The Function and Regulation of Photobodies in Phytochrome Signaling

Light is a critical environmental signal that regulates every phase of the plant life cycle, from germination to floral initiation. Of the many light receptors in the model plant Arabidopsis thaliana, the red- and far-red light-sensing phytochromes (phys) are arguably the best studied, but the earliest events in the phy signaling pathway remain poorly understood. One of the earliest phy signaling events is the translocation of photoactivated phys from the cytoplasm to the nucleus, where they localize to subnuclear foci termed photobodies; in continuous light, photobody localization correlates closely with the light-dependent inhibition of embryonic stem growth. Despite a growing body of evidence supporting the biological significance of photobodies in light signaling, photobodies have also been shown to be dispensable for seedling growth inhibition in continuous light, so their physiological importance remains controversial; additionally, the molecular components that are required for phy localization to photobodies are largely unknown. The overall goal of my dissertation research was to gain insight into the early steps of phy signaling by further defining the role of photobodies in this process and identifying additional intragenic and extragenic requirements for phy localization to photobodies.

Even though the domain structure of phys has been extensively studied, not all of the intramolecular requirements for phy localization to photobodies are known. Previous studies have shown that the entire C-terminus of phys is both necessary and sufficient for their localization to photobodies. However, the importance of the individual subdomains of the C-terminus is still unclear. For example a truncation lacking part of the most C-terminal domain, the histidine kinase-related domain (HKRD), can still localize to small photobodies in the light and behaves like a weak allele. However, a point mutation within the HKRD renders the entire molecule completely inactive. To resolve this discrepancy, I explored the hypothesis that this point mutation might impair the dimerization of the HKRD; dimerization has been shown to occur via the C-terminus of phy and is required for more efficient signaling. I show that this point mutation impairs nuclear localization of phy as well as its subnuclear localization to photobodies. Additionally, yeast-two-hybrid analysis shows that the wild-type HKRD can homodimerize but that the HKRD containing the point mutation fails to dimerize with both itself and with wild-type HKRD. These results demonstrate that dimerization of the HKRD is required for both nuclear and photobody localization of phy.

Studies of seedlings grown in diurnal conditions show that photoactivated phy can persist into darkness to repress seedling growth; a seedling's growth rate is therefore fastest at the end of the night. To test the idea that photobodies could be involved in regulating seedling growth in the dark, I compared the growth of two transgenic Arabidopsis lines, one in which phy can localize to photobodies (PBG), and one in which it cannot (NGB). Despite these differences in photobody morphology, both lines are capable of transducing light signals and inhibiting seedling growth in continuous light. After the transition from red light to darkness, the PBG line was able to repress seedling growth, as well as the accumulation of the growth-promoting, light-labile transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3), for eighteen hours, and this correlated perfectly with the presence of photobodies. Reducing the amount of active phy by either reducing the light intensity or adding a phy-inactivating far-red pulse prior to darkness led to faster accumulation of PIF3 and earlier seedling growth. In contrast, the NGB line accumulated PIF3 even in the light, and seedling growth was only repressed for six hours; this behavior was similar in NGB regardless of the light treatment. These results suggest that photobodies are required for the degradation of PIF3 and for the prolonged stabilization of active phy in darkness. They also support the hypothesis that photobody localization of phys could serve as an instructive cue during the light-to-dark transition, thereby fine-tuning light-dependent responses in darkness.

In addition to determining an intragenic requirement for photobody localization and further exploring the significance of photobodies in phy signaling, I wanted to identify extragenic regulators of photobody localization. A recent study identified one such factor, HEMERA (HMR); hmr mutants do not form large photobodies, and they are tall and albino in the light. To identify other components in the HMR-mediated branch of the phy signaling pathway, I performed a forward genetic screen for suppressors of a weak hmr allele. Surprisingly, the first three mutants isolated from the screen were alleles of the same novel gene, SON OF HEMERA (SOH). The soh mutations rescue all of the phenotypes associated with the weak hmr allele, and they do so in an allele-specific manner, suggesting a direct interaction between SOH and HMR. Null soh alleles, which were isolated in an independent, tall, albino screen, are defective in photobody localization, demonstrating that SOH is an extragenic regulator of phy localization to photobodies that works in the same genetic pathway as HMR.

In this work, I show that dimerization of the HKRD is required for both the nuclear and photobody localization of phy. I also demonstrate a tight correlation between photobody localization and PIF3 degradation, further establishing the significance of photobodies in phy signaling. Finally, I identify a novel gene, SON OF HEMERA, whose product is necessary for phy localization to photobodies in the light, thereby isolating a new extragenic determinant of photobody localization. These results are among the first to focus exclusively on one of the earliest cellular responses to light - photobody localization of phys - and they promise to open up new avenues into the study of a poorly understood facet of the phy signaling pathway.

