Exposure of oligodendrocytes to media from injured astrocytes resulted in an increase in oligodendrocyte NADPH oxidase activity. Inhibition of NADPH oxidase activation was sufficient to attenuate oligodendrocyte death in vitro and at 1 week following spinal cord injury, suggesting that excitotoxicity of oligodendrocytes after trauma is dependent on the intrinsic activation of the NADPH oxidase enzyme. Acute administration of the NADPH oxidase inhibitor apocynin and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate channel blocker 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione significantly improved locomotor behavior and preserved descending axon fibers following spinal cord injury. These studies lead to a better understanding of oligodendrocyte death after trauma and identify potential therapeutic targets in disorders involving demyelination and oligodendrocyte death. Spinal cord injury is a debilitating neurological disorder that initiates a cascade of cellular events that result in a period of secondary damage that can last for months after the initial trauma. The ensuing outcome of these prolonged cellular perturbations is the induction of neuronal and glial cell death through excitotoxic mechanisms and subsequent free radical production. We have previously shown that astrocytes can directly induce oligodendrocyte death following trauma, but the mechanisms regulating this process within the oligodendrocyte remain unclear. Here we provide evidence demonstrating that astrocytes directly regulate oligodendrocyte death after trauma by inducing activation of NADPH oxidase within oligodendrocytes. Spinal cord injury resulted in a significant increase in oxidative damage which correlated with elevated expression of the gp91 phox subunit of the NADPH oxidase enzyme. Immunohistochemical analysis confirmed the presence of gp91 phox in oligodendrocytes  Oxidative stress and reactive oxygen species (ROS) production play a predominant role in the secondary injury mechanisms of spinal cord injury (SCI) . Under nOligodendrocytes are particularly susceptible to oxidative stress as a result of their poor antioxidant defense system -11. The Previous work by our laboratory demonstrated that astrocytes can directly influence glutamate-mediated oligodendrocyte toxicity after injury through activation of the astroglial nuclear factor – κB (NF – κB) transcription factor . Using aCybb-/- mice lacking gp91 phox (B6.129S6-Cybbtm1Din/J) were obtained from the Jackson Laboratory . Mice were housed in a 12 h light/dark cycle in a virus/antigen free environment. All mice had free access to food and water throughout the course of the study.Female mice expressing a dominant negative form of the IκBα protein in GFAP expressing cells (GFAP-IκBα-dn) were produced and characterized as previously described -20. WildAll surgical procedures were performed in the Surgical Core Facility of The Miami Project to Cure Paralysis, according to the protocols and guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Miami. The studies shown here were approved by the University of Miami IACUC. Adult female mice 3-5 months of age (20-24g in weight) underwent a moderate to severe contusion injury to the T9 segment of spinal cord using the Electromagnetic Spinal Cord Injury Device (Ohio State University). In brief, mice received a laminectomy between T8 and T10 vertebral segments. Mice were injured with a spinal cord displacement of 0.5 - 0.6 cm. Following injury, mice were treated with topical antibiotics and subcutaneous Ringer’s solution to reduce fluid loss. Daily administration of Ringer’s solution to prevent dehydration and gentamicin to prevent urinary tract infections was given for the 5 days after injury or as needed. Bladder expressions were performed twice daily until the reacquisition of bladder control.2 for 10 days, at which time the fetal bovine serum was replaced with horse serum. After 14 days, astrocyte cultures were treated with dibutyryl cyclic adenosine monophosphate  to enhance cell proliferation and maturation . Slides were treated with species-specific fluorescent secondary antibodies  and mounted using DAPI mounting medium . Sections lacking primary antibody treatment were used as negative controls.Serial spinal cord sections spaced 500 µM apart were counted using Stereo Investigator (MBF Biosciences) software with a 63x objective under blind conditions. For each section, a 40 x 40 µM counting frame was used to count cells at 200 µM intervals. Dc Protein Assay kit . Equal amounts of protein (30 µg) was separated by SDS-PAGE and transferred to a nitrocellulose membrane (BioRad). The blots probed with primary antibody [gp91 phox  β-tubulin ] in blocking solution overnight at 4°C, and for 1 hr at room temperature with a species-specific horseradish peroxidase (HRP)-conjugated secondary antibody . Protein densitometry analysis was performed using Quantity One (BioRad) software.A 1 cm segment of spinal cord centered on the lesion site was homogenized in lysis buffer  supplemented with Complete protease inhibitor (Roche). Protein concentrations were determined by Lowry assay using the The Oxyblot was conducted using the Oxyblot Oxidizied Protein Assay Kit  according to manufacturer’s specifications. Apocynin was dissolved in 1% EtOH and saline. Mice received intraperitoneal injections of vehicle  or 10 mg/kg apocynin 1 hr after SCI. Mice received injections of 10 mg/kg apocynin or vehicle daily for 1 week. NBQX and apocynin were dissolved in water and apocynin was heated to 65°C until completely in solution. Mice received intraperitoneal injections of vehicle (water), 30 mg/kg NBQX, 10 mg/kg apocynin, or NBQX + apocynin 1 hr after SCI, and continued to receive daily doses for 1 week.Mice were placed in an open field for 4 minutes, and their hind limb locomotor activity was scored according to the Basso Mouse Scale . BehavioSets of cross-sections at a spacing of 500 µm and ranging from 3.5 mm rostral to 3.5 mm caudal of the lesion epicenter were stained with Luxol Fast Blue and analyzed using Stereo Investigator (MBF Biosciences) software. Intact white matter and the lesion site were contoured on each section, and the total volume was calculated using stereological principles.Spinal cord sections 3 mm caudal to the lesion site were imaged using confocal microscopy, and the relative fluorescence intensity was quantified by ImageJ software. The maximum background threshold was determined for each image and set as the minimum threshold for intensity quantification.Statistical assessment was performed using one or two-way ANOVA, followed by appropriate post-tests. Student’s T test was used for single comparisons. Chronic behavioral evaluations were calculated using the Kruskal-Wallis significance test to assess if the treatments resulted in an overall significant change from controls. P values equal to or less than 0.05 will be considered statistically significant. Error bars indicate the standard error of the mean. Previously we determined that mice lacking functional NF-κB in astrocytes possessed a significant reduction in the amount of oligodendrocyte death at 1 week following a moderate contusion SCI . We dete NADPH oxidase activation has been identified as a key mediator of free radical production and subsequent cell death ,31. To dExcessive AMPA receptor activation on oligodendrocytes induces cell death through excitotoxic mechanisms and subsequent ROS production ,32,33. HTo further confirm the function of the NADPH oxidase enzyme within oligodendrocytes after injury, we treated oligodendrocyte cultures with media from injured astrocyte cultures known to induce glutamate excitotoxic oligodendrocyte death  and perfCybb-/- mice, which are deficient in the gp91 phox subunit of NADPH oxidase, were treated with injured WT or GFAP-IκBα-dn astrocyte media. Cytotoxicity was assessed using a trypan blue exclusion assay in which dying cells will be permeable to the dye  from controls, suggesting that injured astrocyte-mediated oligodendrocyte death is dependent upon NADPH oxidase activation within oligodendrocytes.To determine if NADPH oxidase activation within oligodendrocyte is necessary for glutamate excitotoxicity induced by injured astrocytes , oligode the dye . Injured-/- mice with elevated extracellular glutamate concentrations in the presence of varying levels of zinc  for 30 min followed by treatment with injured astrocyte media for 3 hrs . Apocyniin vivo models of neurological disorders using apocynin, dosages ranging from 4 mg/kg to 300 mg/kg were shown to have significant improvements on behavior and survival [To determine if apocynin treatment is capable of reducing oligodendrocyte death following SCI, WT mice were subjected to a moderate spinal cord contusion at the thoracic level T9. One hour after injury, mice received a 10 mg/kg intraperitoneal injection of apocynin, and continued to receive daily treatments with apocynin for one week. In other survival -40. In oSeveral reports have shown that axonal AMPA receptor activation is necessary for axon dysfunction and degeneration ,42. StudThis data shows that combined AMPA receptor and NADPH oxidase inhibition results in a significant recovery in hind limb locomotor behavior following SCI. Apocynin was the only single treatment that was able to induce recovery in the BMS assessment, providing confirmatory data demonstrating that targeting oligodendrocyte preservation is important for the recovery of motor movement but alone is not sufficient to induce large changes in motor improvement. 3) when compared to uninjured controls . Total spinal cord volume was not significantly changed between treatment groups, suggesting that the reduction in myelin volume was the result of myelin loss and not a reduction in spinal cord volume in the vehicle treated condition. Quantification of lesion volume revealed a significantly reduced lesion in combined treatment animals (.577 ± .19 mm3) compared to vehicle controls , suggesting that any behavioral recovery following NBQX or apocynin treatment was probably due to the preservation of myelin and not a reduction in overall lesion size. Surprisingly, NBQX treated animals had a lesion volume comparable to vehicle treated controls, indicating, at least in this study, NBQX alone did not reduce necrotic damage. The combined treatment effectively reduced white matter damage and lesion volume, indicating that axon damage was potentially reduced.  