Single-cell analysis of yeast, mammalian cells, and fungal spores with a microfluidic pressure-driven chip-based system.

BACKGROUND: Cytomics aims at understanding the function of cellular systems by analysis of single cells. Recently, there has been a growing interest in single cell measurements being performed in microfluidic systems. These systems promise to integrate staining, measurement, and analysis in a single system. One important aspect is the limitation of allowable cell sizes due to microfluidic channel dimensions. Here we want to demonstrate the broad applicability of microfluidic chip technology for the analysis of many different cell types. METHODS: We have developed a microfluidic chip and measurement system that allows flow cytometric analysis of fluorescently stained cells from different organisms. In this setup, the cells are moved by pressure-driven flow inside a network of microfluidic channels and are analyzed individually by fluorescence detection. RESULTS: We have successfully applied the system to develop a methodology to detect viable and dead cells in yeast cell populations. Also, we have measured short interfering RNA (siRNA) mediated silencing of protein expression in mammalian cells. In addition, we have characterized the infection state of Magnaportae grisea fungal spores. CONCLUSIONS: Results obtained with the microfluidic system demonstrate a broad applicability of microfluidic flow cytometry to measurement of various cell types.     

Time-course of changes in amounts of specific proteins upon exposure to hyper-g, 2-D clinorotation, and 3-D random positioning of Arabidopsis cell cultures

In previous studies it has been shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by altered gene expression. In this study an investigation was carried out into how different g conditions affect the proteome of such cells. For this purpose, callus cells were exposed to 8 g (centrifugation) and simulated microgravity (2-D clinorotation: fast rotating clinostat, yielding 0.0016 g at maximum; and 3-D random positioning) for up to 16 h. Extracts containing total soluble protein were subjected to 2-D SDS-PAGE. Image analysis of Sypro Ruby-stained gels showed that approximately 28 spots reproducibly and significantly (P <0.05) changed in amount after 2 h of hypergravity (18 up- and 10 down-regulated). These spots were analysed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the case of 2-D clinorotation, 19 proteins changed in a manner similar to hypergravity, while random positioning affected only eight spots. Identified proteins were mainly stress related, and are involved in detoxification of reactive oxygen species, signalling, and calcium binding. Surprisingly, centrifugation and clinorotation showed homologies which were not detected for random positioning. The data indicate that simulation of weightlessness is different between clinorotation and random positioning.     

Incorporating anthropogenic effects into trophic ecology: predator–prey interactions in a human-dominated landscape

Apex predators perform important functions that regulate ecosystems worldwide. However, little is known about how ecosystem regulation by predators is influenced by human activities. In particular, how important are top-down effects of predators relative to direct and indirect human-mediated bottom-up and top-down processes? Combining data on species'' occurrence from camera traps and hunting records, we aimed to quantify the relative effects of top-down and bottom-up processes in shaping predator and prey distributions in a human-dominated landscape in Transylvania, Romania. By global standards this system is diverse, including apex predators (brown bear and wolf), mesopredators (red fox) and large herbivores (roe and red deer). Humans and free-ranging dogs represent additional predators in the system. Using structural equation modelling, we found that apex predators suppress lower trophic levels, especially herbivores. However, direct and indirect top-down effects of humans affected the ecosystem more strongly, influencing species at all trophic levels. Our study highlights the need to explicitly embed humans and their influences within trophic cascade theory. This will greatly expand our understanding of species interactions in human-modified landscapes, which compose the majority of the Earth''s terrestrial surface.     

Increased Summer Temperatures Reduce the Growth and Regeneration of Larix sibirica in Southern Boreal Forests of Eastern Kazakhstan

The larch forests at the southern limit of the Siberian boreal forest in Central Asia have repeatedly experienced strong recent growth declines attributed to decreasing summer precipitation in the course of climate warming. Here, we present evidence from the southernmost Larix sibirica forests in eastern Kazakhstan that these declines are primarily caused by a decrease in effective moisture due to increasing summer temperatures, despite constant annual, and summer precipitation. Tree-ring chronologies (>800 trees) showed a reduction by 50–80% in mean ring width and an increase in the frequency of missing rings since the 1970s. Climate-response analysis revealed a stronger (negative) effect of summer temperature (in particular of the previous year’s June and July temperature) on radial growth than summer precipitation (positive effect). It is assumed that a rise in the atmospheric vapor pressure deficit, which typically increases with temperature, is negatively affecting tree water status and radial growth, either directly or indirectly through reduced soil moisture. Larch rejuvenation ceased in the 1950s, which is partly explained by increasing topsoil desiccation in a warmer climate and a high drought susceptibility of larch germination, as was demonstrated by a germination experiment with variable soil moisture levels. The lack of regeneration and the reduced annual stem increment suggest that sustainable forest management aiming at timber harvesting is no longer feasible in these southern boreal forests. Progressive climate warming is likely to cause a future northward shift of the southern limit of the boreal forest.     

Stable nitrogen isotope analysis of dentine serial sections elucidate sex differences in weaning patterns of wild chimpanzees (Pan troglodytes)

Offspring provisioning is one of the most energetically demanding aspects of reproduction for female mammals. Variation in lactation length and weaning strategies between chimpanzees (Pan troglodytes), our closest living relative, and modern human societies have been reported. When and why these changes occurred is frequently debated. Our study used stable nitrogen isotope data of tooth root dentine from wild Western chimpanzees (Pan troglodytes verus) in Taï National Park, Côte d'Ivoire, to quantify weaning in these chimpanzees and explore if infant sex plays a role in maternal investment. We analyzed serial sections of deciduous lateral incisor root dentine from four Taï chimpanzees to establish the δ¹⁵N signal of nursing infants; we then analyzed serial sections of first permanent mandibular molar root dentine from 12 Taï chimpanzees to provide quantitative δ¹⁵N data on weaning in this population. Up to 2 years of age both sexes exhibited dentine δ¹⁵N values ≈2–3‰ higher than adult female Taï chimpanzees, consistent with a nursing signal. Thereafter a steady decrease in δ¹⁵N values consistent with the onset, and progression, of weaning, was visible. Sex differences were also evident, where male δ¹⁵N values decreased at a significantly slower rate compared to females. Confirmation of sex differences in maternal investment among Taï chimpanzees, demonstrates the viability of using isotope analysis to investigate weaning in non‐human primates. Additionally, assuming that behaviors observed in the Taï chimpanzees are illustrative of the ancestral pattern, our results provide a platform to enable the trajectory of weaning in human evolution to be further explored. Am J Phys Anthropol 153:635–642, 2014. © 2013 Wiley Periodicals, Inc.     

Proteome-wide Analysis of Lysine Acetylation Suggests its Broad Regulatory Scope in Saccharomyces cerevisiae

Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S. cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved compared with nonacetylated lysines. A large fraction of the conserved acetylation sites are present on proteins involved in cellular metabolism, protein synthesis, and protein folding. Furthermore, quantification of the Rpd3-regulated acetylation sites identified several previously known, as well as new putative substrates of this deacetylase. Rpd3 deficiency increased acetylation of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex subunit Sgf73 on K33. This acetylation site is located within a critical regulatory domain in Sgf73 that interacts with Ubp8 and is involved in the activation of the Ubp8-containing histone H2B deubiquitylase complex. Our data provides the first global survey of acetylation in budding yeast, and suggests a wide-ranging regulatory scope of this modification. The provided dataset may serve as an important resource for the functional analysis of lysine acetylation in eukaryotes.Lysine acetylation is a dynamic and reversible post-translational modification. Acetylation of lysines on their ε-amino group is catalyzed by lysine acetyltransferases (KATs¹, also known as histone acetyltrasferases (HATs)), and reversed by lysine deacetylases (KDACs, also known as histone deacetylases (HDACs)) (1). The enzymatic machinery involved in lysine acetylation is evolutionary conserved in all forms of life (2–4). The role of acetylation has been extensively studied in the regulation of gene expression via modification of histones (5). Acetylation also plays important roles in controlling cellular metabolism (6–10), protein folding (11), and sister chromatid cohesion (12). Furthermore, acetylation has been implicated in regulating the beneficial effects of calorie restriction (13), a low nutrient diet without starvation, and aging. Based on these findings, it is proposed that the functional roles of acetylation in these processes are evolutionary conserved from yeast to mammals.Advancements in mass spectrometry (MS)-based proteomics have greatly facilitated identification of thousands of post-translational modification (PTM) sites in eukaryotic cells (14–18). Proteome-wide mapping of PTM sites can provide important leads for analyzing the functional relevance of individual sites and a systems-wide view of the regulatory scope of post-translational modifications. Also, large-scale PTM datasets are an important resource for the in silico analysis of PTMs, which can broaden the understanding of modification site properties and their evolutionary trajectories.The budding yeast Saccharomyces cerevisiae is a commonly used unicellular eukaryotic model organism. Yeast has been used in many pioneering “-omics” studies, including sequencing of the first eukaryotic genome (19), systems-wide genetic interactions analysis (20, 21), MS-based comprehensive mapping of a eukaryotic proteome (22), and proteome-wide analysis of protein-protein interactions (23, 24). In addition, S. cerevisiae has been extensively used to study the molecular mechanisms of acetylation. Many lysine acetyltransferases and deacetylases were discovered in this organism (2, 25), and their orthologs were subsequently identified in higher eukaryotes. Furthermore, the functional roles of many well-studied acetylation sites on histones are conserved from yeast to mammals. Recent data from human and Drosophila cells show that acetylation is present on many highly conserved metabolic enzymes (26–28). However, only a few dozen yeast acetylation sites are annotated in the Uniprot database. Given the presence of a well-conserved and elaborate acetylation machinery in yeast, we reasoned that many more acetylation sites exist in this organism that remained to be identified.Here we used high resolution mass spectrometry-based proteomics to investigate the scope of acetylation in S. cerevisiae. We identified about 4000 unique acetylation sites in this important model organism. Bioinformatic analysis of yeast acetylation sites and comparison with previously identified human and Drosophila acetylation sites indicates that many acetylation sites are evolutionary conserved. Furthermore, quantitative analysis of the Rpd3-regulated acetylation sites identified several nuclear proteins that showed increased acetylation in rpd3 knockout cells. Our results provide a systems-wide view of acetylation in budding yeast, and a rich dataset for functional analysis of acetylation sites in this organism.     

Determinants of elevated carbohydrate antigen 125 in patients with severe symptomatic aortic valve stenosis referred for transcatheter aortic valve implantation

Purpose: Elevated carbohydrate antigen 125 (CA125) predicts adverse outcome after transcatheter aortic valve implantation (TAVI). While known underlying pathophysiological mechanisms of elevated CA125 include serosal effusions and inflammatory stimuli, clinical determinants associated with elevated CA125 in patients referred for TAVI remain unknown. Therefore, we investigated clinical, laboratory and echocardiographic determinants of elevated CA125 in patients with severe aortic valve stenosis referred for TAVI.

Methods: This study includes 650 patients with severe aortic stenosis referred for TAVI. Baseline CA125 was determined by an immunoassay and dichotomized (elevated versus normal) based on the manufacturer cutoff value (>35?U/mL).

Results: CA125 elevation was present in 28% (181/650). Patients with elevated CA125 had an overall worse clinical profile and were more symptomatic with a higher rate of NYHA class III/IV (80% versus 58%; p?Conclusion: Elevated CA125 levels in patients referred for TAVI summarize a subset of patients with an overall worse clinical profile who are more symptomatic.