis rate constants k(deg) and k(syn) confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144 h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.

Temporal profiling of the coding and noncoding murine cytomegalovirus transcriptomes.

The global transcriptional program of murine cytomegalovirus (MCMV), involving coding, noncoding, and antisense transcription, remains unknown. Here we report an oligonucleotide custom microarray platform capable of measuring both coding and noncoding transcription on a genome-wide scale. By profiling MCMV wild-type and immediate-early mutant strains in fibroblasts, we found rapid activation of the transcriptome by 6.5 h postinfection, with absolute dependency on ie3, but not ie1 or ie2, for genomic programming of viral gene expression. Evidence is also presented to show, for the first time, genome-wide noncoding and bidirectional transcription at late stages of MCMV infection.

Surviving Starvation: Proteomic and Lipidomic Profiling of Nutrient Deprivation in the Smallest Known Free-Living Eukaryote.

Marine phytoplankton, comprising cyanobacteria, micro- and pico-algae are key to photosynthesis, oxygen production and carbon assimilation on Earth. The unicellular green picoalga Ostreococcus tauri holds a key position at the base of the green lineage of plants, which makes it an interesting model organism. O. tauri has adapted to survive in low levels of nitrogen and phosphorus in the open ocean and also during rapid changes in the levels of these nutrients in coastal waters. In this study, we have employed untargeted proteomic and lipidomic strategies to investigate the molecular responses of O. tauri to low-nitrogen and low-phosphorus environments. In the absence of external nitrogen, there was an elevation in the expression of ammonia and urea transporter proteins together with an accumulation of triglycerides. In phosphate-limiting conditions, the expression levels of phosphokinases and phosphate transporters were increased, indicating an attempt to maximise scavenging opportunities as opposed to energy conservation conditions. The production of betaine lipids was also elevated, highlighting a shift away from phospholipid metabolism. This finding was supported by the putative identification of betaine synthase in O. tauri. This work offers additional perspectives on the complex strategies that underpin the adaptive processes of the smallest known free-living eukaryote to alterations in environmental conditions.

Characterisation of the Bax-nucleophosmin interaction: the importance of the Bax C-terminus.

The molecular chaperone nucleophosmin has been identified as a novel Bax binding protein with this interaction proposed to be a key event in the activation and translocation of Bax in mitochondrial dysfunction and apoptotic cell death. Using a proximity assay, we have quantitatively defined the high affinity and saturable interaction between Bax and nucleophosmin indicative of a competitive and specific mechanism. Binding of full length Bax to nucleophosmin was only observed after conformational change was induced using non-ionic detergents (e.g., NP-40). The Bax-nucleophosmin interaction was inhibited by a Bax C-terminal antibody (IC(50) = 1 nM) but minimally affected by antibodies directed against either the N-terminus or alpha-helices 4 and 5. Bcl-2 and p53 inhibited the interaction between full length activated Bax and nucleophosmin. The proximity assay based on the Bax-nucleophosmin interaction was robust and reproducible (Z' = 0.50) facilitating its use for screening a small chemical library. A low molecular weight non-peptide compound, 2-(5-methyl-2-phenyl-1,3-thiazol-4-yl)ethanohydrazide, partially inhibited the Bax-nucleophosmin interaction (IC(50) = 100 nM) and also attenuated UV-induced cell death of HEK293 cells. The present investigations demonstrate the importance of exposure of the C-terminus of Bax for its interaction with nucleophosmin. These protein-protein interaction assays provide a technical approach both for the study of Bax-interacting proteins and for the discovery of novel anti-apoptotic agents.

Bovine telomere dynamics and the association between telomere length and productive lifespan.

Average telomere length (TL) in blood cells has been shown to decline with age in a range of vertebrate species, and there is evidence that TL is a heritable trait associated with late-life health and mortality in humans. In non-human mammals, few studies to date have examined lifelong telomere dynamics and no study has estimated the heritability of TL, despite these being important steps towards assessing the potential of TL as a biomarker of productive lifespan and health in livestock species. Here we measured relative leukocyte TL (RLTL) in 1,328 samples from 308 Holstein Friesian dairy cows and in 284 samples from 38 female calves. We found that RLTL declines after birth but remains relatively stable in adult life. We also calculated the first heritability estimates of RLTL in a livestock species which were 0.38 (SE = 0.03) and 0.32 (SE = 0.08) for the cow and the calf dataset, respectively. RLTL measured at the ages of one and five years were positively correlated with productive lifespan (p < 0.05). We conclude that bovine RLTL is a heritable trait, and its association with productive lifespan may be used in breeding programmes aiming to enhance cow longevity.

Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format.

The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 µg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed (samples homogenised, RNA purified and quantified) in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments.