Measurement of H₂O₂ within living drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H<sub>2</sub>O<sub>2</sub> in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H<sub>2</sub>O<sub>2</sub> levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H<sub>2</sub>O<sub>2</sub> to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H<sub>2</sub>O<sub>2</sub> that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H<sub>2</sub>O<sub>2</sub> with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H<sub>2</sub>O<sub>2</sub> correlates with aging, it may not be causative

Labelling and Family Resemblance in the discrimination of polymorphous categories by pigeons

publication-status: Acceptedtypes: Article© 2011 Springer Verlag. This is a post print version of the article published in Animal Cognition, 2011, 14 (1), pp 21-34. The final publication is available at link.springer.comTwo experiments examined whether pigeons discriminate polymorphous categories on the basis of a single highly predictive feature or overall similarity. In the first experiment, pigeons were trained to discriminate between categories of photographs of complex real objects. Within these pictures, single features had been manipulated to produce a highly salient texture cue. Either the picture or the texture provided a reliable cue for discrimination during training, but in probe tests, the picture and texture cues were put into conflict. Some pigeons showed a significant tendency to discriminate on the basis of the picture cue (overall similarity or family resemblance), whereas others appeared to rely on the manipulated texture cue. The second experiment used artificial polymorphous categories in which one dimension of the stimulus provided a completely reliable cue to category membership, whereas three other dimensions provided cues that were individually unreliable but collectively provided a completely reliable basis for discrimination. Most pigeons came under the control of the reliable cue rather than the unreliable cues. A minority, however, came under the control of single dimensions from the unreliable set. We conclude that cue salience can be more important than cue reliability in determining what features will control behavior when multiple cues are available

Heterogeneous Response to a Quorum-Sensing Signal in the Luminescence of Individual Vibrio fischeri

The marine bacterium Vibrio fischeri regulates its bioluminescence through a quorum sensing mechanism: the bacterium releases diffusible small molecules (autoinducers) that accumulate in the environment as the population density increases. This accumulation of autoinducer (AI) eventually activates transcriptional regulators for bioluminescence as well as host colonization behaviors. Although V.fischeri quorum sensing has been extensively characterized in bulk populations, far less is known about how it performs at the level of the individual cell, where biochemical noise is likely to limit the precision of luminescence regulation. We have measured the time-dependence and AI-dependence of light production by individual V.fischeri cells that are immobilized in a perfusion chamber and supplied with a defined concentration of exogenous AI. We use low-light level microscopy to record and quantify the photon emission from the cells over periods of several hours as they respond to the introduction of AI. We observe an extremely heterogeneous response to the AI signal. Individual cells differ widely in the onset time for their luminescence and in their resulting brightness, even in the presence of high AI concentrations that saturate the light output from a bulk population. The observed heterogeneity shows that although a given concentration of quorum signal may determine the average light output from a population of cells, it provides far weaker control over the luminescence output of each individual cell

A Salmonella toxin promotes persister formation through acetylation of tRNA

The recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth

Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H₂O₂ in living <i>Drosophila</i>

The role of hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; in vivo. Here we describe a method for assessing changes in H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; within the mitochondrial matrix of living &lt;i&gt;Drosophila&lt;/i&gt;. We use a ratiometric mass spectrometry probe, MitoB ((3-hydroxybenzyl)triphenylphosphonium bromide), which contains a triphenylphosphonium cation component that drives its accumulation within mitochondria. The arylboronic moiety of MitoB reacts with H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; to form a phenol product, MitoP. On injection into the fly, MitoB is rapidly taken up by mitochondria and the extent of its conversion to MitoP enables the quantification of H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;. To assess MitoB conversion to MitoP, the compounds are extracted and the MitoP/MitoB ratio is quantified by liquid chromatography–tandem mass spectrometry relative to deuterated internal standards. This method facilitates the investigation of mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; production in mouse and cell culture studies

Strategic Plan for Lung Vascular Research: An NHLBI-ORDR Workshop Report

The Division of Lung Diseases of the National Heart, Lung, and Blood Institute, with the Office of Rare Diseases Research, held a workshop to identify priority areas and strategic goals to enhance and accelerate research that will result in improved understanding of the lung vasculature, translational research needs, and ultimately the care of patients with pulmonary vascular diseases. Multidisciplinary experts with diverse experience in laboratory, translational, and clinical studies identified seven priority areas and discussed limitations in our current knowledge, technologies, and approaches. The focus for future research efforts include the following: (1) better characterizing vascular genotype–phenotype relationships and incorporating systems biology approaches when appropriate; (2) advancing our understanding of pulmonary vascular metabolic regulatory signaling in health and disease; (3) expanding our knowledge of the biologic relationships between the lung circulation and circulating elements, systemic vascular function, and right heart function and disease; (4) improving translational research for identifying disease-modifying therapies for the pulmonary hypertensive diseases; (5) establishing an appropriate and effective platform for advancing translational findings into clinical studies testing; and (6) developing the specific technologies and tools that will be enabling for these goals, such as question-guided imaging techniques and lung vascular investigator training programs. Recommendations from this workshop will be used within the Lung Vascular Biology and Disease Extramural Research Program for planning and strategic implementation purposes

C. elegans metallothioneins : response to and defence against ROS toxicity

The genome of the nematode Caenorhabditis elegans encodes for two multifunctional metal binding metallothioneins (MTs), CeMT-1 and CeMT-2. Here we applied qPCR to identify a transcriptional up-regulation following the exposure to free radical generators (ROS) paraquat or hydrogen peroxide, a trend that was confirmed with Pmtl::GFP expressing alleles. The deletion of the MT loci resulted in an elevation of in vivo levels of hydrogen peroxide and exposure to ROS caused a reduction in total egg production, growth and life span in wild type nematodes, effects that were particularly pronounced in the CeMT-2 and double knockout. Moreover, in vitro incubation of recombinant MTs with hydrogen peroxide demonstrated the presence of direct oxidation of the CeMTs, with zinc released from both isoforms and concomitant formation of intra-molecular disulfides. Finally, metabolic profiling (metabolomic) analysis of wild type and MT knockouts in the presence/absence of oxidative stressors, confirmed the overall trend described by the other experiments, and identified 2-aminoadipate as a potential novel small-molecule marker of oxidative stress. In summary, this study highlights that C. elegans metallothioneins scavenge and protect against reactive oxygen species and potentially against oxidative stress, with CeMT-2 being more effective than CeMT-1