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1.  Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart 
The Journal of Physiology  2014;592(Pt 21):4715-4731.
Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l−1 NaCl (as control) or 0.7 mmol l−1 NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.
doi:10.1113/jphysiol.2014.275263
PMCID: PMC4253472  PMID: 25172947
2.  A Model for the Evolution of Biological Specificity: a Cross-Reacting DNA-Binding Protein Causes Plasmid Incompatibility 
Journal of Bacteriology  2014;196(16):3002-3011.
Few biological systems permit rigorous testing of how changes in DNA sequence give rise to adaptive phenotypes. In this study, we sought a simplified experimental system with a detailed understanding of the genotype-to-phenotype relationship that could be altered by environmental perturbations. We focused on plasmid fitness, i.e., the ability of plasmids to be stably maintained in a bacterial population, which is dictated by the plasmid's replication and segregation machinery. Although plasmid replication depends on host proteins, the type II plasmid partitioning (Par) machinery is entirely plasmid encoded and relies solely on three components: parC, a centromere-like DNA sequence, ParR, a DNA-binding protein that interacts with parC, and ParM, which forms actin-like filaments that push two plasmids away from each other at cell division. Interactions between the Par operons of two related plasmids can cause incompatibility and the reduced transmission of one or both plasmids. We have identified segregation-dependent plasmid incompatibility between the highly divergent Par operons of plasmids pB171 and pCP301. Genetic and biochemical studies revealed that the incompatibility is due to the functional promiscuity of the DNA-binding protein ParRpB171, which interacts with both parC DNA sequences to direct plasmid segregation, indicating that the lack of DNA binding specificity is detrimental to plasmid fitness in this environment. This study therefore successfully utilized plasmid segregation to dissect the molecular interactions between genotype, phenotype, and fitness.
doi:10.1128/JB.01811-14
PMCID: PMC4135640  PMID: 24914185
3.  Oral Coenzyme Q10 Supplementation Does Not Prevent Cardiac Alterations During a High Altitude Trek to Everest Base Camp 
High Altitude Medicine & Biology  2014;15(4):459-467.
Abstract
Holloway, Cameron J., Andrew J. Murray, Kay Mitchell, Daniel S. Martin, Andrew W. Johnson, Lowri E. Cochlin, Ion Codreanu, Sundeep Dhillon, George W. Rodway, Tom Ashmore, Denny Z.H. Levett, Stefan Neubauer, Hugh E. Montgomery, Michael P.W. Grocott, and Kieran Clarke, on behalf of the Caudwell Xtreme Everest 2009 Investigators. Oral Coenzyme Q supplementation does not prevent cardiac alterations during a high altitude trek to Everest Base Camp. High Alt Med Biol 15:000—000, 2014.—Exposure to high altitude is associated with sustained, but reversible, changes in cardiac mass, diastolic function, and high-energy phosphate metabolism. Whilst the underlying mechanisms remain elusive, tissue hypoxia increases generation of reactive oxygen species (ROS), which can stabilize hypoxia-inducible factor (HIF) transcription factors, bringing about transcriptional changes that suppress oxidative phosphorylation and activate autophagy. We therefore investigated whether oral supplementation with an antioxidant, Coenzyme Q10, prevented the cardiac perturbations associated with altitude exposure. Twenty-three volunteers (10 male, 13 female, 46±3 years) were recruited from the 2009 Caudwell Xtreme Everest Research Treks and studied before, and within 48 h of return from, a 17-day trek to Everest Base Camp, with subjects receiving either no intervention (controls) or 300 mg Coenzyme Q10 per day throughout altitude exposure. Cardiac magnetic resonance imaging and echocardiography were used to assess cardiac morphology and function. Following altitude exposure, body mass fell by 3 kg in all subjects (p<0.001), associated with a loss of body fat and a fall in BMI. Post-trek, left ventricular mass had decreased by 11% in controls (p<0.05) and by 16% in Coenzyme Q10-treated subjects (p<0.001), whereas mitral inflow E/A had decreased by 18% in controls (p<0.05) and by 21% in Coenzyme Q10-treated subjects (p<0.05). Coenzyme Q10 supplementation did not, therefore, prevent the loss of left ventricular mass or change in diastolic function that occurred following a trek to Everest Base Camp.
doi:10.1089/ham.2013.1053
PMCID: PMC4273181  PMID: 24661196
altitude; Coenzyme Q10; cardiac metabolism; heart function; hypoxia
4.  Skeletal muscle energy metabolism in environmental hypoxia: climbing towards consensus 
Skeletal muscle undergoes metabolic remodelling in response to environmental hypoxia, yet aspects of this process remain controversial. Broadly, environmental hypoxia has been suggested to induce: (i) a loss of mitochondrial density; (ii) a substrate switch away from fatty acids and towards other substrates such as glucose, amino acids and ketone bodies; and (iii) a shift from aerobic to anaerobic metabolism. There remains a lack of a consensus in these areas, most likely as a consequence of the variations in degree and duration of hypoxic exposure, as well as the broad range of experimental parameters used as markers of metabolic processes. To attempt to resolve some of the controversies, we performed a comprehensive review of the literature pertaining to hypoxia-induced changes in skeletal muscle energy metabolism. We found evidence that mass-specific mitochondrial function is decreased prior to mass-specific mitochondrial density, implicating intra-mitochondrial changes in the response to environmental hypoxia. This loss of oxidative capacity does not appear to be matched by a loss of glycolytic capacity, which on the whole is not altered by environmental hypoxia. Environmental hypoxia does however induce a selective attenuation of fatty acid oxidation, whilst glucose uptake is maintained or increased, perhaps to support glycolysis in the face of a downregulation of oxidative metabolism, optimising the pathways of ATP synthesis for the hypoxic environment.
Electronic supplementary material
The online version of this article (doi:10.1186/2046-7648-3-19) contains supplementary material, which is available to authorized users.
doi:10.1186/2046-7648-3-19
PMCID: PMC4253994  PMID: 25473486
Hypoxia; High altitude; Skeletal muscle; Mitochondria; Metabolism
5.  Evolving a 24-hr oscillator in budding yeast 
eLife  null;3:e04875.
We asked how a new, complex trait evolves by selecting for diurnal oscillations in the budding yeast, Saccharomyces cerevisiae. We expressed yellow fluorescent protein (YFP) from a yeast promoter and selected for a regular alternation between low and high fluorescence over a 24-hr period. This selection produced changes in cell adhesion rather than YFP expression: clonal populations oscillated between single cells and multicellular clumps. The oscillations are not a response to environmental cues and continue for at least three cycles in a constant environment. We identified eight putative causative mutations in one clone and recreated the evolved phenotype in the ancestral strain. The mutated genes lack obvious relationships to each other, but multiple lineages change from the haploid to the diploid pattern of gene expression. We show that a novel, complex phenotype can evolve by small sets of mutations in genes whose molecular functions appear to be unrelated to each other.
DOI: http://dx.doi.org/10.7554/eLife.04875.001
eLife digest
In living things, many important behaviors—including animal heartbeats and sleep patterns—happen in cycles. Machines called oscillators, which are found inside cells, control these behaviors. There are many different oscillators and they share some common features, despite involving different genes.
Each oscillator is formed of a set of genes that interact with each other to drive regular cycles lasting seconds, hours, or even months. The oscillators do not need any cues from the environment to maintain these cycles. However, cues such as light or temperature can keep the oscillator synchronized with the environment.
To ask how complex machines like oscillators could evolve, Wildenberg and Murray inserted a gene that makes a fluorescent protein into budding yeast, a single-celled species that does not have an oscillator with a period of 24 hr. These yeast cells were then selectively grown through a few hundred generations to experimentally evolve a yeast strain where the levels of protein fluorescence regularly alternated over 24-hr periods.
Wildenberg and Murray then carried out further experiments to discover the cause of the pattern of protein fluorescence. These revealed that the pattern was due to the yeast cells alternating between forming clumps of multiple cells and living separately. The genes that mutated to create the cycles of cell clumping in the yeast all appear to have unrelated roles.
The 24-hr oscillator that evolved in the yeast has some of the features of the biological oscillators found in nature. It maintains regular cycles even without any cues from the environment and it can control a cell behavior, but the oscillator appears to be unable to accept cues from the environment, a universal property of naturally evolved circadian clocks. Further work to understand how the genes work together in the oscillator will help to better understand how 24-hr oscillators in nature can evolve from genes that lack any 24-hr behavior.
DOI: http://dx.doi.org/10.7554/eLife.04875.002
doi:10.7554/eLife.04875
PMCID: PMC4270073  PMID: 25383925
experimental evolution; circadian rhythm; yeast; genome; evolutionary novelty; complex traits; S. cerevisiae
6.  Conservation Weighting Functions Enable Covariance Analyses to Detect Functionally Important Amino Acids 
PLoS ONE  2014;9(11):e107723.
The explosive growth in the number of protein sequences gives rise to the possibility of using the natural variation in sequences of homologous proteins to find residues that control different protein phenotypes. Because in many cases different phenotypes are each controlled by a group of residues, the mutations that separate one version of a phenotype from another will be correlated. Here we incorporate biological knowledge about protein phenotypes and their variability in the sequence alignment of interest into algorithms that detect correlated mutations, improving their ability to detect the residues that control those phenotypes. We demonstrate the power of this approach using simulations and recent experimental data. Applying these principles to the protein families encoded by Dscam and Protocadherin allows us to make testable predictions about the residues that dictate the specificity of molecular interactions.
doi:10.1371/journal.pone.0107723
PMCID: PMC4224327  PMID: 25379728
7.  A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution 
PLoS ONE  2014;9(10):e109780.
Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.
doi:10.1371/journal.pone.0109780
PMCID: PMC4199604  PMID: 25329559
8.  Mapping sensory circuits by anterograde trans-synaptic transfer of recombinant rabies virus 
Neuron  2014;81(4):766-778.
Summary
Primary sensory neurons convey information from the external world to relay circuits within the central nervous system (CNS), but the identity and organization of the neurons that process incoming sensory information remains sketchy. Within the CNS viral tracing techniques that rely on retrograde trans-synaptic transfer provide a powerful tool for delineating circuit organization. Viral tracing of the circuits engaged by primary sensory neurons has, however, been hampered by the absence of a genetically tractable anterograde transfer system. In this study we demonstrate that rabies virus can infect sensory neurons in the somatosensory system, is subject to anterograde trans-synaptic transfer from primary sensory to spinal target neurons, and can delineate output connectivity with third-order neurons. Anterograde trans-synaptic transfer is a feature shared by other classes of primary sensory neurons, permitting the identification and potentially the manipulation of neural circuits processing sensory feedback within the mammalian CNS.
doi:10.1016/j.neuron.2013.12.033
PMCID: PMC3988472  PMID: 24486087
9.  Tethering Sister Centromeres to Each Other Suggests the Spindle Checkpoint Detects Stretch within the Kinetochore 
PLoS Genetics  2014;10(8):e1004492.
The spindle checkpoint ensures that newly born cells receive one copy of each chromosome by preventing chromosomes from segregating until they are all correctly attached to the spindle. The checkpoint monitors tension to distinguish between correctly aligned chromosomes and those with both sisters attached to the same spindle pole. Tension arises when sister kinetochores attach to and are pulled toward opposite poles, stretching the chromatin around centromeres and elongating kinetochores. We distinguished between two hypotheses for where the checkpoint monitors tension: between the kinetochores, by detecting alterations in the distance between them, or by responding to changes in the structure of the kinetochore itself. To distinguish these models, we inhibited chromatin stretch by tethering sister chromatids together by binding a tetrameric form of the Lac repressor to arrays of the Lac operator located on either side of a centromere. Inhibiting chromatin stretch did not activate the spindle checkpoint; these cells entered anaphase at the same time as control cells that express a dimeric version of the Lac repressor, which cannot cross link chromatids, and cells whose checkpoint has been inactivated. There is no dominant checkpoint inhibition when sister kinetochores are held together: cells expressing the tetrameric Lac repressor still arrest in response to microtubule-depolymerizing drugs. Tethering chromatids together does not disrupt kinetochore function; chromosomes are successfully segregated to opposite poles of the spindle. Our results indicate that the spindle checkpoint does not monitor inter-kinetochore separation, thus supporting the hypothesis that tension is measured within the kinetochore.
Author Summary
The spindle checkpoint monitors tension on chromosomes to distinguish between chromosomes that are correctly and incorrectly attached to the spindle. Tension is generated across a correctly attached chromosome as microtubules from opposite poles attach to and pull kinetochores apart, but are resisted by the cohesin that holds sister chromatids together. This tension generates separation between kinetochores as pericentric chromatin stretches and it also elongates the kinetochores. To monitor tension, the checkpoint could measure the separation between kinetochores or the stretch within them. We inhibited the ability of pericentric chromatin to stretch by tethering sister centromeres to each other, and we asked whether the resulting reduction in inter-kinetochore separation artificially activated the spindle checkpoint. Inhibiting inter-kinetochore separation does not delay anaphase, and the timing of mitosis was the same in cells with or without the spindle checkpoint, showing that the checkpoint is not activated. Inhibiting chromatin stretch does not alter the function of kinetochores as chromosomes are still segregated correctly, nor does it hinder the checkpoint. Cells whose sister kinetochores are held together can still activate the checkpoint in response to microtubule depolymerization. Our results indicate the spindle checkpoint does not monitor inter-kinetochore separation and likely monitors tension within kinetochores.
doi:10.1371/journal.pgen.1004492
PMCID: PMC4125069  PMID: 25101645
10.  Mitochondria at the extremes: pioneers, protectorates, protagonists 
The engulfment of a proto-mitochondrion by a primitive unicellular organism gave rise to the first eukaryotic cell, and ever since, mitochondrial function has been a vital aspect of eukaryotic life. Under conditions of physiological stress, the mitochondrion is far from a passive bystander, instead playing a key role in signalling pathways and the cellular responses they elicit. In this thematic series of Extreme Physiology & Medicine, the role of the mitochondrion in the response to physiological stress will be considered anew, through research articles, reviews, viewpoints and methodology papers that aim to reposition the organelle as a key player in the human response to a wide range of extreme conditions.
doi:10.1186/2046-7648-3-10
PMCID: PMC4017221  PMID: 24822089
11.  HATL5: A Cell Surface Serine Protease Differentially Expressed in Epithelial Cancers 
PLoS ONE  2014;9(2):e87675.
Over the last two decades, cell surface proteases belonging to the type II transmembrane serine protease (TTSP) family have emerged as important enzymes in the mammalian degradome, playing critical roles in epithelial biology, regulation of metabolic homeostasis, and cancer. Human airway trypsin-like protease 5 (HATL5) is one of the few family members that remains uncharacterized. Here we demonstrate that HATL5 is a catalytically active serine protease that is inhibited by the two Kunitz type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and 2, as well as by serpinA1. Full-length HATL5 is localized on the cell surface of cultured mammalian cells as demonstrated by confocal microscopy. HATL5 displays a relatively restricted tissue expression profile, with both transcript and protein present in the cervix, esophagus, and oral cavity. Immunohistochemical analysis revealed an expression pattern where HATL5 is localized on the cell surface of differentiated epithelial cells in the stratified squamous epithelia of all three of these tissues. Interestingly, HATL5 is significantly decreased in cervical, esophageal, and head and neck carcinomas as compared to normal tissue. Analysis of cervical and esophageal cancer tissue arrays demonstrated that the squamous epithelial cells lose their expression of HATL5 protein upon malignant transformation.
doi:10.1371/journal.pone.0087675
PMCID: PMC3912027  PMID: 24498351
12.  Coding-sequence determinants of gene expression in Escherichia coli 
Science (New York, N.Y.)  2009;324(5924):255-258.
Synonymous mutations do not alter the encoded protein, but they can influence gene expression. To investigate the mechanisms, we engineered a synthetic library of 154 genes that vary randomly at synonymous sites, but all encode the same green fluorescent protein. When expressed in E. coli, GFP protein levels varied 250-fold across the library. GFP mRNA levels, mRNA degradation patterns, and bacterial growth rates also varied, but codon bias did not correlate with gene expression. Rather, the stability of mRNA folding near the ribosomal binding site explained over half the variation in protein levels. In our analysis, mRNA folding and associated rates of translation initiation play a predominant role in shaping expression levels of individual genes, whereas codon bias influences global translation efficiency and cellular fitness.
doi:10.1126/science.1170160
PMCID: PMC3902468  PMID: 19359587
13.  Heterothallism in Saccharomyces cerevisiae isolates from nature: effect of HO locus on the mode of reproduction 
Molecular ecology  2009;19(1):121-131.
Understanding the evolution of sex and recombination, key factors in the evolution of life, is a major challenge in biology. Studies of reproduction strategies of natural populations are important to complement the theoretical and experimental models. Fungi with both sexual and asexual life cycles are an interesting system for understanding the evolution of sex. In a study of natural populations of yeast Saccharomyces cerevisiae, we found that the isolates are heterothallic, meaning their mating type is stable, while the general belief is that natural S. cerevisiae strains are homothallic (can undergo mating-type switching). Mating-type switching is a gene-conversion process initiated by a site-specific endonuclease HO; this process can be followed by mother–daughter mating. Heterothallic yeast can mate with unrelated haploids (amphimixis), or undergo mating between spores from the same tetrad (intratetrad mating, or automixis), but cannot undergo mother–daughter mating as homothallic yeasts can. Sequence analysis of HO gene in a panel of natural S. cerevisiae isolates revealed multiple mutations. Good correspondence was found in the comparison of population structure characterized using 19 microsatellite markers spread over eight chromosomes and the HO sequence. Experiments that tested whether the mating-type switching pathway upstream and downstream of HO is functional, together with the detected HO mutations, strongly suggest that loss of function of HO is the cause of heterothallism. Furthermore, our results support the hypothesis that clonal reproduction and intratetrad mating may predominate in natural yeast populations, while mother–daughter mating might not be as significant as was considered.
doi:10.1111/j.1365-294X.2009.04436.x
PMCID: PMC3892377  PMID: 20002587
heterothallism; HO; reproduction; Saccharomyces cerevisiae
14.  Establishment of anti-tumor memory in humans using in vitro-educated CD8+ T cells 
Science translational medicine  2011;3(80):10.1126/scitranslmed.3002207.
While advanced stage melanoma patients have a median survival of less than a year, adoptive T cell therapy can induce durable clinical responses in some patients. Successful adoptive T cell therapy to treat cancer requires engraftment of anti-tumor T lymphocytes that not only retain specificity and function in vivo but also display an intrinsic capacity to survive. To date, adoptively transferred anti-tumor CD8+ T lymphocytes (CTL) have had limited life spans unless the host has been manipulated. To generate CTL that possess an intrinsic capacity to persist in vivo, we developed a human artificial antigen presenting cell system that can educate anti-tumor CTL to acquire both a central memory and effector memory phenotype as well as the capacity to survive in culture for prolonged periods of time. In the present report, we examined whether anti-tumor CTL generated using this system could function and persist in patients. Here, we showed that MART1-specific CTL, educated and expanded using our artificial antigen presenting cell system, could survive for prolonged periods in advanced stage melanoma patients without previous conditioning or cytokine treatment. Moreover, these CTL trafficked to the tumor, mediated biological and clinical responses, and established anti-tumor immunologic memory. Therefore, this approach may broaden the availability of adoptive cell therapy to patients both alone and in combination with other therapeutic modalities.
doi:10.1126/scitranslmed.3002207
PMCID: PMC3861895  PMID: 21525398
15.  Oral 28-day and developmental toxicity studies of (R)-3-hydroxybutyl (R)-3-hydroxybutyrate 
Regulatory toxicology and pharmacology : RTP  2012;63(2):10.1016/j.yrtph.2012.04.001.
(R)-3-Hydroxybutyl (R)-3-hydroxybutyrate (ketone monoester) has been developed as an oral source of ketones, which may be utilized for energy. In a 28-day toxicity study, Crl:WI (Wistar) rats received diets containing, as 30% of the calories, ketone monoester (12 and 15 g/kg body weight/day for male and female rats, respectively). Control groups received either carbohydrate- or fat-based diets. Rats in the test group consumed less feed and gained less weight than control animals; similar findings have been documented in studies of ketogenic diets. Between-group differences were noted in selected hematology, coagulation, and serum chemistry parameters; however, values were within normal physiological ranges and/or were not accompanied by other changes indicative of toxicity. Upon gross and microscopic evaluation, there were no findings associated with the ketone monoester. In a developmental toxicity study, pregnant Crl:WI (Han) rats were administered 2 g/kg body weight/day ketone monoester or water (control) via gavage on days 6 through 20 of gestation. No Caesarean-sectioning or litter parameters were affected by the test article. The overall incidence of fetal alterations was higher in the test group; however, there were no specific alterations attributable to the test substance. The results of these studies support the safety of ketone monoester.
doi:10.1016/j.yrtph.2012.04.001
PMCID: PMC3809901  PMID: 22504461
(R)-3-Hydroxybutyl (R)-3-hydroxybutyrate; Developmental toxicity; Ketones; Toxicity; Safety
16.  No evidence for a local renin-angiotensin system in liver mitochondria 
Scientific Reports  2013;3:2467.
The circulating, endocrine renin-angiotensin system (RAS) is important to circulatory homeostasis, while ubiquitous tissue and cellular RAS play diverse roles, including metabolic regulation. Indeed, inhibition of RAS is associated with improved cellular oxidative capacity. Recently it has been suggested that an intra-mitochondrial RAS directly impacts on metabolism. Here we sought to rigorously explore this hypothesis. Radiolabelled ligand-binding and unbiased proteomic approaches were applied to purified mitochondrial sub-fractions from rat liver, and the impact of AngII on mitochondrial function assessed. Whilst high-affinity AngII binding sites were found in the mitochondria-associated membrane (MAM) fraction, no RAS components could be detected in purified mitochondria. Moreover, AngII had no effect on the function of isolated mitochondria at physiologically relevant concentrations. We thus found no evidence of endogenous mitochondrial AngII production, and conclude that the effects of AngII on cellular energy metabolism are not mediated through its direct binding to mitochondrial targets.
doi:10.1038/srep02467
PMCID: PMC3747509  PMID: 23959064
17.  Selective sweeps in growing microbial colonies 
Physical Biology  2012;9(2):026008.
Evolutionary experiments with microbes are a powerful tool to study mutations and natural selection. These experiments, however, are often limited to the well-mixed environments of a test tube or a chemostat. Since spatial organization can significantly affect evolutionary dynamics, the need is growing for evolutionary experiments in spatially structured environments. The surface of a Petri dish provides such an environment, but a more detailed understanding of microbial growth on Petri dishes is necessary to interpret such experiments. We formulate a simple deterministic reaction-diffusion model, which successfully predicts the spatial patterns created by two competing species during colony expansion. We also derive the shape of these patterns analytically without relying on microscopic details of the model. In particular, we find that the relative fitness of two microbial strains can be estimated from the logarithmic spirals created by selective sweeps. The theory is tested with strains of the budding yeast Saccharomyces cerevisiae, for spatial competitions with different initial conditions and for a range of relative fitnesses. The reaction-diffusion model also connects the microscopic parameters like growth rates and diffusion constants with macroscopic spatial patterns and predicts the relationship between fitness in liquid cultures and on Petri dishes, which we confirmed experimentally. Spatial sector patterns therefore provide an alternative fitness assay to the commonly used liquid culture fitness assays.
doi:10.1088/1478-3975/9/2/026008
PMCID: PMC3359763  PMID: 22476106
Fisher waves; wave velocity; selective sweep; competition at the front; spatial assay; relative fitness
18.  Improved use of a public good selects for the evolution of undifferentiated multicellularity 
eLife  2013;2:e00367.
We do not know how or why multicellularity evolved. We used the budding yeast, Saccharomyces cerevisiae, to ask whether nutrients that must be digested extracellularly select for the evolution of undifferentiated multicellularity. Because yeast use invertase to hydrolyze sucrose extracellularly and import the resulting monosaccharides, single cells cannot grow at low cell and sucrose concentrations. Three engineered strategies overcame this problem: forming multicellular clumps, importing sucrose before hydrolysis, and increasing invertase expression. We evolved populations in low sucrose to ask which strategy they would adopt. Of 12 successful clones, 11 formed multicellular clumps through incomplete cell separation, 10 increased invertase expression, none imported sucrose, and 11 increased hexose transporter expression, a strategy we had not engineered. Identifying causal mutations revealed genes and pathways, which frequently contributed to the evolved phenotype. Our study shows that combining rational design with experimental evolution can help evaluate hypotheses about evolutionary strategies.
DOI: http://dx.doi.org/10.7554/eLife.00367.001
eLife digest
Life first appeared on Earth more than 3 billion years ago in the form of single-celled microorganisms. The diverse array of complex life forms that we see today evolved from these humble beginnings, but it is not clear what triggered the evolution of multicellular organisms from single cells.
One of the simplest multicellular eukaryotes is the yeast, Saccharomyces cerevisiae—a fungus that has been used for centuries in baking and brewing and, more recently, as a model organism in molecular biology. Yeast cells feed on sugar (sucrose), but are unable to absorb it directly from their surroundings. Instead they secrete an enzyme called invertase, which breaks down the sucrose into simpler components that cells can take up with the help of sugar transporters.
However, single yeast cells living in a low-sucrose environment face a problem: most of the simple sugars that they produce diffuse out of reach. To overcome this difficulty, the cells could form multicellular clumps, which would enable each cell to consume the sugars that drift away from its neighbours. Alternatively, the cells could increase their production of invertase, or they could begin to take up sucrose directly.
Using genetic engineering, Koschwanez et al. produced three strains of yeast, each with one of these traits, and confirmed that all three strategies do indeed help fungi to grow in low sucrose. But could any of these traits evolve spontaneously? To test this possibility, Koschwanez et al. introduced wild-type yeast cells into a low-sucrose environment and studied any populations of cells that managed to survive. Of 12 that did, 11 had acquired the ability to form multicellular clumps, while 10 had increased their expression of invertase. Surprisingly, none had evolved the ability to import sucrose. However, 11 of the populations that survived also displayed an adaptation that the researchers had not predicted beforehand: they all expressed higher levels of the sugar transporters that take up sucrose breakdown products.
The work of Koschwanez et al. suggests that the benefits of being able to share invertase and, therefore, simple sugars, may have driven the evolution of multicellularity in ancient organisms. Moreover, their use of rational design (engineered mutations) combined with experimental evolution (allowing colonies to grow under selection pressure and studying the strategies that they adopt) offers a new approach to studying evolution in the lab.
DOI: http://dx.doi.org/10.7554/eLife.00367.002
doi:10.7554/eLife.00367
PMCID: PMC3614033  PMID: 23577233
Multicellularity; Experimental evolution; Evolution of cooperation; S. cerevisiae
19.  Seeing Mutations in Living Cells 
Current biology : CB  2010;20(16):1432-1437.
Summary
Background
Evolution depends on mutations: rare errors in the transmission of genetic information. Experimentally, mutations have been found by detecting altered phenotypes or sequencing complete genomes, but most mutations do not have overt phenotypes, and sequencing is expensive and has limited time resolution. The major source of mutations is DNA replication errors. Nearly all mistakes in DNA replication are detected and repaired by the mismatch repair machinery.
Results
We use a functional, fluorescently labeled derivative of one of the key mismatch repair proteins (MutL) to see and count the small fraction of errors in Escherichia coli that does not get repaired and is converted into stable mutations by the next round of DNA replication. Over a 300-fold range, there is a linear relationship between the frequency of fluorescent foci and the genetically measured mutation frequency, and the mean frequency of fluorescent foci agrees well with estimates of the global mutation rate.
Conclusion
We describe a method for detecting the majority of genomic mutations emerging in living cells, independently of their potential phenotype. The distribution of emerging mutations per cell is roughly Poisson distributed, suggesting that all the cells in the population have roughly the same mutation rate.
doi:10.1016/j.cub.2010.06.071
PMCID: PMC3612896  PMID: 20674359
20.  Don't Make Me Mad, Bub! 
Developmental cell  2012;22(6):1123-1125.
The history of Bub1, a spindle checkpoint component, reveals a spectacular case of parallel evolution. In this issue of Developmental Cell, Suijkerbuijk et al. (2012) provide evidence that Bub1 has duplicated and diverged many times during eukaryotic evolution, dividing the functions of its ancestor between the two duplicated copies.
doi:10.1016/j.devcel.2012.05.020
PMCID: PMC3587729  PMID: 22698277
21.  Mad2 and Mad3 Cooperate to Arrest Budding Yeast in Mitosis 
Current Biology  2011;22(3):180-190.
Summary
Background
The spindle checkpoint ensures accurate chromosome transmission by delaying chromosome segregation until all chromosomes are correctly aligned on the mitotic spindle. The checkpoint is activated by kinetochores that are not attached to microtubules or are attached but not under tension and arrests cells at metaphase by inhibiting the anaphase-promoting complex (APC) and its co-activator Cdc20. Despite numerous studies, we still do not understand how the checkpoint proteins coordinate with each other to inhibit APCCdc20 activity.
Results
To ask how the checkpoint components induce metaphase arrest, we constructed fusions of checkpoint proteins and expressed them in the budding yeast, Saccharomyces cerevisiae, to mimic possible protein interactions during checkpoint activation. We found that expression of a Mad2-Mad3 protein fusion or non-covalently linked Mad2 and Mad3, but not the overexpression of the two separate proteins, induces metaphase arrest that is independent of functional kinetochores or other checkpoint proteins. We further showed that artificially tethering Mad2 to Cdc20 also arrests cells in metaphase independently of other checkpoint components.
Conclusion
Our results suggest that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, which is sufficient to inhibit APC activity and is the most downstream event in spindle checkpoint activation.
doi:10.1016/j.cub.2011.12.029
PMCID: PMC3277655  PMID: 22209528
22.  Metabolic differentiation in retinal cells 
Nature cell biology  2012;14(8):859-864.
Unlike healthy adult tissues, cancers produce energy mainly by aerobic glycolysis instead of oxidative phosphorylation1. This adaptation, called the Warburg effect, may be a feature of all dividing cells, both normal and cancerous2, or it may be specific to cancers3. Whether in a normally growing tissue during development, proliferating and postmitotic cells produce energy in fundamentally different ways is not known. Here we show in the embryonic Xenopus retina in vivo, that dividing progenitor cells depend less on oxidative phosphorylation for ATP production than non-dividing differentiated cells, and instead use glycogen to fuel aerobic glycolysis. The transition from glycolysis to oxidative phosphorylation is connected to the cell differentiation process. Glycolysis is indispensable for progenitor proliferation and biosynthesis, even when it is not used for ATP production. These results suggest that the Warburg effect can be a feature of normal proliferation in vivo, and that the regulation of glycolysis and oxidative phosphorylation is critical for normal development.
doi:10.1038/ncb2531
PMCID: PMC3442239  PMID: 22750943
23.  Suppression of Mitochondrial Electron Transport Chain Function in the Hypoxic Human Placenta: A Role for miRNA-210 and Protein Synthesis Inhibition 
PLoS ONE  2013;8(1):e55194.
Fetal growth is critically dependent on energy metabolism in the placenta, which drives active exchange of nutrients. Placental oxygen levels are therefore vital, and chronic hypoxia during pregnancy impairs fetal growth. Here we tested the hypothesis that placental hypoxia alters mitochondrial electron transport chain (ETS) function, and sought to identify underlying mechanisms. We cultured human placental cells under different oxygen concentrations. Mitochondrial respiration was measured, alongside levels of ETS complexes. Additionally, we studied placentas from sea-level and high-altitude pregnancies. After 4 d at 1% O2 (1.01 KPa), complex I-supported respiration was 57% and 37% lower, in trophoblast-like JEG3 cells and fibroblasts, respectively, compared with controls cultured at 21% O2 (21.24 KPa); complex IV-supported respiration was 22% and 30% lower. Correspondingly, complex I levels were 45% lower in placentas from high-altitude pregnancies than those from sea-level pregnancies. Expression of HIF-responsive microRNA-210 was increased in hypoxic fibroblasts and high-altitude placentas, whilst expression of its targets, iron-sulfur cluster scaffold (ISCU) and cytochrome c oxidase assembly protein (COX10), decreased. Moreover, protein synthesis inhibition, a feature of the high-altitude placenta, also suppressed ETS complex protein levels. Our results demonstrate that mitochondrial function is altered in hypoxic human placentas, with specific suppression of complexes I and IV compromising energy metabolism and potentially contributing to impaired fetal growth.
doi:10.1371/journal.pone.0055194
PMCID: PMC3559344  PMID: 23383105
24.  Water and sodium intake habits and status of ultra-endurance runners during a multi-stage ultra-marathon conducted in a hot ambient environment: an observational field based study 
Nutrition Journal  2013;12:13.
Background
Anecdotal evidence suggests ultra-runners may not be consuming sufficient water through foods and fluids to maintenance euhydration, and present sub-optimal sodium intakes, throughout multi-stage ultra-marathon (MSUM) competitions in the heat. Subsequently, the aims were primarily to assess water and sodium intake habits of recreational ultra-runners during a five stage 225 km semi self-sufficient MSUM conducted in a hot ambient environment (Tmax range: 32°C to 40°C); simultaneously to monitor serum sodium concentration, and hydration status using multiple hydration assessment techniques.
Methods
Total daily, pre-stage, during running, and post-stage water and sodium ingestion of ultra-endurance runners (UER, n = 74) and control (CON, n = 12) through foods and fluids were recorded on Stages 1 to 4 by trained dietetic researchers using dietary recall interview technique, and analysed through dietary analysis software. Body mass (BM), hydration status, and serum sodium concentration were determined pre- and post-Stages 1 to 5.
Results
Water (overall mean (SD): total daily 7.7 (1.5) L/day, during running 732 (183) ml/h) and sodium (total daily 3.9 (1.3) g/day, during running 270 (151) mg/L) ingestion did not differ between stages in UER (p < 0.001 vs. CON). Exercise-induced BM loss was 2.4 (1.2)% (p < 0.001). Pre- to post-stage BM gains were observed in 26% of UER along competition. Pre- and post-stage plasma osmolality remained within normal clinical reference range (280 to 303 mOsmol/kg) in the majority of UER (p > 0.05 vs. CON pre-stage). Asymptomatic hyponatraemia (<135 mmol/L) was evident pre- and post-stage in n = 8 UER, corresponding to 42% of sampled participants. Pre- and post-stage urine colour, urine osmolality and urine/plasma osmolality ratio increased (p < 0.001) as competition progressed in UER, with no change in CON. Plasma volume and extra-cellular water increased (p < 0.001) 22.8% and 9.2%, respectively, from pre-Stage 1 to 5 in UER, with no change in CON.
Conclusion
Water intake habits of ultra-runners during MSUM conducted in hot ambient conditions appear to be sufficient to maintain baseline euhydration levels. However, fluid over-consumption behaviours were evident along competition, irrespective of running speed and gender. Normonatraemia was observed in the majority of ultra-runners throughout MSUM, despite sodium ingestion under benchmark recommendations.
doi:10.1186/1475-2891-12-13
PMCID: PMC3554439  PMID: 23320854
Water; Drinking; Beverages; Total body water; Dehydration; Euhydration; Hyponatraemia; Carbohydrate
25.  ASYMMETRY IN SEXUAL PHEROMONES IS NOT REQUIRED FOR ASCOMYCETE MATING 
Current biology : CB  2011;21(16):1337-1346.
SUMMARY
Background
We investigated the determinants of sexual identity in the budding yeast, Saccharomyces cerevisiae. The higher fungi are divided into the Ascomycetes and the Basidiomycetes. Most Ascomycetes have two mating types: one (called α in yeasts and MAT1-1 in filamentous fungi) produces a small, unmodified, peptide pheromone, and the other (a in yeasts and MAT1-2 in filamentous fungi) produces a peptide pheromone conjugated to a C terminal farnesyl group that makes it very hydrophobic. In the Basidiomycetes, all pheromones are lipid-modified, and this difference is a distinguishing feature between the phyla. We asked whether the asymmetry in pheromone modification is required for successful mating in Ascomycetes.
Results
We cloned receptor and pheromone genes from a filamentous Ascomycete and a Basidiomycete and expressed these in the budding yeast, Saccharomyces cerevisiae, to generate novel, alternative mating pairs. We find that two yeast cells can mate even when both cells secrete a-like or α-like peptides. Importantly, this is true regardless of whether the cells express the a- or α-mating type loci, which control the expression of other, sex-specific genes, in addition to the pheromones and pheromone receptors.
Conclusions
We demonstrate that the asymmetric pheromone modification is not required for successful mating of ascomycete fungi and confirm that, in budding yeast, the primary determinants of mating are the specificity of the receptors and their corresponding pheromones.
doi:10.1016/j.cub.2011.06.054
PMCID: PMC3159855  PMID: 21835624

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