The ROS wheel: refining ROS transcriptional footprints

In the last decade, microarray studies have delivered extensive inventories of transcriptome-wide changes in messenger RNA levels provoked by various types of oxidative stress in Arabidopsis (Arabidopsis thaliana). Previous cross-study comparisons indicated how different types of reactive oxygen species (ROS) and their subcellular accumulation sites are able to reshape the transcriptome in specific manners. However, these analyses often employed simplistic statistical frameworks that are not compatible with large-scale analyses. Here, we reanalyzed a total of 79 Affymetrix ATH1 microarray studies of redox homeostasis perturbation experiments. To create hierarchy in such a high number of transcriptomic data sets, all transcriptional profiles were clustered on the overlap extent of their differentially expressed transcripts. Subsequently, meta-analysis determined a single magnitude of differential expression across studies and identified common transcriptional footprints per cluster. The resulting transcriptional footprints revealed the regulation of various metabolic pathways and gene families. The RESPIRATORY BURST OXIDASE HOMOLOG F-mediated respiratory burst had a major impact and was a converging point among several studies. Conversely, the timing of the oxidative stress response was a determining factor in shaping different transcriptome footprints. Our study emphasizes the need to interpret transcriptomic data sets in a systematic context, where initial, specific stress triggers can converge to common, aspecific transcriptional changes. We believe that these refined transcriptional footprints provide a valuable resource for assessing the involvement of ROS in biological processes in plants

Opposing effects of TIGAR- and RAC1-derived ROS on Wnt-driven proliferation in the mouse intestine

Reactive oxygen species (ROS) participate in numerous cell responses, including proliferation, DNA damage, and cell death. Based on these disparate activities, both promotion and inhibition of ROS have been proposed for cancer therapy. However, how the ROS response is determined is not clear. We examined the activities of ROS in a model of Apc deletion, where loss of the Wnt target gene Myc both rescues APC loss and prevents ROS accumulation. Following APC loss, Myc has been shown to up-regulate RAC1 to promote proliferative ROS through NADPH oxidase (NOX). However, APC loss also increased the expression of TIGAR, which functions to limit ROS. To explore this paradox, we used three-dimensional (3D) cultures and in vivo models to show that deletion of TIGAR increased ROS damage and inhibited proliferation. These responses were suppressed by limiting damaging ROS but enhanced by lowering proproliferative NOX-derived ROS. Despite having opposing effects on ROS levels, loss of TIGAR and RAC1 cooperated to suppress intestinal proliferation following APC loss. Our results indicate that the pro- and anti-proliferative effects of ROS can be independently modulated in the same cell, with two key targets in the Wnt pathway functioning to integrate the different ROS signals for optimal cell proliferation

The interplay of reactive oxygen species, hypoxia, inflammation, and sirtuins in cancer initiation and progression

The presence of ROS is a constant feature in living cells metabolizing O2. ROS concentration and compartmentation determine their physiological or pathological effects. ROS overproduction is a feature of cancer cells and plays several roles during the natural history of malignant tumor. ROS continuously contribute to each step of cancerogenesis, from the initiation to the malignant progression, acting directly or indirectly. In this review, we will (a) underline the role of ROS in the pathway leading a normal cell to tumor transformation and progression, (b) define the multiple roles of ROS during the natural history of a tumor, (c) conciliate many conflicting data about harmful or beneficial effects of ROS, (d) rethink the importance of oncogene and tumor suppressor gene mutations in relation to the malignant progression, and (e) collocate all the cancer hallmarks in a mechanistic sequence which could represent a "physiological" response to the initial growth of a transformed stem/pluripotent cell, defining also the role of ROS in each hallmark. We will provide a simplified sketch about the relationships between ROS and cancer. The attention will be focused on the contribution of ROS to the signaling of HIF, NFκB, and Sirtuins as a leitmotif of cancer initiation and progressi

Coordinated transcriptional regulation between a reactive oxygen species-responsive gene network and the circadian clock in Arabidopsis thaliana : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Plant Biology, New Zealand