To investigate whether AMPA and NADPH oxidase inhibition reduces white matter damage and lesion volume after SCI, spinal cords were stained for Luxol Fast Blue to visualize intact myelin and lesion size at 12 weeks after injury. Volume analysis revealed a significant reduction of intact myelin in vehicle treated animals (4.64 ± .83 mmTract tracing studies have demonstrated the projection of serotonergic axons from the raphe nuclei in the brainstem throughout the spinal cord along the raphespinal tract adjacent to the gray matter lateral horn . These dIn a previous study, we showed that injured astrocytes can influence oligodendrocyte survival through the regulation of extracellular glutamate and zinc levels . We showIn order to follow-up with these initial studies, we investigated the oxidative stress response in WT and GFAP-IκBα-dn animals following a moderate contusion SCI. Peak expression of gp91 phox, the catalytic subunit of the NADPH oxidase enzyme, following SCI occurs at 1 week after injury and coincides with the second wave of apoptotic oligodendrocyte death -50. In oDespite possessing significantly lower levels of gp91 phox protein expression than WT mice, SCI resulted in a several fold increase in the amount of gp91 phox in GFAP-IκBα-dn animals. The reason for this increase in unclear, however, the expression of gp91 phox is not limited to astrocytes. Microglia, neurons, endothelial cells, fibroblasts, and macrophages all express NADPH oxidase as well. It is possible that the increase in gp91 phox observed in the GFAP-IκBα-dn group is a compensatory mechanism influenced by these additional cell types, or it could be that the astrocytes were able to increase the expression of gp91 phox through an alternative signaling pathway not involving NF-κB. Additional studies would need to be performed in order to elucidate these complex cellular interactions.2+-permeable AMPA receptors. Additional studies have shown that high levels of extracellular glutamate induce excitotoxic cell death involving the production of ROS ,51. SincCybb-/- oligodendrocytes devoid of gp91 phox with injured astrocyte media. Oligodendrocytes lacking gp91 phox were protected from injured astrocyte-mediated cell death. Excitotoxicity in neurons stimulates a rapid production of superoxide resulting in cell death . Brennanin vitro results were verified in vivo following a moderate contusion SCI. Daily administration of 10 mg/kg apocynin was able to reduce oligodendrocyte death by 75% at 1 week following SCI. Experiments using a vascular clamp model of spinal cord injury showed that administration of a 5 mg/kg apocynin at 1 and 6 hrs after injury completely blocked cell death within the spinal cord and induced a significant increase in locomotor behavior by 4 days after injury [These r injury . Using ar injury  and our Contrary to the NF-κB transcription factor, inhibition of the NADPH oxidase enzyme alone did not result in drastic improvements in functional recovery. The reason for this result is unclear, but NF-κB regulates a variety of proteins involved in the inflammatory response which could be detrimental to axon function . InteresRecognizing that oligodendrocyte preservation alone is likely insufficient to promote functional recovery after contusive SCI, we employed a combinatorial treatment strategy to reduce oligodendrocyte death and block axonal damage. Axonal AMPA receptor activation has been identified as a mediator of axon damage ,42, and Contrary to the individual treatments, combined treatment of apocynin and NBQX significantly reduced lesion size, in addition to preserving white matter, potentially due to an additive effect of AMPA receptor and NADPH oxidase inactivation in reducing free radical production and axonal damage as a result of calpain inhibition . In addiOf particular note is the preservation of serotonergic fibers below the level of the lesion by the combined treatment of NBQX and apocynin. Serotonin has previously been linked to the regulation of patterned walking in adult animals ,52 and iin vitro and in vivo. This form of combinatorial strategy could lead to novel therapeutics in the field of medicinal research. This concept of targeting two proteins in similar signaling pathways has previously been established as vertical inhibition. Previous work performed by Werzoma et al.  showed tThese studies demonstrate that oligodendrocytes express NADPH oxidase, and activation of this enzyme is triggered by injured astrocytes. Pharmacological and genetic inhibition of NADPH oxidase rendered oligodendrocytes immune to the cytotoxic effects of injured astrocytes. Combinatorial strategies inhibiting NADPH oxidase and the AMPA receptor resulted in significant improvements in functional recovery, reduced white matter damage, and improved axon sparing following SCI. These studies provide a novel understanding of the ability of astrocytes to regulate oligodendrocyte fate following SCI, and provide valuable insight into previously unknown mechanisms governing oligodendrocyte death following trauma.


Schwann cells (SCs) are the cell type responsible for the formation of the myelin sheath in the peripheral nervous system (PNS). As retinoic acid (RA) and other retinoids have a profound effect as regulators of the myelination program, we sought to investigate how their nuclear receptors levels were regulated in this cell type.de novo protein synthesis and did not involve changes in mRNA stability. On the other hand, RAR-β mRNA levels were only slightly modulated by axonal contact mimickers, while RA produced a strong transcriptional upregulation that was independent of de novo protein synthesis without changes in mRNA stability.In the present study, by using Schwann cells primary cultures from neonatal Wistar rat pups, as well as myelinating cocultures of Schwann cells with embryonic rat dorsal root ganglion sensory neurons, we have found that sustained expression of RXR-γ depends on the continuous presence of a labile activator, while axonal contact mimickers produced an increase in RXR-γ mRNA and protein levels, increment that could be prevented by RA. The upregulation by axonal contact mimickers and the transcriptional downregulation by RA were dependent on All together, our results show that retinoid receptors are regulated in a complex manner in Schwann cells, suggesting that they could have a prominent role as regulators of Schwann cell physiology. All-trans-retinoic acid (RA) and other retinoids are potent regulators of morphogenesis, cell growth and differentiation, especially in the nervous system, where their abundance is comparatively very high Most of the studies on RA functions on the nervous system relate to its function as a morphogen during neural tube development or as a differentiation factor for central nervous system (CNS) neurons, but RA signaling also occurs in the peripheral nervous system (PNS). In the sciatic nerve of adult rats, all the required components of the RA signaling pathway  are expressed in vitro by using a myelinating DRGN/SC coculture system, as well as in vivo during the development of the sciatic nerve, RA is a strong inhibitor of PNS myelin formation through two different mechanisms, one involving Egr2  upregulation, which is dependent on retinoid X receptor (RXR) activation, and another one that implies MAG downregulation through binding of the retinoid to retinoic acid receptor (RAR).On the other hand, RA has been shown to induce differentiation of neuroblastoma cells towards a Schwann cell-resembling phenotype All-trans-RA binds with high affinity to RARs and, with a lower affinity, to RXRs, each one being codified by three separate genes . Another retinoid, 9-cis-RA presents a similar affinity in its binding to both receptors, while the so called rexinoids are able to differentially activate RXRs. RARs function only as heterodimers with RXRs, regulating the transcription of the genes that present retinoic acid response elements (RAREs) in their promoters. Gene regulation by rexinoids through RXR can be mediated by retinoid X response elements (RXREs) present in their promoters to which they bind as RXR-RXR homodimers, as well as by acting as heterodimers of RXR in combination with several members of the nuclear receptor superfamily The members of the nuclear receptor superfamily act as ligand-dependent transcription factors that regulate the transcription of the genes that contain specific hormone response elements (HREs) in their promoters. The retinoids exert their actions through their binding to two groups of nuclear receptors. In addition to the concentration of retinoids and rexinoids, the ability of a particular cell type to respond to these hormones also depends on the relative expression levels of the different receptors present at this particular cell type. The pleiotropic effects of retinoids in development can partially be explained by the patterns of expression of the various RAR and RXR genes As SC differentiation is under the control of retinoids, we sought to investigate if retinoid receptors levels were also regulated in this cell type during its differentiation, as well as by the retinoid itself. Using both purified SC cultures, as well as myelinating cocultures of SCs with sensory DRGNs, we have been able to show that RXR-γ is transcriptionally upregulated by axonal contact, while RAR-β, as well as RXR-γ levels are differentially regulated by RA in SCs. RA produces a strong upregulation of RAR-β, while inhibiting RXR-γ expression.All animal work was done in compliance with the European Community Law (86/609/EEC) and the Spanish law (R.D. 1201/ 2005). All experimental protocols were approved by the Ethics Committee of the Consejo Superior de Investigaciones Cientificas .SCs were isolated and purified as previously indicated Purified DRGN and SC cultures were prepared by using methods previously described SCs were isolated from the sciatic nerves of 4-day-old rat pups as previously described Treatments with the different retinoids  were initiated at the time of ascorbic acid addition , and replenished with every feeding, together with new ascorbic acid, every 2 to 3 days. Sister control cultures received an equivalent amount of vehicle (0.01% ethanol for the 1 µM RA treatments).http://rsbweb.nih.gov/ij/).Samples were prepared for Western blot analysis by homogenization in radioimmunoprecipitation assay (RIPA) buffer [PBS with 1% Nonidet P-40/0.5% deoxycholate/0.1% sodium dodecyl sulfate (SDS)/1 mM PMSF/Complete protease inhibitor tablets (Roche)] followed by high-speed centrifugation. Protein determination was made by using the BCA™ Protein Assay Kit (Pierce). Equivalent amounts of total protein extract from each sample were electrophoretically separated on 10–15% discontinuous acrylamide gels, transferred to pure nitrocellulose membranes , and the equal loading and transfer of the samples was monitored by Ponceau staining of the membranes. The different proteins were visualized by incubation with specific antibodies overnight at 4°C, followed by incubation with a secondary antibody for two hours at room temperature. The rabbit polyclonal antibody against RXR-γ (Anaspec) was used at a dilution of 1∶1,000. Secondary horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoResearch) were used at a dilution of 1∶10,000. The blots were developed by chemiluminescence  as indicated by the manufacturer. Blots were quantitated with the imaging and analysis software ImageJ 1.42d  and quantitated with a Nanodrop™ spectrophotometer (Thermo Scientific). 1 µg total RNA from each sample was reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad) and the relative levels of the genes of interest determined by quantitative real time PCR using FastStart Universal SYBR Green Master (ROX) mix (Roche) in a Mx3005P instrument (Stratagene). Every measure was always performed in duplicate (two PCR reactions from the same RT sample) and the average of those technical replicates was considered as the Chttp://www.macvector.com/) coupled with Amplify software (http://engels.genetics.wisc.edu/amplify/). Primers were designed from common sequences in the rat and mouse genes and, when possible (multiexonic genes), spanning two consecutives exons to avoid interference from possible genomic DNA contamination. For monoexonic genes, a DNase treatment of the samples was performed prior to cDNA sysnthesis. In all cases, primers were first tested by conventional RT-PCR to produce a single amplicon of the correct size, followed by Q-RT-PCR to avoid primer-dimer artifacts and ensure a lineal range of quantitation. When several mRNA splice variants were present for a given gene, the primers were always designed to recognize all known isoforms. The primers used are indicated in Supp. Info. The primers were designed by using the MacVector suite  using specific oligonucleotides for the genes of interest as primers (the same reverse primer used in the subsequent PCR). hnRNA levels were determined by using primers from an exon and a contiguous intron (exon 3 and intron III for RAR-β and exon 2 and intron II for RXR-γ), so DNA amplification only could occur from newly formed, immature non-spliced RNA.t-test (unpaired) analysis using StatPlus:mac LE2009 coupled with Microsoft Excel:mac 2008. Unless indicated otherwise, all comparisons were made against their respective controls. Throughout the test, differences were considered as significant when p<0.05. In the Figures, three different levels of significance  were considered.Throughout the whole article, data shown is representative of at least two separate experiments performed each time at least in duplicate . Significance was determined by Student's We have recently shown that retinoic acid regulates myelin formation in the peripheral nervous system. In order to analyze which receptors were present in SCs, we performed real time RT-Q-PCR from primary Schwann cell cultures compared to primary DRG sensory neurons and total brain. In general, with the exception of RAR-β, all retinoid receptors, RARs and RXRs, were expressed by isolated Schwann cells at levels comparable or superior to DRG sensory neurons or total brain, which is known to express all retinoid receptors at relatively high levels .Interestingly, not all retinoid receptors were homogenously maintained during the different stages of myelin formation or during sciatic nerve development in vitro by the exposition to cAMP-elevating agents These results suggest that cell-to-cell contact upregulates RXR-γ expression, either in the SCs, in the DRGNs or in both cell types. As SCs express higher levels of retinoid receptors than DRGNs, and it is well known that neuronal contact strongly influence its physiology, we decided to analyze whether axonal contact could upregulate RXR-γ expression in SCs. DRGNs are pseudo-unipolar neurons; they present a bifurcated axon with central and peripheral branches and no dendrites. Axonal contact in SCs can be partially mimicked Although forskolin is used frequently to mimic axonal contact, it also influences Hedgehog (Hg) signaling. As several studies show cross-regulations during embryonic development between RA and Hg signaling pathways, we decided to analyze if forskolin treatment could be able to influence Hg signaling in SC cultures, which, in turn, might be responsible for changes in RAR-β and RXR-γ expression levels and not the mimicked axonal contact effect by forskolin. For this purpose, Hh signaling was blocked by treatment with the specific Hh receptor smoothened inhibitor cyclopamine, and the RAR-β and RXR-γ mRNA levels were determined. While treatment with forskolin alone produced an increase in RXR-γ and a reduction in RAR-β mRNA levels, cyclopamine treatment failed to mimic these effects . Cyclopade novo protein synthesis was required for RXR-γ induction by axonal contact mimickers. Interestingly, the presence of the protein synthesis inhibitor ciycloheximide produced an almost complete suppression of RXR-γ steady state mRNA levels , their half-lives were identical in both circumstances , indicatFinally, we sought to analyze if the changes in RXR-γ steady state mRNAs levels by RA and axonal contact translate into increased receptor abundance at the protein level. Similarly to what occurs at the mRNA level, RA treatment of isolated SCs produced a small decrease in RXR-γ protein levels in the absence of axonal contact mimickers. The combined treatment with BPE and forskolin produced an increase in RXR-γ protein levels, which was prevented by treatment with the retinoid .Previous work from our lab and others has shown that retinoids play an important role during Schwann cell development and function. The results shown in this work demonstrate that retinoid receptors are indeed present in Schwann cells and that their expression levels are tightly regulated by retinoic acid itself as well as by agents that mimic axonal contact.in vivo surge of cAMP in Schwann cells, which allows for Oct 6 activation at the promyelinating stage It has been described that retinoic acid signaling components are present in the peripheral nervous system and that after sciatic nerve injury there is an upregulation in retinoic acid signaling as measured by a RARE reporter in a transgenic reporter mouse Schwann cell behavior depends on the combination of extrinsic growth factors and hormones along with juxtacrine signaling coming from the neurons. A clear example is the induction of Egr2, the major myelinogenic transcription factor, whose expression depends mainly on axonal contact in vivo in neural crest-derived cells of chick embryos AmphiRAR) exists, retinoic acid is able to induce its expression in the nerve cord during development On the other hand, retinoids themselves are also potent regulators of their own receptors in Schwann cells. RAR-β is highly upregulated by RA in a similar fashion to what has been described in other cell types, including PC12 cells Interestingly, although cAMP has been described to be able to synergize with the induction of RAR-β by RA in embrionary carcinoma cells de novo protein synthesis, while in Schwann cells, the presence of the protein synthesis inhibitor cycloheximide not only abolishes RXR-γ regulation but, in fact, it produces a strong decrease in mRNA levels, uncovering the existence of a labile activator of RXR-γ expression in Schwann cells. Additionally, this de novo protein synthesis dependence indicates that the effect of the retinoid is not due to the direct binding of RAR on RXR-γ promoter region. In line with this, no putative RARE can be found in the promoter sequences upstream of RXR-γ rat gene. Unfortunately, neither the upregulation by axonal contact mimickers nor the downregulation by RA can be found in promoter assays with constructs that compass up to 3 Kb upstream of the transcription starting site of rat RXR-γ gene (data not shown).Much less is known about the control of RXR expression levels by retinoids. The down-regulation of some RXR-γ transcripts by retinoic acid during differentiation of F9 mouse teratocarcinoma cells has already been described in situ hybridization technique does not allow discrimination on the nature of the cell types that express RXR-γ in the ganglia, although the same authors already reported a comparatively very high RXR-γ expression level in 7-day-old rats. The detection onset of RXR-γ in these ganglia coincides with the period during which they begin to innervate their peripheral and central targets. Interestingly, until 10.5 dpc, Egr2 expression in peripheral nerves is restricted to the proximities of the ganglia, but at 15.5 there is a dramatic activation of Egr2 expression being reported along the whole sciatic nerve Egr2RXR-γ expression is developmentally regulated during peripheral nervous system development in rodents +/−/RXR-β−/−/RXR-γ−/− mutant mice are viable and almost indistinguishable from RXR-α+/− mice in vivo. Animal models of forced overexpression after birth, as well as conditional knock-outs of the different RXR isoforms on Schwann cells should provide further mechanistic insights into the role of RXRs on Schwann cell differentiation and subsequent peripheral myelin formation.RXR belongs to the Class II subfamily of nuclear receptors and, besides being able to act by itself as a ligand-dependent transcription factor, its major role is as the heterodimerizing partner of the Class I subfamily of nuclear receptors, which is formed by RAR, as well as the vitamin D3 (VDR), the thyroid hormone (TR) or the peroxisome proliferator activated receptors (PPAR) Table S1Q-RT-PCR primer list.(DOC)Click here for additional data file.


Mycobacterium tuberculosis and their role in regulatory networks is still poorly understood. Here we report a genome-wide characterization of sRNAs in M. tuberculosis integrating experimental and computational analyses. Global RNA-seq analysis of exponentially growing cultures of M. tuberculosis H37Rv had previously identified 1373 sRNA species. In the present report we show that 258 (19%) of these were also identified by microarray expression. This set included 22 intergenic sRNAs, 84 sRNAs mapping within 5′/3′ UTRs, and 152 antisense sRNAs. Analysis of promoter and terminator consensus sequences identified sigma A promoter consensus sequences for 121 sRNAs (47%), terminator consensus motifs for 22 sRNAs (8.5%), and both motifs for 35 sRNAs (14%). Additionally, 20/23 candidates were visualized by Northern blot analysis and 5′ end mapping by primer extension confirmed the RNA-seq data. We also used a computational approach utilizing functional enrichment to identify the pathways targeted by sRNA regulation. We found that antisense sRNAs preferentially regulated transcription of membrane-bound proteins. Genes putatively regulated by novel cis-encoded sRNAs were enriched for two-component systems and for functional pathways involved in hydrogen transport on the membrane.Only few small RNAs (sRNAs) have been characterized in  Recent research has shed some light on the relevance of sRNAs in bacterial pathogenesis, including modulation of expression of virulence factors in response to environmental and host signals Regulatory RNA species can modulate transcription, translation, mRNA stability, DNA maintenance, and gene silencing. They function through a variety of mechanisms, including changes in RNA conformation, protein binding, base pairing with other RNAs, and interaction with DNA Mycobacterium tuberculosis (MTB) results from the ability of this pathogen to sense the host environment and to switch to a non-replicating, persistent form that is resistant to host insult and to treatment with most antibiotics. Multiple genes associated with persistence have been identified in MTB. However, the complex post-transcriptional regulation underlying the phenotypic switch to persistence remains poorly understood. In particular, our knowledge of the regulatory networks involving sRNAs is still in its infancy. Understanding how sRNAs control bacterial virulence may open a new prospective in the fight against tuberculosis (TB) and contribute to the goal of TB eradication Intracellular survival of About 40 species of sRNAs have been reported so far in mycobacteria (including MTB) by cloning approaches and computational predictions M. tuberculosis H37Rv. We used a set of 1373 sRNA species previously identified by RNA-seq . The microarray analysis validated 258 (18.8%) species  than the positive control genes  and 5′/3′ UTRs (32.6%); of the latter class, most were 5′UTRs (5′UTRs: 90%). RNA species identified within the 5′ UTRs could represent tabolism .To show that the different RNAs validated by microarray expression analysis were transcribed, we compared the frequency of the nucleotide’s distributions at specific positions within the −10 region between sRNA candidates, annotated coding sequences (CDSs), and random sequences. We could confirm the effective transcription of sRNAs identified by microarrays by showing a clear non-random distribution of nucleotides within the −10 region. In particular, we showed that our candidates show a −10 region more similar to the one of CDSs rather than to random genomic sequences not annotated for transcripts. To achieve this goal we used a statistical approach previously described by Dornenburg Microarray data were further validated by Northern blot analysis. We selected 23 candidate sRNAs  from allThe candidates chosen were:four intergenic ;eight 5′/3′ UTRs of genes ;ten AS .Rv2165c gene, since it is highly expressed by microarray analysis. As a negative control we used a 5′UTR sRNA not validated by microarray (#76). Since Northern blot analysis allows relative comparison of transcript abundance, RNA was extracted from exponential and stationary phase cultures in order to evaluate differential expression associated with growth state. Total RNA was extracted from the strain H37Rv. For a smaller pool  we also tested the attenuated strain H37Ra.We also included candidate 1359, although it was mapped within the M. tuberculosis H37Rv than in H37Ra.The results of the Northern blot showed that in most cases (20 out of 23), one or more specific bands were detected . The Nor14–18–NNGNNG).Since computationally predicted length and that observed by Northern blot analysis differed in almost all species, we set out to identify the 5′ ends of eight selected sRNAs  by primer extension. In all cases, we could identify one or