Constitutive activation of the Raf-MAPK pathway causes negative feedback inhibition of Ras-PI3K-AKT and cellular arrest through the EphA(2) receptor

The Raf-mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K)-AKT pathways are two downstream effectors of the small GTPase Ras. Although both pathways are positively regulated by Ras, the Raf-MAPK and PI3K-AKT pathways have been shown to control opposing functions within the cell, suggesting a need for cross-talk regulation. The PI3K -AKT pathway can inhibit the Raf-MAPK pathway directly during processes such as muscle differentiation. Here we describe the ability of the Raf-MAPK pathway to negatively regulate the PI3K-AKT pathway during cellular arrest. Constitutive activation of Raf or methyl ethyl ketone 1 (MEK1) leads to inhibition of AKT and cellular arrest. Furthermore, we show that activation of Raf-MEK1 signaling causes negative feedback inhibition of Ras through the ephrin receptor EphA(2). EphA(2)-mediated negative feedback inhibition is required for Raf-induced AKT inhibition and cell cycle arrest, therefore establishing the inhibition of the Ras-PI3K-AKT pathway as a necessary event for the Raf-MEK1-regulated cellular arrest.

Calbindin 2 (CALB2) Regulates 5-Fluorouracil Sensitivity in Colorectal Cancer by Modulating the Intrinsic Apoptotic Pathway

The role of the calcium binding protein, Calbindin 2 (CALB2), in regulating the response of colorectal cancer (CRC) cells to 5-Fluorouracil (5-FU) was investigated. Real-time RT-PCR and Western blot analysis revealed that CALB2 mRNA and protein expression were down-regulated in p53 wild-type and p53 null isogenic HCT116 CRC cell lines following 48 h and 72 h 5-FU treatment. Moreover, 5-FU-induced apoptosis was significantly reduced in HCT116 and LS174T CRC cell lines in which CALB2 expression had been silenced. Further investigation revealed that CALB2 translocated to the mitochondria following 5-FU treatment and that 5-FU-induced loss of mitochondrial membrane potential (Delta psi(m)) was abrogated in CALB2-silenced cells. Furthermore, CALB2 silencing decreased 5-FU-induced cytochrome c and smac release from the mitochondria and also decreased 5-FU-induced activation of caspases 9 and 3/7. Of note, co-silencing of XIAP overcame 5-FU resistance in CALB2-silenced cells. Collectively, these results suggest that following 5-FU treatment in CRC cell lines, CALB2 is involved in apoptosis induction through the intrinsic mitochondrial pathway. This indicates that CALB2 may be an important mediator of 5-FU-induced cell death. Moreover, down-regulation of CALB2 in response to 5-FU may represent an intrinsic mechanism of resistance to this anti-cancer drug.

Sirolimus Stimulates Vascular Stem/Progenitor Cell Migration and Differentiation Into Smooth Muscle Cells via Epidermal Growth Factor Receptor/Extracellular Signal-Regulated Kinase/β-Catenin Signaling Pathway

Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown.

Association of common gene variants in the WNT/β-catenin pathway with colon cancer recurrence

Wnt/β-catenin signaling has a central role in the development and progression of most colon cancers (CCs). Germline variants in Wnt/β-catenin pathway genes may result in altered gene function and/or activity, thereby causing inter-individual differences in relation to tumor recurrence capacity and chemoresistance. We investigated germline polymorphisms in a comprehensive panel of Wnt/β-catenin pathway genes to predict time to tumor recurrence (TTR) in patients with stage III and high-risk stage II CC. A total of 234 patients treated with 5-fluorouracil-based chemotherapy were included in this study. Whole-blood samples were analyzed for putative functional germline polymorphisms in SFRP3, SFRP4, DKK2, DKK3, Axin2, APC, TCF7L2, WNT5B, CXXC4, NOTCH2 and GLI1 genes by PCR-based restriction fragment-length polymorphism or direct DNA sequencing. Polymorphisms with statistical significance were validated in an independent study cohort. The minor allele of WNT5B rs2010851 T>G was significantly associated with a shorter TTR (10.7 vs 4.9 years; hazard ratio: 2.48; 95% CI, 0.96-6.38; P=0.04) in high-risk stage II CC patients. This result remained significant in multivariate Cox's regression analysis. This study shows that the WNT5B germline variant rs2010851 was significantly identified as a stage-dependent prognostic marker for CC patients after 5-fluorouracil-based adjuvant therapy.