Most organisms have evolved endogenous biological clocks as internal timekeepers to fine-tune physiological processes to the external environment. Energetic cycles such as photosynthesis and glycolytic cycles are physiological processes that have been shown to be under clock control. This work sought to understand the mechanism of the synchrony between the circadian oscillator and products of energetic cycles. The fact that plants rely on photosynthesis for survival,and that photosynthesis relies on the sun, this would have meant that oxygen levels would have fluctuated across the day. A common by-product of oxygen metabolism and photosynthesis is the Reactive Oxygen Species(ROS). Evidence has proposed ROS as regulators of cellular signaling and plant development. However, if ROS levels are left unmanaged, it may cause oxidative stress in organisms, which could damage cellular components and disrupt normal mechanisms of cellular signaling. Therefore, it is advantageous for plants to be able to anticipate such periodic burst in ROS. My research investigates the role of the circadian clock in regulating ROS homeostasis in the model plant Arabidopsis thaliana. I found that ROS production and scavenging wax and wane in a periodic manner under diurnal and circadian conditions. Not only that, at the transcriptional level, ROS7 responsive genes exhibited time-of-day specific phases under diurnal and circadian conditions,suggesting the role of the circadian clock in ROS signaling. Mutations in the core-clock regulator, CIRCADIAN3 CLOCK3 ASSOCIATED3 1 (CCA1), affect both the transcriptional regulation of ROS genes and ROS homeostasis. Furthermore, mis- expressions of other clock genes such as EARLY3 FLOWERING3 33 (ELF3), LUX3 ARRHYTHMO3 (LUX) and TIMING3 OF3 CAB3 EXPRESSION3 13 (TOC1) also have profound effects on ROS signaling and homeostasis, thus suggesting a global clock effect on ROS networks. Taken together, CCA1 is proposed as a master regulator of ROS signaling where the response to oxidative stress is dependent on the time of CCA1 expression. Plants exhibit the strongest response at dawn, the time when CCA1 peaks. Moreover, CCA1 can associate to the Evening Element or CCA17Binding Site on promoters of ROS genes in vivo to coordinate transcription. A common feature of circadian clocks is the presence of multiple interlocked transcriptional feedback loops. It is shown here that the oscillator incorporates ROS as a component of the loop where ROS signals could feed back to affect circadian behavior by changing CCA1 and TOC1 transcription. The clock regulates a plethora of output pathways; particularly the transcription of an output gene FLAVIN3BINDING3KELCH3REPEAT3FHBOX31(FKF1) is affected by ROS signals. Temporal coordination of ROS signaling by CCA1 and the reciprocal control of circadian behavior by ROS revealed a mechanistic link of which plants match their physiology to the environment to confer fitness.Page v (Acknowledgements) has been removed from the published version at the author's request

Inside the labyrinth : the thematics of space in the fiction of Paola Capriolo

In Capriolo's fiction we see, above all, the centrality of place in the minds and lives of her protagonists, often linked with the idea of the labyrinth: labyrinth as endless tortuous passageways, enclosed place, puzzle, quest. The stories, built around the obsessions of their protagonists, transcend normal temporal and spatial boundaries, and reflect the labyrinth in various forms: as a physical maze; as mirror reflections, sometimes infinitely receding; as a remote, closed-off place; and as a metaphor suggesting confusion of the mind or of ideas. As well as a considerable degree of overlap within these areas, there is a pervading, underlying sense of ambiguity, and the idea of permanence and of eternal 'being'

Regulation of intracellular cyclic GMP concentration by light and calcium in electropermeabilized rod photoreceptors.