more bands, one of which corresponded to the predicted 5′ end with +1 or −1 nt difference (data not shown). A −10 consensus sequence for sigma factor A (TANNNT) was identified for 7 out of 8 sRNAs mapped, suggesting that transcription of the sRNAs is under sigA control. Upstream of the candidate 1359 a good consensus sequence for −10 and −35 sigma B is present  within −50 and +15 nucleotides from the 5′-end mapped according to RNA-seq results. At the same time, we searched for putative terminators downstream of the predicted sRNAs. The 3′-end region (−20/+270 nucleotides) was sought for intrinsic terminators using the GeSTer algorithm Classification of all 1373 candidates, promoter and terminator consensus sequence analyses, and genomic annotation are summarized in in silico analysis to identify pathways regulated by the set of novel sRNAs discovered in MTB. We focused on AS RNAs because the direct targets for this class of sRNAs are known. When we performed microarray analysis we found 152 AS sRNAs that overlap the same number of unique target genes in the opposite strand  as reported by other studies based on RNA-seq approach Eukarya and in Archaeacis-acting RNAs may inhibit stress-responsive modules, such as the two-component systems Most of the sRNAs identified are antisense. This is in agreement with recent findings that extensive antisense transcription occurs in bacteria, as it does in ilvB  suggests that also in mycobacteria, as in most bacteria investigated so far leuA. This candidate  pstS2 knockout strains showed attenuated virulence pstS2 and pknD genes are encoded within the same operon, and pknD knockout strains do not survive in inorganic phosphate poor-growing conditions pstS2-pknD operon. Interestingly, the candidate 135 mapped antisense to the 5′ region of the pstS2 gene  groES, encoding for a co-chaperonin protein groES gene. We suggest that this sRNA may be a thermosensor, as frequently found in other bacteria upstream of genes with a function similar to groES.Other putative riboswitches we have identified are candidate 161, controlling expression of a nitrate reductase . However, by comparing the polypeptide encoded by MTB with the protein encoded by M. bovis BCG we found that homology starts 53 amino acids downstream of the indicated start codon, where a GTG codon is present (nucleotide 2429447) preceded by a Shine-Dalgarno sequence (GGGGAGG)  . Thus, cet al.  classified this sRNA as a 3′UTR to Rv3321A. However, the length observed by Northern blot, the presence of both a sigma A consensus sequence and a possible stem-loop structure support the hypothesis that the candidate 498 is a trans-acting sRNA, whose targets have to be identified, rather than a sRNA overlapping to the Rv3321A-3′ end.Candidate 498 is transcribed in opposite direction to both neighboring genes mcr3 sRNA gene mcr3 no promoter consensus sequences were present. On the contrary, upstream of the 5′end of 190, which is internal to mcr3, we found −10 and −35 sigA consensus sequences, that can be identified with the most active rRNA operon promoter, P3 cis-acting regulatory elements controlling unique MTB rRNA operon.Candidate 190 mapped upstream of 16S rRNA gene being entirely contained in rrl and rrf genes, respectively). According to the positive signal obtained by Northern blot, this putative sRNA might be a residual RNA derived from ribosomal RNA processing. Additional bands at higher and lower molecular weight observed by Northern blot support this hypothesis. Very recently, ribosomal noncoding RNAs have been described in eukaryotic cells The candidate 193 (predicted length: 60 nucleotides) mapped between the ribosomal RNAs 23S and 5S . Exponential growth phase culture was harvested at OD600 between 0.5 and 0.8 and washed twice in PBS. The small-size RNA (approx. <200 nucleotides) enriched fraction was extracted using the mirVana™ miRNA Isolation Kit  according to manufacturer’s instruction. Both small-size RNA enriched and total RNA (depleted sRNAs) fractions were collected and analysed with Agilent 2100 Bioanalyzer .For analysis of sRNAs, et al.A custom analysis pipeline was developed for analyzing Illumina reads  and relative p-value  were taken into account.The application of such pipeline , 16S , and 5S . As negative controls we considered the same number of probes of the positive controls, made of random sequences not mapping in the M. tuberculosis H37Rv genome. Each array contained two complete probe sets and relative mismatches, each in duplicate. Small-size RNA fraction and total RNA fraction depleted of small-size RNAs were prepared using mirVana miRNA Isolation kit . The ULS labeling kit for CombiMatrix arrays  was used to label RNA samples with biotin and for hybridization on a 12K array followed by electrochemical detection on the ElectraSense reader performed according to manufacturer’s instructions. Two independent experiments were carried out starting from two biological replicates each . Microarray hybridization was carried out twice  for each biological replicate; every microarray was acquired at least twice. Results were analyzed applying Robust Multichip Analysis (RMA) procedure on the logarithm of the background (mismatches) corrected perfect match electrical current intensities.CombiMatrix microarray technology  has been adopted to validate putative sRNA candidates by expression evidence. Probe sets for candidates have been designed according to the following criteria: the probes were non-overlapping sequences of 30 nt, spanning all the candidate length. Probes were located at least 5 nt from each other and from the candidate boundaries. As result, probe sets were made of one single probe when candidates were shorter than 75 nt of length; two probes when candidates length were between 75 and 110 nt; three probes when candidates length were between 110 and 180 nt, and four probes for longer candidates. To each probe, a control mismatch probe was provided (mismatch: two bases). As positive controls, probes targeting the following genes have been included: We compared the candidates expression with controls by means of a t-type statistic test. P-values were computed by means of permutations, to avoid any distributional assumption, and adjusted for multiplicity by means of the Holm-Bonferroni method We decided to consider approximately 60 nucleotides at the 5′ end of a gene as putative promoter region. As reported by Gardner and colleagues 32P-labeled riboprobes or oligonucleotides, as described previously 10 µg of total RNA were separated on a 6% denaturing polyacrilamyde gel, blotted on a positively charged membrane , and hybridized to specific 2), 5 U RNasin (Promega) and 10 U AMV Reverse Transcriptase (Finnzymes) and incubated 30 min at 50°C for the extension. RNA was hydrolyzed with NaOH and all samples were neutralized with HCl and then precipitated and dissolved in 6 µl stop mix . Reaction products were separated on a 6% denaturing polyacrylamide gel along with sequencing reactions made with the same labeled oligonucleotides used for the primer extensions.The oligonucleotide probes were 5′ end labeled with T4 polynucleotide kinase (Promega). 5′ ends were identified by primer extension analysis, as described in Boorstein and Craig We used a statistical approach to further validate sRNA candidates. The approach described by Dornenburg In summary, we compared the frequency of specific nucleotides at fixed positions within the −10 consensus region. Due to the lack of consistent published data on −10 consensus sequences in MTB, we considered only the TANNNT hexamer recognized by the sigma factor sigA. Therefore, we analyzed the frequency of T in position −13, A in position −12 and T in position −8 for our sRNA candidates, for annotated CDSs in the genome of MTB and for a set random sequences, all of the same sample size. Random sequences were generated and used as control. A score of 3 was attributed when all the 3 nucleotides TAT were found at the expected position ; a score of 2, 1 or 0 was attributed when 2, 1 or 0 nucleotides, respectively were found at expected positions. An average of the scores was then calculated for each of the three categories . A Mann-Whitney test statistic was used to compare scores and p-values computed by permutation methods. For comparison we considered a number of random sequences equal to the sample size considered by the permutation. Sample sizes were as follow: 1948 candidates , 1373 type A candidates, 260 validated candidates, 4048 CDSs. All statistical analyses were performed in R and Bioconductor environment i) GO terms from the DAVID database ii) metabolic pathways from the KEGG database iii) functional categories from the TubercuList database (http://genolist.pasteur.fr/TubercuList). We then applied the Fisher’s Exact Test to determine biological terms that are enriched among antisense-regulated genes, based on the contingency table shown in a-1) before computing the Fisher exact probability; this modification removes the effect of terms based on single genes only We obtained functional annotation for MTB genes from three different sources: ; thin arrows indicate sRNAs; dotted arrows the distance from the 5′/3′ ends of CDS to the putative 5′/3′end of the sRNA; AS: antisense.(TIF)Click here for additional data file.Figure S2Schematic representation of the distance between the −10 consensus hexamer in candidate sRNAs, annotated coding region and random sequences. Picture of the relative mean score differences among sequences groups. To preserve as best as possible these dissimilarity, we obtained the plot coordinates from a non-linear multidimensional scaling of the pairwise absolute differences, taken as inter-groups distance matrix.(TIF)Click here for additional data file.Figure S3Schematic representation of the mapping position of sRNA candidates validated by Northern blot (from 54 to 1029). Empty arrows represent coding sequences (CDS), whereas sRNA candidates are reported as grey arrows. Dotted arrow (candidate 1359) represents the possibly erroneous annotation of the CDS Rv2165c. Dotted lines represent UTRs.(TIF)Click here for additional data file.Figure S4Schematic representation of the mapping position of sRNA candidates validated by Northern blot (from 1080 to 1565).(TIF)Click here for additional data file.Figure S5Northern blot for candidate 1938 in M. tuberculosis H37Rv and H37Ra .(TIF)Click here for additional data file.Figure S6Schematic representation for the mapping position of the sRNA 1359 validated by Northern blot.(TIF)Click here for additional data file.Table S1Dataset including all the candidates  representing the starting point of the present work.(XLSX)Click here for additional data file.Table S2Functional categories of genes showing RNA species within the 5′ UTR. Classification was performed according to TubercuList.(XLSX)Click here for additional data file.Table S3Functional enrichment of cis-regulated genes.(XLS)Click here for additional data file.Table S4List of oligonucleotides used in the Northern blot. * Oligo: 32P-labelled oligonucleotides used as probes in the Northern blot assays; FWD or REV: primers used to amplify the genomic regions to be used as templates in the in vitro transcription of the riboprobes.(XLSX)Click here for additional data file.Table S5List of other oligonucleotides used for the primer extension. DAVID database and TubercuList have been used for functional annotation of MTB genes. GO: gene onthology; mtu: KEGG pathway. TBL: TubercuList.(XLSX)Click here for additional data file.Table S6Contingency table for evaluating functional enrichment of antisense-regulated genes. a = number of AS-regulated genes annotated with term j; b = number of AS-regulated genes not annotated with term j. The quantities c, and d are similarly defined for the remaining genes not overlapped by AS sRNAs. N is the total number of M. tuberculosis genes for which annotation is available.(XLSX)Click here for additional data file.


The Yellowstone geothermal complex contains over 10,000 diverse geothermal features that host numerous phylogenetically deeply rooted and poorly understood archaea, bacteria, and viruses. Microbial communities in high-temperature environments are generally less diverse than soil, marine, sediment, or lake habitats and therefore offer a tremendous opportunity for studying the structure and function of different model microbial communities using environmental metagenomics. One of the broader goals of this study was to establish linkages among microbial distribution, metabolic potential, and environmental variables. Twenty geochemically distinct geothermal ecosystems representing a broad spectrum of Yellowstone hot-spring environments were used for metagenomic and geochemical analysis and included approximately equal numbers of: (1) phototrophic mats, (2) “filamentous streamer” communities, and (3) archaeal-dominated sediments. The metagenomes were analyzed using a suite of complementary and integrative bioinformatic tools, including phylogenetic and functional analysis of both individual sequence reads and assemblies of predominant phylotypes. This volume identifies major environmental determinants of a large number of thermophilic microbial lineages, many of which have not been fully described in the literature nor previously cultivated to enable functional and genomic analyses. Moreover, protein family abundance comparisons and in-depth analyses of specific genes and metabolic pathways relevant to these hot-spring environments reveal hallmark signatures of metabolic capabilities that parallel the distribution of phylotypes across specific types of geochemical environments. The Yellowstone hotspot is responsible for an enormous number  and diversity of thermal features that cover a wide range in pH (2–10), temperature (40–92°C), and geochemical properties  is intertwined with major geological events that have shaped the planet , and trace element cycling . However, the microbial complexity typical of temperate soil and aquatic habitats  archaeal-dominated sediments. These studies are the outgrowth of one of the major collaborative activities of an NSF Research Coordination Network, established to promote and coordinate research focused on geothermal biology and geochemistry in YNP. Importantly, the research presented here represents the first comprehensive and coordinated non-PCR based survey of microorganisms distributed across a broad spectrum of high-temperature habitats in YNP.Metagenome sequencing of environmental DNA has provided a useful tool for studying microbial community structure and function in numerous environments . While sites chosen for this study  strategically cover the bimodal pH ranges observed in YNP  show that most geothermal systems in YNP exhibit EC values ranging from 0.1–3 dS/m (or mmho/cm) , and other saline environments, the geothermal habitats sampled herein have significantly lower levels of dissolved ions, which would not be expected to impart significant environmental selection toward halophilic  or alkaliphilic (high-pH) microorganisms.A geo-referenced geothermal database developed by the Yellowstone Center for Resources  contains information for numerous geothermal features including pH, temperature, electrical conductivity (EC), and site context . A version of this database (∼8000 entries) is available at the RCN YNP websiteP Figure , the mic) Figure , which cdecision-tree  versus more neutral (pH 5–9) environments. It is well established that the upper temperature limit for oxygenic phototrophs is ∼70–74°C Brock, , but cya2 influx from the atmosphere will influence the resulting microbial community, but systems with plentiful sulfide and/or elemental S will (i) contain little to no detectable dissolved oxygen, and (ii) contain species of S that can serve as potential electron donors or acceptors. Utilization of an O2 variable in the decision-tree does not offer the same utility; low O2 does not imply the presence of reduced S species. Some geothermal springs discharge non-sulfidic, hypoxic waters that contain other reduced constituents such as ferrous Fe .Both low- and high-pH sites are further separated based on the presence or absence of sulfide and/or elemental S Figure . This isyes or no, they represent important physical determinants of microbial community structure. Gradients in oxygen, light quantity, and quality, and sulfide are substantial as a function of mat depth and influence the distribution of microorganisms  outflow channels, as well as site OSP_8 (Fe-oxide mat), which is immediately down-gradient of OSP_14. Members of the deeply rooted bacterial Aquificales are known for assuming filamentous morphology in turbulent environments where the discharge of hot reduced waters occurs in the presence of air or oxygenated waters  as well as the ingassing of O2(g)  are crucial for defining the microenvironmental context of microorganisms in geothermal settings. All Aquificales “streamer” communities  were obtained within the primary flow path of high-velocity (0.1–0.3 ms2. The in-channel flow environments promote equilibration with atmospheric conditions and encourage O2 influx. The acidic Fe-oxide mat (0.5–1 cm depth) sampled in Norris Geyser Basin (OSP_8) occurs within a high-velocity channel where oxygenation results in O2(aq) concentrations of ∼ 40–60 μM, which are 20–30% of theoretical saturation at this temperature and pressure. The concentration of Fe(II) does not appear in the decision-tree, because pH and the inference of oxygen  are already included as major determinants . Consequently, it is not the concentration of Fe per se that determines whether Fe(II) is oxidized, but whether the hydrodynamic or physical setting promotes oxygenation. For example, the more acidic CH_1 site contains ∼5 times more soluble Fe(II) than OSP_8 (250 versus 45 μM), but due to the consistent geothermal delivery of low levels of H2S(g) and the ubiquity of solid-phase elemental sulfur in CH_1, the consumption of any O2 is likely driven by reduced phases of sulfur rather than by Fe(II). Consequently, Fe-oxides are not present in the thermal pool of CH_1, although evidence of these phases was observed where the air-water interface meets rocks along the edge of the pool.The oxidation of aqueous Fe(II) to solid-phase ferric oxyhydroxide is an exergonic reaction under most hydrothermal conditions  glass slide suspended in the water column at 56°C (pH = 5.7). OPP is a large pool (∼0.5 ha) receiving mixed water inputs, and is the only sample in the study to be collected from the photic zone of a large water-body, albeit heavily influenced by geothermal inputs. Although the physical context is considerably different than other springs and pools included in the study, characteristics of OPP_17 place it near the phototrophic sites in the decision-tree  per site ranged from 20 to 60 Mb  reflects the collapse of redundant sequence reads into contigs that correspond to the predominant populations present .The predominant phylotypes present in these three sample groups include expected microorganisms that have been the focus of prior studies at these exact locations Table , as wells Figure . The onls Figure . HoweverMetallosphaera sp.) and novel archaeal populations observed only in the absence of significant levels of dissolved sulfide  and contributed an extensive diversity of archaeal protein families (TIGRFAMS) not currently represented in environmental metagenome data, with the exception of replicate sites included in a smaller study of YNP chemotrophic communities  of the TIGRFAM abundance data Figure  showed tComparing groups, the Aquificales “streamer” communities and archaeal-rich habitats yielded broader functional diversity than phototrophic sites because the chemotrophic sites included both bacteria and archaea while the phototrophic sites were dominated by bacteria. The phototrophic mats contained very few sequences attributable to archaea or members of the Aquificales. Component 1 reflects the amount of Aquificales versus phototrophs  as well as the amount of sequence data attributable to members of the Sulfolobales , in which the content of Sulfolobales decreased from left to right . Consequently, the distribution of different electron transport proteins across sites did not result in identical site clustering obtained using broader TIGRFAM categories  database  with specific physicochemical properties of the habitats they occupy.2+/Fe3+; total dissolved sulfide; dissolved O2) were determined using field methods (with some exceptions given the number and diversity of sites studied) as described in more detail in previous reports  were determined using closed headspace gas chromatography  is extremely favorable when geothermal waters are exposed to atmospheric O2, or in some cases using alternate electron acceptors such as nitrate, ferric Fe, sulfate, or elemental S0. Dissolved Fe(II) and ammonium (NH4) concentrations can be very high in certain YNP geothermal systems, and offer yet another set of exergonic reactions that serve as a potential geochemical niche for chemotrophic organisms.Twenty geothermal sites in Yellowstone National Park Table  were sam2) with lysozyme  for 1.5 h at 37°C. Proteinase K  and SDS [final concentration 0.3% (w/v)] were then added and incubated for 0.5 h at 37°C. This first lysate was removed and the samples were re-extracted using bead-beating protocols. The two lysates were combined and extracted with phenol-chloroform, and the resulting DNA was re-precipitated in ethanol, treated with RNAase and quantified by gel electrophoresis and staining. Our intent was to avoid biasing the samples against organisms that may be difficult to lyse with chemical methods. The extraction kits  used on several of the sulfur-containing sediment samples also included a modest physical lysis step. Across all 20 sites, the extracted DNA ranged in size from ∼4 to 12 kbp, and for many samples these procedures resulted in higher MW DNA ranging from 9 to 20 kbp.A standard DNA extraction protocol was used for the majority of samples, however, several sulfur sediments were ultimately subjected to various extraction kits to generate sufficient DNA yields for library construction. Our main emphasis was to obtain representative, unbiased environmental DNA for construction of small-insert libraries. Briefly, 3–25 g wet samples were extracted with 1 ml of Buffer A  pUC18 libraries were constructed from all sites, and preliminary sequencing of 5–10 megabases (Sanger), was performed as a quality control to determine if the initial sequencing results [via MEGAN , which resulted in reasonably similar overall assembly statistics. Due to the slightly better scaffold construction and prior history using the Celera assembler  received both Sanger sequence and one-half plate of 454 titanium pyro-sequencing . Due to the success of Sanger sequencing in generating significant assemblies from these four sites, the additional pyro-sequence is not discussed in great detail in the subsequent manuscripts. In all four of these sites, contig coverage increased significantly when the pyro-sequence reads were included. However, the analyses discussed here are mostly derived from the Sanger sequence data, in order to maintain a consistent analytical approach for the data from all 20 sites.http://gos.jcvi.org/openAccess/scatterPlotViewer.html.Phylogenetic analysis of random shotgun sequence reads was performed using a number of different approaches, and these yielded convergent information regarding the predominant phylotypes present in these geothermal sites. Individual sequence reads were analyzed using G + C content (mol%) distribution coupled with blastx and MEGAN  in high-temperature geothermal systems. Query DNA sequences known to code for proteins important in the oxidation of reduced chemical constituents or the reduction of a terminal acceptor  to query sequences were then reanalyzed using blastp, and assessed individually using phylogenetic analysis of deduced protein sequences against known relatives, as well as fragment length relative to query length. False positives were minimized using this screening process. This included (i) sequences matching the correct protein family of the query sequence, but not the exact query sequence ; (ii) sequences matching a query sequence due to regions of sequence similarity, but were clearly associated with a gene or gene cluster with different function; and (iii) sequences that returned mis-annotated blastp relatives. It is also possible that our inventory of metabolic potential missed sequences related to a specific query gene. For example, some genes found in the metagenome data were of insufficient length relative to a specific query sequence (<40%) to make a definitive assignment. Moreover, the lower depth of coverage (<1×) of sub-dominant phylotypes precluded a complete functional analysis of these organisms.The assembled metagenome sequence data was also screened for specific functional genes corresponding to known or putative pathways in material and energy transfer. We were specifically interested in assessing metabolic potential for chemolithoautotrophy (COAll annotated metagenome sequence assemblies (Celera/PGA) are available through the DOE-JGI IMG/M websiteThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Microbial_Physiology_and_Metabolism/10.3389/fmicb.2013.00067/abstractThe Supplementary Material for this article can be found online at Supplementary Table S1Contributions of NSF Research Coordination Network Steering Committee and Working Group members to the Yellowstone Metagenome Community Sequencing Project (DOE_JGI CSP 787081).Click here for additional data file.Supplementary Table S2Geochemical parameters measured in the bulk aqueous (<0.2 μm) phase in 20 different geothermal systems sampled for metagenome analysis.Click here for additional data file.Supplementary Table S3List of gene sequences and corresponding accession numbers used to query the assembled environmental sequence data for assessing potential metabolic attributes associated with the predominant phylotypes found within these geothermal sites.Click here for additional data file.Supplementary Figure S1Additional site photographs emphasizing landscape context of geothermal habitats and field sampling efforts (included as a separate file containing 53 annotated photographs).Click here for additional data file.Supplementary Figure S2Top ranked TIGRFAM categories based on their ability to differentiate the three types of sites discussed in this study: aquificales “streamer” communities (blue), archaeal-dominated sediments (yellow), and phototrophic mats (green). (A) Signal transduction; (B) Regulatory functions (ANOVA test with Benjamini-Hochberg FDR multiple testing correction).Click here for additional data file.Supplementary Figure S3Principal components analysis of normalized TIGRFAM protein family abundance data across all 20 sites as well as a subset of diverse metagenomic datasets from IMG. The metagenomes are colored based on rough classification of the environments from which the sample originates: Yellowstone sites in this study = red; Yellowstone sites from other studies = orange; sediments = turquoise; soils = pink; aquatic = light-blue; fungus-garden = green; animal-gut = brown; enrichment community = light-orange; other samples = gray.Click here for additional data file.


Salmo salar L.) after transfer to seawater. IPN vaccines have been available for a long time but their efficacy has been variable. The reason for the varying immune response to these vaccines has not well defined and studies on the importance of using vaccine trains homologous to the virulent field strain has not been conclusive. In this study we prepared one vaccine identical to the virulent Norwegian Sp strain NVI-015 (NCBI: 379740) (T217A221T247 of VP2) and three other vaccine strains developed using the same genomic backbone altered by reverse genetics at three residues yielding variants, T217T221T247, P217A221A247, P217T221A247. These 4 strains, differing in these three positions only, were used as inactivated, oil-adjuvanted vaccines while two strains, T217A221T247 and P217T221A247, were used as live vaccines. The results show that these three residues of the VP2 capsid play a key role for immunogenicity of IPNV vaccines. The virulent strain for inactivated vaccines elicited the highest level of virus neutralization (VN) titers and ELISA antibodies. Interestingly, differences in immunogenicity were not reflected in differences in post challenge survival percentages (PCSP) for oil-adjuvanted, inactivated vaccines but clearly so for live vaccines (TAT and PTA). Further post challenge viral carrier state correlated inversely with VN titers at challenge for inactivated vaccines and prevalence of pathology in target organs inversely correlated with protection for live vaccines. Overall, our findings show that a few residues localized on the VP2-capsid are important for immunogenicity of IPNV vaccines.Infectious pancreatic necrosis virus (IPNV) is a member of the family Birnaviridae that has been linked to high mortalities in juvenile salmonids and postsmolt stages of Atlantic salmon ( Salmo salar L.) after transfer to seawater, and the disease has a worldwide distribution Birnaviridae and it is a double stranded RNA virus made of segment A which codes for VP2, VP3, VP4 and VP5 while segment B encodes for VP1, the RNA-dependent RNA polymerase Infectious pancreatic necrosis (IPN) is a highly contagious disease causing high mortality in juvenile salmonids and in postsmolt stages of Atlantic salmon  gonad cells  at 15°C in Leibowitz L-15 media supplemented with 10% fetal bovine serum (FBS)(Sigma), 2% L-glutamine (Sigma) and gentamycin (Sigma) 25 μg ml−1. All viruses used in this study were produced by reverse genetics as derivatives of the highly virulent Norwegian IPNV Sp strain NVI-015 (GenBank accession nos. AY379740) using the method described below.Viruses used for vaccine production, challenge, ELISA antigens and virus neutralization test (VNT) were all propagated on rainbow trout (217A221T247) to the parent IPNV strain NVI-015 while progeny rNVI-15PAA Generation of full-length cDNA clones of the entire coding and non-coding regions of NVI-015 RNA segment A and B was performed according to procedures described by Yao and Vakharia −3 to 10−8) of cell culture supernatants and adsorbed for 1hr at room temperature (RT). After adsorption the inoculum was removed and cells were overlaid with 0.8% SeaPlaque Agarose (BioWhittaker) in L-15 medium containing 5% FBS and 1% L-glutamine. Cells were incubated at 15°C for 4 days and plaques formed by cytopathic effect (CPE) were removed from the wells using punch biopsy equipment. Plaques were inoculated in RTG-2 monolayers and incubated for 7 days at 15°C. When full CPE was observed, the supernatant was harvested after 2500 rpm centrifugation and sterile filtration as described above. RNA was extracted using the QIAamp Viral RNA mini kit following the manufacturer's recommendations. Complete nucleotide sequences of segment A and B of each virus were determined as described before 221 in the virus isolate used while variant genotypes used in the study are shown in To generate additional viruses with a mutation in residue 221 of VP2, we took advantage of previously observed cell culture adaptation attenuation characteristics of the rNVI-15TAT and rNVI-15PAA strains where attenuation is seen in CHSE-214 but not in RTG-2 cells after 10 passages in culture 9 Tissue Culture Infectious Doses50/ml (TCID50/ml) of vaccine. In this ELISA we used the monoclonal antibody AS-1 Inactivated vaccines were made by inoculating the different recombinant viruses TAT, TTT, PAA and PTA onto RTG-2 cells to ensure that no mutations occurred during virus culture Two viral strains encoding the TAT and PTA motifs produced as described above were used to prepare two live vaccines.To ensure scientific validity and consistency of our findings, we carried out two independent vaccine efficacy trials using an inactivated and a live vaccine approach. In study I we used four oil-based inactivated vaccines based on viral strains in ad libitum . After vaccination, fish were kept at 12°C (12 hrs darkness and 12 hrs light) for smoltification (preparation for transfer to seawater). Post vaccination samples were collected at 10 weeks post vaccination  and 12 fish were sampled per group. Fish were anaesthetized using methiomidate during immunization and sampling.After 840 degree days of immune induction (10 weeks), fish were transferred to sea water and challenged using a cohabitation model by adding 30 virus shedders per tank . Virus s5TCID50/fish, 46 fish were transferred to each of the three parallel tanks. Another 138 fish were injected with PBS as controls and were distributed equally into parallel tanks  using automated equipment.In study II, two live vaccines were used based on the virulent TAT and avirulent PTA strains. Efficacy trials for live vaccines were carried out at the Aquaculture Research Station of the University of Tromsø, Norway using a standard IPN-susceptible AquaGen breed of Atlantic salmon parr. Virulent strains can be used for immunization at parr stage since post-exposure mortality will not occur at this physiological stage. For identification, each fish was intraperitoneally implanted with a pit-tag number using a pit-tag applicator. Each tag number was electronically registered