Elucidation of the Retinoid Signalling Pathway Involved in Axon Guidance in Lymnaea stagnalis and Xenopus laevis

The vitamin A metabolite, retinoic acid (RA), is known to play a crucial role in several developmental processes including axial patterning and differentiation. More recently, RA has been implicated in the regenerative process acting through its classical signaling pathway, the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), to mediate gene transcription. Moreover, RA has been shown to act as a guidance molecule for growth cones of regenerating motorneurons of the pond snail, Lymnaea stagnalis. Our lab has recently shown that RA can induce this morphological response independent of nuclear transcription, however, the role of the retinoid receptors in RA-induced chemoattraction is still unknown. Here, I show that the retinoid receptors, RXR and RAR, may mediate the growth cones response to the metabolically active retinoic acid isomers, all-trans and 9-cis RA, in Lymnaea stagnalis. Data presented here show that both an RXR and RAR antagonist can block growth cone turning in response to application of both isomers. Because no prior investigations have shown growth cone turning of individual vertebrate neurons, I aimed to show that both retinoic acid isomers were capable of inducing growth cone turning of embryonic spinal cord neurons in the frog, Xenopus laevis. For the first time in Xenopus, I showed that both all-trans and 9-cis RA were able to induce significantly more neurite outgrowth from cultured embryonic spinal cord neurons and induce positive growth cone turning of individual growth cones. In addition, I showed that the presence of the RXR antagonist, HX531, blocked 9-cis RA-induced growth cone turning and the RARβ antagonist, LE135, blocked all-trans RA-induced growth cone turning in this species. Evidence provided here shows for the first time, conservation of retinoic acid-induced growth cone turning in a vertebrate model system. In addition, these data show that the receptors involved in this morphological response may be the same in vertebrates and invertebrates.

Histone H2B-R95A mutant identifies the pheromone pathway that signals cell cycle arrest during rapamycin response

La rapamycine est un immunosuppresseur utilisé pour traiter plusieurs types de maladies dont le cancer du rein. Son fonctionnement par l’inhibition de la voie de Tor mène à des changements dans des processus physiologiques, incluant le cycle cellulaire. Chez Saccharomyces cerevisiae, la rapamycine conduit à une altération rapide et globale de l’expression génique, déclenchant un remodelage de la chromatine. Nous proposons que les modifications des histones peuvent jouer un rôle crucial dans le remodelage de la chromatine en réponse à la rapamycine. Notre objectif principal est d’identifier d’une banque de mutants d’histone les variantes qui vont échouer à répondre à la rapamycine dans une tentative de réaliser une caractérisation des modifications d’histone critiques pour la réponse à cette drogue. Ainsi, nous avons réalisé un criblage d’une banque de mutants d’histone et identifié plusieurs mutants d‘histone dont la résistance à la rapamycine a été altérée. Nous avons caractérisé une de ces variantes d’histone, à savoir H2B, qui porte une substitution de l’alanine en arginine en position 95 (H2B-R95A) et démontré que ce mutant est extrêmement résistant à la rapamycine, et non à d’autres drogues. Des immunoprécipitations ont démontré que H2B-R95A est défectueux pour former un complexe avec Spt16, un facteur essentiel pour la dissociation de H2A et H2B de la chromatine, permetant la réplication et la transcription par les ADN et ARN polymérases, respectivement. Des expériences de ChIP-Chip et de micropuce ont démontré que l’arginine 95 de H2B est requise pour recruter Spt16 afin de permettre l’expression d’une multitude de gènes, dont certains font partie de la voie des phéromones. Des évidences seront présentées pour la première fois démontrant que la rapamycine peut activer la voie des phéromones et qu’une défectuosité dans cette voie cause la résistante à cette drogue.

Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway

We investigated the ability of a selection of human influenza A viruses, including recent clinical isolates, to induce IFN-beta production in cultured cell lines. In contrast to the well-characterized laboratory strain A/PR/8/34, several, but not all, recent isolates of H3N2 viruses resulted in moderate IFN-beta stimulation. Through the generation of recombinant viruses, we were able to show that this is not due to a loss of the ability of the NS1 genes to suppress IFN-beta induction; indeed, the NS1 genes behaved similarly with respect to their abilities to block dsRNA signaling. Interestingly, replication of A/Sydney/5/97 virus was less Susceptible to pre-treatment with IFN-alpha than the other viruses. In contrast to the universal effect on dsRNA signaling, we noted differences in the effect of NS1 proteins on expression of interferon stimulated genes and also genes induced by a distinct pathway. The majority of NS1 proteins blocked expression From both IFN-dependent and TNF-dependent promoters by an apparent post-transcriptional mechanism. The NS1 gene of A/PR/8/34 NS1 did not confer these blocks. We noted striking differences in the Cellular localization of different influenza A virus NS1 proteins during infection, which might explain differences in biological activity. (C) 2005 Elsevier Inc. All rights reserved.

Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2β). Both PtdIns-3-P and PI3K-C2β are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2β as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2β- PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.

CB1 receptor allosteric modulators display both agonist and signaling pathway specificity

We have previously identified allosteric modulators of the cannabinoid CB1 receptor (Org 27569, PSNCBAM-1) which display a contradictory pharmacological profile: increasing the specific binding of the CB1 receptor agonist [3H]CP55940 but producing a decrease in CB1 receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signalling endpoints linked to CB1 receptor activation. We assessed the effect of these compounds on CB1 receptor agonist-induced [35S]GTPγS binding, inhibition and stimulation of forskolin stimulated cAMP production, phosphorylation of ERK, and β arrestin recruitment. We also investigated the effect of these allosteric modulators on CB1 agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signalling as compared to WIN55212 and having little effect on [3H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced [35S]GTPγS binding, simulation (Gαs mediated) and inhibition (Gαi mediated) of cAMP production and β arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphoryation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high affinity CB1 agonist binding sites. The receptor conformation stabilised by the allosterics appears to induce signalling and also selectively traffics orthosteric agonist signalling via the ERK phosphorylation pathway.

Tityus discrepans scorpion venom activates platelets through GPVI and a novel Src-dependent signaling pathway

In humans and other mammals, Tityus discrepans (Td) scorpion envenomation produces a variety of systemic effects including respiratory distress, a generalized inflammatory reaction, modulation of blood pressure, fibrin formation, and platelet activation. For many of these effects, the venom components and underlying mechanisms are not known. In the present study, we demonstrate that Td venom (TdV) stimulates integrin αIIbβ3-dependent aggregation of washed human and mouse platelets downstream of Src kinase activation. The pattern of increase in tyrosine phosphorylation induced by TdV in human platelets is similar to that induced by the collagen receptor GPVI, and includes FcR γ-chain, Syk, and PLC γ 2. Confirmation of GPVI activation by TdV was achieved by expression of human GPVI in chicken DT40 B cells and use of a reporter assay. To our surprise, TdV was able to activate mouse platelets deficient in the GPVI-FcR γ-chain complex through a pathway that was also dependent on Src kinases. TdV therefore activates platelets through GPVI and a second, as yet unidentified Src kinase-dependent pathway.

Palmitate Activates Insulin Signaling Pathway in Pancreatic Rat Islets

Objective: To investigate the action of palmitate on insulin receptor (IR) signaling pathway in rat pancreatic islets. The following proteins were studied: IR substrate-1 and -2 (IRS1 and IRS2), phosphatidylinositol 3-kinase, extracellular signal-regulated protein kinase-1 and -2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3). Methods: Immunoblotting and immunoprecipitation assays were used to evaluate the phosphorylation states of IRS1 and IRS2 (tyrosine [Tyr]), ERK1/2 (threonine 202 [Thr202]/Tyr204), and STAT3 (serine [Ser727]). Results: The exposure of rat pancreatic islets to 0.1-mmol/L palmitate for up to 30 minutes produced a significant increase of Tyr phosphorylation in IRS2 but not in IRS1. The association of phosphatidylinositol 3-kinase with IRS2 was also upregulated by palmitate. Exposure to 5.6-mmol/L glucose caused a gradual decrease in ERK1/2 (Thr202/Tyr204) and STAT3 (serine [Ser727]) phosphorylations after 30-minute incubation. The addition of palmitate (0.1 mmol/L), associated with 5.6-mmol/L glucose, abolished these latter effects of glucose after 15-minute incubation. Conclusions: Palmitate at physiological concentration associated with 5.6-mmol/L glucose activates IR signaling pathway in pancreatic A cells.

Amyloid beta-peptide activates nuclear factor-kappa B through an N-methyl-D-aspartate signaling pathway in cultured cerebellar cells

Amyloid P-peptide (A beta) likely causes functional alterations in neurons well prior to their death. Nuclear factor-kappa B (NF-kappa B), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by A beta in neurons and glia, but the mechanism is unknown. Because A beta has also been shown to enhance activation of N-methyl-D-aspartate (NMDA) receptors, we investigated the role of NMDA receptor-mediated intracellular signaling pathways in A beta-induced NF-kappa B activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of A beta 1-40 (1 or 2 mu M) for different periods (6, 12, or 24 hr). MK-801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src-family tyrosine kinase inhibitor), PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3-k) inhibitor] were added 20 min before A beta treatment of the cells. A beta induced a time- and concentration-dependent activation of NF-kappa B (1 mu M, 12 hr); both p50/p65 and p50/p50 NF-kappa B dimers were involved. This activation was abolished by MK-801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. AP at 1 mu M increased the expression of inhibitory protein I kappa B, brain-derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1 beta as shown by RTPCR assays. Collectively, these findings suggest that AP activates NF-kappa B by an NMDA-Src-Ras-like protein through MAPK and PI3-k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to A beta. (c) 2007 Wiley-Liss, Inc.

Endurance training improves responsiveness to insulin and modulates insulin signal transduction through the phosphatidylinositol 3-kinase/Akt-1 pathway

Background: Endurance training increases insulin-stimulated muscle glucose transport and leads to improved metabolic control in diabetic patients.Objective: To analyze the effects of endurance training on the early steps of insulin action in muscle of rats. Design: Male rats submitted to daily swimming for 6 weeks were compared with sedentary controls. At the end of the training period, anesthetized animals received an intravenous (i.v.) injection of insulin and had a fragment of their gastrocnemius muscle excised for the experiments.Methods: Associations between insulin receptor, insulin receptor substrates (IRS)-1 and -2 and phosphatidylinositol 3-kinase (PI3-kinase) were analyzed by immunoprecipitation and immunoblotting. Akt-1 serine phosphorylation and specific protein quantification were detected by immunoblotting of total extracts, and IRS-1/IRS-2-associated PI3-kinase activity were determined by thin-layer chromatography.Results: Insulin-induced phosphorylation of IRS-1 and IRS-2 increased respectively by 1.8-fold (P < 0.05) and 1.5-fold (P < 0.05), whereas their association with PI3-kinase increased by 2.3-fold (P < 0.05) and 1.9-fold (P < 0.05) in trained rats as compared with sedentary controls, respectively. The activity of PI3-kinase associated with IRS-1 and IRS-2 increased by 1.8-fold (P < 0.05) and 1.7-fold (P < 0.05) respectively, in trained rats as compared with their untrained counterparts. Serine phosphorylation of Akt-1/PKB increased 1.7-fold (P < 0.05) in trained rats in response to insulin. These findings were accompanied by increased responsiveness to insulin as demonstrated by a reduced area under the curve for insulin during an i.v. glucose tolerance test, by increased glucose disappearance rate during an insulin tolerance test, and by increased expression of glucose transporter-4.Conclusions: the increased responsiveness to insulin induced by chronic exercise in rat skeletal muscle may result, at least in part, from the modulation of the insulin signaling pathway at different molecular levels.

Acute exercise decreases PTP-1B protein level and improves insulin signaling in the liver of old rats

It is now commonly accepted that chronic inflammation associated with obesity during aging induces insulin resistance in the liver. In the present study, we investigated whether the improvement in insulin sensitivity and insulin signaling, mediated by acute exercise, could be associated with modulation of protein-tyrosine phosphatase 1B (PTP-1B) in the liver of old rats. Aging rats were subjected to swimming for two 1.5-h long bouts, separated by a 45 min rest period. Sixteen hours after the exercise, the rats were sacrificed and proteins from the insulin signaling pathway were analyzed by immunoblotting. Our results show that the fat mass was increased in old rats. The reduction in glucose disappearance rate (Kitt) observed in aged rats was restored 16 h after exercise. Aging increased the content of PTP-1B and attenuated insulin signaling in the liver of rats, a phenomenon that was reversed by exercise. Aging rats also increased the IRβ/PTP-1B and IRS-1/PTP-1B association in the liver when compared with young rats. Conversely, in the liver of exercised old rats, IRβ/PTP-1B and IRS-1/PTP-1B association was markedly decreased. Moreover, in the hepatic tissue of old rats, the insulin signalling was decreased and PEPCK and G6Pase levels were increased when compared with young rats. Interestingly, 16 h after acute exercise, the PEPCK and G6Pase protein level were decreased in the old exercised group. These results provide new insights into the mechanisms by which exercise restores insulin signalling in liver during aging. © 2013 Moura et al; licensee BioMed Central Ltd.