Abstract This study examines the regulation of cGMP by illumination and by calcium during signal transduction in vertebrate retinal photoreceptor cells. We employed an electropermeabilized rod outer segment (EP-ROS) preparation which permits perfusion of low molecular weight compounds into the cytosol while retaining many of the features of physiologically competent, intact rod outer segments (ROS). When nucleotide-depleted EP-ROS were incubated with MgGTP, time- and dose-dependent increases in intracellular cGMP levels were observed. The steady state cGMP concentration in EP-ROS (0.007 mol cGMP per mol rhodopsin) approached the cGMP concentration in intact ROS. Flash illumination of EP-ROS in a 250-nM free calcium medium resulted in a transient decrease in cGMP levels; this occurred in the absence of changes in calcium concentration. The kinetics of the cGMP response to flash illumination of EP-ROS were similar to that of intact ROS. To further examine the effects of calcium on cGMP metabolism, dark-adapted EP-ROS were incubated with MgGTP containing various concentrations of calcium. We observed a twofold increase in cGMP steady state levels as the free calcium was lowered from 1 μM to 20 nM; this increase was comparable to the behavior of intact ROS. Measurements of guanylate cyclase activity in EP-ROS showed a 3.5-fold increase in activity over this range of calcium concentrations, indicating a retention of calcium regulation of guanylate cyclase in EP-ROS preparations. Flash illumination of EP-ROS in either a 50- or 250-nM free calcium medium revealed a slowing of the recovery time course at the lower calcium concentration. This observation conflicts with any hypothesis whereby a reduction in free calcium concentration hastens the recovery of cytoplasmic cGMP levels, either by stimulating guanylate cyclase activity or by inhibiting phosphodiesterase activity. We conclude that changes in the intracellular calcium concentration during visual transduction may have more complex effects on the recovery of the photoresponse than can be accounted for solely by guanylate cyclase activation

Siglec-E promotes β2-integrin-dependent NADPH oxidase activation to suppress neutrophil recruitment to the lung

Siglec-E is a sialic acid-binding Ig-like lectin expressed on murine myeloid cells. It has recently been shown to function as a negative regulator of β2-integrin-dependent neutrophil recruitment to the lung following exposure to lipopolysaccharide (LPS). Here, we demonstrate that siglec-E promoted neutrophil production of reactive oxygen species (ROS) following CD11b β2-integrin ligation with fibrinogen in a sialic acid-dependent manner, but it had no effect on ROS triggered by a variety of other stimulants. Siglec-E promotion of ROS was likely mediated via Akt activation, because siglec-E-deficient neutrophils plated on fibrinogen exhibited reduced phosphorylation of Akt, and the Akt inhibitor, MK2206, blocked fibrinogen-induced ROS. In vivo imaging showed that siglec-E also promoted ROS in acutely inflamed lungs following exposure of mice to LPS. Importantly, siglec-E-promoted ROS were required for its inhibitory function, as the NADPH oxidase inhibitor, apocynin, reversed the siglec-E-mediated suppression of neutrophil recruitment and blocked neutrophil ROS production in vitro. Taken together, these results demonstrate that siglec-E functions as an inhibitory receptor of neutrophils via positive regulation of NADPH oxidase activation and ROS production. Our findings have implications for the inhibitory role of siglec-9 on human neutrophils in sepsis and acute lung injury

Mitochondrial ROS cause motor deficits induced by synaptic inactivity:implications for synapse pruning

Developmental synapse pruning refines burgeoning connectomes. The basic mechanisms of mitochondrial reactive oxygen species (ROS) production suggest they select inactive synapses for pruning: whether they do so is unknown. To begin to unravel whether mitochondrial ROS regulate pruning, we made the local consequences of neuromuscular junction (NMJ) pruning detectable as motor deficits by using disparate exogenous and endogenous models to induce synaptic inactivity en masse in developing Xenopus laevis tadpoles. We resolved whether: (1) synaptic inactivity increases mitochondrial ROS; and (2) antioxidants rescue synaptic inactivity induced motor deficits. Regardless of whether it was achieved with muscle (α-bugarotoxin), nerve (α-latrotoxin) targeted neurotoxins or an endogenous pruning cue (SPARC), synaptic inactivity increased mitochondrial ROS in vivo. The manganese porphyrins MnTE-2-PyP5+ and/or MnTnBuOE-2-PyP5+ blocked mitochondrial ROS to significantly reduce neurotoxin and endogenous pruning cue induced motor deficits. Selectively inducing mitochondrial ROS—using mitochondria-targeted Paraquat (MitoPQ)—recapitulated synaptic inactivity induced motor deficits; which were significantly reduced by blocking mitochondrial ROS with MnTnBuOE-2-PyP5+. We unveil mitochondrial ROS as synaptic activity sentinels that regulate the phenotypical consequences of forced synaptic inactivity at the NMJ. Our novel results are relevant to pruning because synaptic inactivity is one of its defining features