in an Excel sheet using an automated electronic reader  linked to a computer. Fish were divided into three groups, the two vaccines and unvaccinated controls . Each vael tanks . To avoi7.0 TCID50/fish and were immediately put to cohabit with vaccinated fish in the tanks. In addition to virus shedders, each tank of the TAT and PTA vaccinated groups was allocated 30 unvaccinated control fish at challenge  and live (study-II) vaccine efficacy trials, samples collected included whole blood in EDTA while head kidney, liver, spleen and exocrine pancreas samples were stored in 10% formalin. Head kidney samples were also stored in transport media for virus cultivation.5′-GAGTCACAGTCCTGAATC-3′) and A-Sp1689R (5′-AGCCTGTTCTTGAGGGCTC-3′). RNA extraction was carried out using the RNAeasy Mini kit (Qiagen) according to manufacturer's recommendation. PCR products were sequenced as described by Santi et alHead kidney tissues were homogenized in a stomacher in transport media followed by a low spin at 2500 rpm for 10 min. 0.1 ml was inoculated on RTG-2 cells grown in 24 well plates to final dilutions of 1 and 0.1%. The plates were incubated at 15°C. After seven days, 0.1 ml of the supernatant was obtained and used to inoculate new monolayers of confluent cells for a second passage. Final reading was carried out after seven days of the second passage. Results were scored based on the presence (positive) or absence (negative) of CPE during the second passage. Supernatants obtained from second passage monolayers were tested for the presence of IPNV using RT-PCR with primers A-Sp500F (2 TCID50/ml) in titration plates. The virus-antisera mixture was incubated at RT for 1h. Growth media was removed from RTG-2 monolayers in 96 well plates and replaced with 0.1 ml of the virus-antisera mixture. All serum samples from the four vaccine and control groups were tested against the four viral strains to yield a cross neutralization assay. Positive IPN virus controls, negative controls (plasma and blanks with L-15 medium) were used on each plate. The plates were incubated at 15°C and were examined for presence of CPE after seven days. Replication patterns and kinetics of the virus strains have been characterized previously and it has been shown that their ability to replicate in vitro does not differ between the recombinant viruses Sera from experimental fish were diluted in a two-fold dilutions starting from 1∶10 up to 1∶1280 in L-15 medium without FBS and were mixed with an equal volume of virus suspension  examination as previously described TM and later transferred to STATA version 12 (http://www.stata.com) for analysis. The Kaplan Meyer's survival analysis was used to generate the post challenge survival percentages (PSCP) Experimental data was entered in Microsoft ExcelFish vaccinated with the TAT and TTT vaccines had the same level of protection while those vaccinated with the PTA strain had slightly higher mortality . HoweverBased on the observation from the challenge study we were first interested in assessing the antibody levels to the challenge virus (TAT) for the 4 different vaccine groups and we examined the neutralizing titers at 10 wpv . VN titers elicited by the TAT-vaccine (against TAT strain) were significantly higher than the PAA and PTA vaccinated fish . The TTT We also examined antibody responses generated by the four vaccines against the challenge strain (TAT) by ELISA. The findings corresponded with VN assay results  showing 2 =  −0.959, p<0.021) at 10 wpc indicating that there was an inverse relationship between antibody levels and post challenge infection. Supplementary data from a low challenge dose (1×106TCID50/ml) trial showed absence of post challenge head kidney infections in fish vaccinated with the TAT vaccine while fish vaccinated with heterologous vaccines showed increasing infection rates that corresponded with increase in amino acid substitution on the vaccine strains  and the most distant strain in terms of amino acid substitutions, the PTA variant. Mortality in the virus shedders started 3 dpc while in the controls and vaccinated groups it started 21 dpc and ceased at 55 dpc . Fish van = 10) in immunized fish during immune induction up to 8 wpv  were five times (4.927) higher than levels detected in the PTA vaccinated fish  when tested against the TAT antigen. These findings show that antibody levels detected at challenge were predictive of the risk of post challenge mortality. In addition, the risk of PTA-controls  dying was about five times higher than TAT-controls  suggesting that there was a protective effect from vaccinated fish on controls, likely a herd immunity effect. The risk of fish dying in the control group that did not cohabit with vaccinated fish was 29  times higher than the TAT vaccinated fish. In summary, these data demonstrate that the risk of post challenge mortality for live vaccines can be predicted by antibody levels detected at challenge using a homologous antigen to the challenge virus.Neutralizing antibodies of TAT vaccinated fish were higher than for the PTA group prior to challenge when tested against the challenge strain . Antibodn = 10) of the PTA vaccinated fish had tissue damage in the liver and pancreas. Put together, data in In order to gain insight on mechanisms of protection rendered by live vaccines we assessed their ability to prevent establishment of pathology in target organs. Viral antigens were only detected in infected tissues after challenge and not before , showing217A221T247 (not shown). Similarly, samples from virus re-isolation at 4 and 10 wpc had the same residues at the defined positions clearly indicating that there was no mutation observed during mortality or in the virus carriers after challenge. Both virus re-isolation and sequencing gave the same results with only one sample of the post challenge studies detected positive on RT-PCR failed to show CPE after the second passage on RTG-2 monolayers (n = 180).In order to demonstrate that the mortality observed was caused by the challenge virus (strain rNVI-15TA) we extracted RNA and prepared cDNA from supernatants obtained from virus re-isolation studies. Chromatograms from cDNA sequences showed clear peaks encoding TOne key finding of this study was that IPN vaccines based on the virulent TAT virus variant elicited the highest antibody responses compared to other, lesser virulent virus strains. This was clearly shown from neutralization antibody tests and ELISA for both inactivated and live vaccines. Our conclusion is that the TAT strain is the most immunogenic of those tested while the PTA strain was the least immunogenic. Yet, fish immunized with different variants of IPNV with adjuvanted, inactivated vaccines were equally protected against lethal challenge, although marginally lower for the PAA variant. Using live vaccines, VN antibody titers correlated with protection against mortality. Although less immunogenic, antibodies elicited by PTA vaccines from both the inactivated and live vaccines were neutralizing against the challenge strain. Epitope mapping for IPNV has been reported n = 10) and PTA  live vaccines (It has been pointed out vaccines . Furthern = 10) compared to the PTA-controls   . AlthougData from inactivated vaccines show that antibodies are correlates of protection . Similar inverse correlations between hemagglutination inhibition (HI) antibody titers with post exposure influenza virus infections have been reported Residues that play a key role in virulence and immunogenicity of IPNV are localized on the central region of the VP2 capsid as indicated from previous studies Figure S1Study design for the inactivated vaccines. Study design I included a three parallel tank system to assess the efficacy of inactivated, water-in-oil adjuvanted vaccines made from the four viral strains listed in 7 TCID50/ml of the TAT strain were put to cohabit with vaccinated and control fish in each tank. For each vaccine group, survivors of the challenge were pooled together and monitored for an additional 7 weeks.(TIF)Click here for additional data file.Figure S2Study design for the live vaccines. A three parallel tanks system including the virulent TAT and avirulent PTA vaccine strains. A total of 138 fish were injected with either of the live vaccines per group or given phosphate buffered saline (PBS control). After immune induction period, 38 vaccinated fish were transferred to each of three parallel tanks. To avoid cross infection since the study involved the use of live vaccines, each vaccine group was assigned its own parallel tank system. Also the PBS injected fish were kept separate from the vaccinated fish to avoid exposure to vaccine virus prior to challenge. At each sampling time point , 4 fish from each group in each parallel tank were sacrificed and sampled. Challenge was carried out by adding eight virus shedders injected with 107TCID50/ml and 30 PBS controls each parallel tank.(TIF)Click here for additional data file.Figure S3Virus neutralization for the inactivated vaccines at 10weeks post challenge. Virus neutralization (VN) antibody titers for the inactivated vaccines at 10 weeks post challenge (wpc) shows that the TTT vaccinated fish are significantly lower than all other vaccine groups while the TAT is not significantly higher than the PAA, PTA and control fish.(TIF)Click here for additional data file.Figure S4Virus neutralization test for the live vaccine groups. Virus neutralization antibody titers against the TAT strain for the TAT- and PTA-live vaccinated fish increase at 4 and 8 wpv.(TIF)Click here for additional data file.Table S1Hazard risk ratios of for inactivated vaccines expressed relative to the TAT vaccine. The data express the relative risk of dying in the TTT, PAA and PTA vaccinated fish relative to the TAT-vaccinated fish.(DOCX)Click here for additional data file.Table S2Relative risk of post challenge IPNV infection in head kidney samples of fish vaccinated with inactivated vaccines.(DOCX)Click here for additional data file.Table S36TCID50/ml).Relative risk of post challenge IPNV infection in head kidney samples of fish vaccinated with inactivated vaccines Click here for additional data file.Table S4Post challenge hazard risk ratios of for live vaccines expressed relative to the TAT vaccine. The data express the relative risk of dying in the PTA, TAT-controls and PTA-controls relative to the TAT-vaccinated fish.(DOCX)Click here for additional data file.Table S5Post challenge virus re-isolation from head kidney samples of fish vaccinated with live vaccines.(DOCX)Click here for additional data file.Table S6Post challenge detection of viral antigens by immunohistochemistry and histopathology of fish vaccinated with live vaccines.(DOCX)Click here for additional data file.


Understanding the mechanisms by which natural populations cope with environmental stress is paramount to predict their persistence in the face of escalating anthropogenic impacts. Reef-building corals are increasingly exposed to local and global stressors that alter nutritional status causing reduced fitness and mortality, however, these responses can vary considerably across species and populations.Symbiodinium ITS-1 type C2) over the course of the experiment indicated that heterotrophic uptake and photosynthesis were limited, creating nutritional deprivation conditions. Regulation of coral host genes involved in metabolism, CO2 transport and oxidative stress could be detected already after five days, whereas PSII activity took twice as long to respond. Opposing expression trajectories of Tgl, which releases fatty acids from the triacylglycerol storage, and Dgat1, which catalyses the formation of triglycerides, indicate that the decline in lipid content can be attributed, at least in part, by mobilisation of triacylglycerol stores. Corals from the inshore location had initially higher lipid content and showed consistently elevated expression levels of two genes involved in metabolism  and calcification (carbonic anhydrase).We compare the expression of 22 coral host genes in individuals from an inshore and an offshore reef location using quantitative Reverse Transcription-PCR (qRT-PCR) over the course of 26 days following translocation into a shaded, filtered seawater environment. Declines in lipid content and PSII activity of the algal endosymbionts (Coral host gene expression adjusts rapidly upon change in nutritional conditions, and therefore can serve as an early signature of imminent coral stress. Consistent gene expression differences between populations indicate that corals acclimatize and/or adapt to local environments. Our results set the stage for analysis of these processes in natural coral populations, to better understand the responses of coral communities to global climate change and to develop more efficient management strategies. The degree to which reef corals will persist into the future depends on the severity of chronic stress and the frequency of acute stress  densities and photosynthesis-related physiological responses. Variation in PSII performance has been documented among symbiont types Corals are able to optimise their fitness under local environmental regimes through physiological and genetic adaptation e.g., . PhysiolThe nutritional needs of corals are met from auto and heterotrophic sources, which relative importance may vary among environmental regimes, microbial symbiont communities and species Gene expression is now used more commonly to understand the stress response and evolutionary ecology of corals 2 transport, oxidative stress, immunity and symbiosis, as well as measure photosynthesis and lipid content, over a finer timescale.In this study, we investigate how gene expression, lipid content of the holobiont and symbiont PSII activity in corals from two source populations responded following translocation into a controlled laboratory environment with reduced potential for auto and heterotrophy for one month. Previously, we used microarray analysis to demonstrate widespread differential expression of metabolic genes and fluorescent proteins between two time points consistent with nutritional limitation C3 and NFKB) and three stress genes . This cluster also contained one distinctive sub-cluster of three genes associated with calcification and metabolism , demonstrating a tendency towards differential expression between corals from different populations . The second cluster contained seven genes that were down-regulated in the laboratory with little differentiation between locations except C-type lectin (Ctl) which appeared to be up-regulated in corals from Orpheus Island in the laboratory but down-regulated in corals from Davies Reef (Rdh1) and four metabolic genes . Triacylglycerol lipase (Tgl) did not belong to any of the two clusters and displayed the greatest differential expression of all genes examined, being uniformly up-regulated in the laboratory.The normalized relative expression of 22 target genes  was compies Reef . The secPLRT<0.013, CA and Catalase (Cat) (Adj. PMCMC<0.018) but not in Aldh3. The significance of model terms and pairwise comparisons for all genes can be found in CA and Aldh3 was greater in Davies Reef corals at several sampling times whereas Cat was expressed at a higher level in field sampled Davies Reef corals but became similar between populations in the laboratory (Adj. PMCMC<0.022). This occurred after an initial increase in expression of Orpheus Island corals and a later decrease in Davies Reef corals (PMCMC<0.018).Population-specific changes in gene expression over time were significant in three genes (Adj. PLRT<0.013). In three of these genes  expression declined and a significant drop in gene expression was identified between the field and the first laboratory sample  but not between corals sourced from different populations . These environmentally driven changes in gene expression corresponded well with changes in physiology and many were statistically significant after five days in the laboratory. Changes in lipid content and PSII activity of symbionts were more variable and our results, therefore, add gene expression to host specific responses that can be detected earlier in the acclimatisation process compared to, for example, changes in PSII function of symbionts The corals examined here displayed both immediate and longer-term responses to changing environmental conditions associated with translocation from two field locations into a shaded laboratory condition with limited potential for heterotropic feeding  and Aldehyde Dehydrogenase 3 (Aldh3) are particularly tempting to interpret as a consequence of genetic divergence between populations. In contrast, the difference in Catalase was only detectable at the initial time-point and therefore most likely reflected short-term acclimatisation to local conditions. Cytosolic α - Carbonic Anhydrase (CA) catalyses the reversible hydration of CO2 to HCO3−  have roles lipid metabolism Acropora species Dgat1 catalyses the formation of triacylglycerols A. millepora decrease or cease storing their lipid during times of low food availability. In contrast, Tgl, which has the inverse role and releases fatty acids from the triacylglycerol storage form The energetic status of corals can be defined by the content of total or specific protein, lipid and carbohydrates and is a good indicator of coral fitness including survival following environmental stress CA did not reflect patterns of PSII acclimation but was relatively constant throughout the laboratory sampling duration. In contrast, Retinol dehydrogenase (Rdh1) was down-regulated in corals from both locations in response to the laboratory environment. This enzyme is involved in vision in higher organisms and can respond to light environments by retinoic acid receptor activation or can act in transcriptional regulation Rdhl responds to changes in ambient light environments in a similar fashion to PSII acclimation.Our results indicated no location-specific difference in photo-physiology prior to or following translocation despite large differences in the average ambient light environments. This suggests that autotrophic capacity was similar between corals from the two environments. A gradual reduction in rETRmax and Ek occurred in corals from both locations and supports acclimatisation to the shaded common laboratory environment. Shade acclimatisation is known to occur in corals exposed to low-light environments over comparable time scales Col) and a Hemolytic Cel-III type lectin (Cel3) showed increased expression after translocation into the laboratory. Col is a major component of connective tissue in animals and the mesoglea in corals Col in Cnidarian symbiosis has been proposed based on expression in symbiotic vs aposymbiotic states Ctl, a C-type lectin was not significantly explained by our model terms, however, this gene is widely hypothesised to play a role in the onset and maintenance of symbiosis possibly through immune regulation Symbiodinium densities, pigment concentrations and symbiont specific gene expression will benefit future attempts to better understand how the physiological adaptation of photosymbionts affect coral gene expression, health and fitness.Collagen  and three genes associated with oxidative stress , supporting the hypothesis that the laboratory treatment did impose some stress on the corals despite the absence of obvious disease or bleaching. The up-regulation of oxidate stress genes could also be a response to Reactive Oxygen Species (ROS) production as a result of increased Tgl activity Mn-SOD and uPAR did not respond to our experimental treatment. Urokinase Plasminogen Activation Receptor (uPAR) is involved in cellular signalling, adhesion, wound healing and tissue regeneration Pocillopora damicornis, possibly because of a trade-off mechanism between growth and stress response Bay Our results demonstrate the utility of gene expression data to detect rapid changes in physiological status as well as to understand long-term acclimatisation and/or adaptation in natural coral populations. As the co-regulation patterns and functional roles of coral genes are increasingly understood, transcriptomic data, combined with physiological and energetic analyses, will play a growing role in revealing how populations and species of corals may vary in their response to local and global stressors Acropora millepora, from two source populations: Pioneer Bay, Orpheus Island  and Davies Reef lagoon  on the Great Barrier Reef, Australia. All necessary permits were obtained for the described field studies (Great Barrier Reef Marine Park Authority Permit number G06/15571.1). At offshore locations shallow corals routinely experience light levels >800 µmol photons/m2/s and SPM<1.5 mg/L compared with 300 µmol photons/m2/s and SPM<1.5 mg/L at inshore locations 2/s) and flow-through seawater was filtered to 1 µm to remove all SPM. Three replicate branches from seven replicate colonies per population were sampled in the field and four times in the laboratory over the following month. Colonies ranged from 15 to 22 cm in diameter (mean diameter = 18.5 cm), which corresponds to ca. 225 branches. Sampled branches were removed from the central section of colonies and constituted ∼7% of colony biomass. All colonies were the same colour morph (pink) and hosted the same type of photosynthetic endosymbionts  to develop and implement a multiplex reverse transcription (RT) qPCR assay to analyse the expression of 29 genes The mRNA was extracted with poly-T magnetic beads using manufacturer's recommendations (Invitrogen) 2 , 1 µL of Forward Primer Plex  and 0.35 µL of Thermo-Start DNA Polymerase (ABgene). The PCR thermal cycling program included a 10 min denaturation step at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C and 1 min at 68.5°C. 1 µL of 1∶20 diluted PCR product was analysed on automated capillary sequencer  with 0.2 µL of ET-400 DNA size standard (Beckman-Coulter). Electrophoregrams were visualized in the GenomeLab™ Genetic Analysis System software (Beckman-Coulter), filtered then imported in GenomeLabTM eXpress Profiler software where peak binning was performed to associate fragment sizes with genes and to quantify the area under the peak used to evaluate the expression level of the associated gene Polymerase Chain Reaction (PCR) was performed on cDNA in 10 µL reaction volumes containing 2 µL of PCR Buffer, 2 µL of MgClH+ and Na+/K+ ATPases and MnSOD as the three most stable genes, but not the a priori control genes (CTG_1319 and Ribol9). We then applied a normalisation factor (NF) based on the geometric mean of the expression values of these three most stable genes. The normalised gene expression ratios of remaining target genes were further analysed and the a priori control genes, as well as the actual normalisation genes were excluded leaving 22 genes for further analysis.We first pre-normalised the data by expressing each gene as a fraction of the internal spike-in control (i.e. Kanamycin). GeNorm was then used to determine the most stable genes for use in normalisation Symbiodinium may be absent or in low abundance in apical polyps v/Fm) and effective quantum yields (ΔF/Fm′) were calculated as described in Bay et al.max, a.u. [maximum relative electron transport rate], and Ek, µmol photons m−2 s−1 [minimum saturating irradiance]) were modeled based on a hyperbolic tangent formula devised by Platt et al.et al.Rapid light curve analysis is a tool to assess photochemical efficiency through PSII electron flow using pulse-amplitude fluorometry (PAM) Two branches from each colony were snap frozen in liquid nitrogen following photo-physiological analysis. Each branch was crushed into a fine powder, divided into two aliquots , freeze-dried overnight in pre-weighed containers, then re-weighed to determine the sample dry weight. Total lipid was extracted using a Dichloromethane/Methanol extraction method, and lipid content was determined gravimetrically LRT = 0.027). The optimality of the selected model was also confirmed by a large weight of the Akaike information criterion. Gene expression was analyzed on a gene-by-gene basis, using log2-transformed deviances from the global mean for the particular gene as the response variable The data were analyzed using linear mixed model approach, with population, time-point, and their interaction as fixed factors, and a specific coral colony as a scalar random factor. This approach allows a more accurate estimation of among-group variance, and is less sensitive to violations of parametric assumptions and missing data compared to repeated measures ANOVA MCMCglmmMCMCglmm, the default prior was used, corresponding to the maximum-likelihood estimates of the parameters. We compared consecutive time-points and conducted four test when only the time factor was significant (FDR corrected αMCMC = 0.024) and eight tests when population specific responses were detected (FDR corrected α MCMC = 0.018). We also compared populations at individual time-points when the time by population interaction was significant .Depending on which fixed effects were significant, we evaluated the pairwise differences between consecutive time-points from the results of Markov Chain Monte Carlo (MCMC) sampling after implementing the optimal linear mixed model in heatmap.2 of the package gplots; plotting of gene expression and physiological time series was done using ggplot function of the ggplot2 package Hierarchical clustering of gene expression profiles and heatmap visualisation was done using function File S1Likelihood ratio tests, Akaike weights and pairwise differences from gene expression and physiological analyses.(PDF)Click here for additional data file.File S2Genes included in the gene expression assay, abbreviations, accession numbers, concentration of primer in Reverse Primer Plex (nM), length of the transcript (bp) and Oligo sequences.(PDF)Click here for additional data file.