Brassinosteroids (BRs) are a class of steroid hormones regulating a wide range of physiological processes during the plant life cycle from seed development to the modulation of flowering and senescence. The last decades, and recent years in particular, have witnessed a significant advance in the elucidation of the molecular mechanisms of BR signaling from perception by the transmembrane receptor complex to the regulation of transcription factors influencing expression of the target genes. Application of the new approaches shed light on the molecular functions of the key players regulating the BR signaling cascade and allowed identification of new factors. Recent studies clearly indicated that some of the components of BR signaling pathway act as multifunctional proteins involved in other signaling networks regulating diverse physiological processes, such as photomorphogenesis, cell death control, stomatal development, flowering, plant immunity to pathogens and metabolic responses to stress conditions, including salinity. Regulation of some of these processes is mediated through a crosstalk between BR signalosome and the signaling cascades of other hormones, including auxin, abscisic acid, ethylene and salicylic acid. Unravelling the complicated mechanisms of BR signaling and its interconnections with other molecular networks may be of great importance for future practical applications in agriculture. Brassica napus and displaying high level of biological activity [Arabidopsis thaliana, led to the elucidation of BR biosynthesis as well as identification and functional analysis of the signaling components [Brassinosteroids (BRs) are plant-specific polyhydroxylated steroid hormones displaying high activity in stimulation of plant growth and development and regulation of a broad spectrum of physiological responses to biotic and abiotic stress conditions. The first identified representative of BRs was steroidal lactone—brassinolide, extracted from pollen grains of activity ,2. BRs hactivity ,4. Extenmponents . It has mponents ,7. BRs hmponents ,8.2 assimilation and Rubisco activity. BRs stimulate the expression of alfa- and beta-tubulin genes and affect reorientation of cortical microtubules, which influence arrangement of cellulose microfibrylls. Leaf senescence proved to be stimulated by this group of hormones as well. When exogenously applied at high concentrations, BRs inhibit root elongation, but promote lateral roots formation. Moreover, BRs regulate the processes of photo- and skotomorphogenesis (etiolation) and are known to have a positive impact on reproductive development and regulation of flowering time [BRs regulate a broad range of physiological processes, such as: seed development and germination, cell division and elongation, what results in highly stimulating impact on plant growth, differentiation of tracheary elements, polarization of cell membrane, proton pumping to apoplast and into a vacuole by stimulation of transmembrane ATPases, as well as increasing the efficiency of photosynthesis by elevating the level of COing time . Many ofing time –13.via phosphorylation/dephosphorylation cascade to the regulation of gene expression by a group of transcription factors. It has been reported that some of the components of BR signaling are directly involved in other molecular pathways regulating various physiological processes. Thus, these protein factors may function as cross-talk points interconnecting BR signaling pathway with the molecular mechanisms regulating various aspects of plant physiology. This paper reviews recent advances in elucidating molecular mechanisms of BR signal transduction pathway including introduction of new key players of this relay and presents molecular mechanisms of interconnections of BR signaling pathway with other molecular networks.Recently BRs have gained more attention as numerous reports indicated that BRs modulate plant metabolic response to environmental biotic and abiotic stresses, and these physiological processes include: salt and drought stress tolerance, thermotolerance, oxidative stress tolerance, pathogen resistance, as well as herbicide and pesticide tolerance . The queArabidopsis thaliana a model of BRs signal transduction pathway has been established. The process is commenced by the perception of the hormone ligand by the cell membrane-associated receptor complex, which initiates a relay mediated by phosphorylation/dephosphorylation cascade leading to changes in target gene expression [−2, and differences in BRI1 density in the plasma membranes of different tissues may be of regulatory importance and contribute to various sensitivities to BR [BRI1 gene have been identified [Recently by deploying genetics, genomics, proteomics and many other approaches performed mainly in pression . In planpression ,17. BRs pression ,18–20. Ipression ,22. An aes to BR ,24. Sincentified . This reentified . It has entified ,28, howeentified . This paentified . There ientified . The parentified ,33 and fentified . Stoichientified . A high-entified ,36. Downentified .i.e., Tyr-1052, Tyr-1057) stimulate its kinase activity only in de-phosphorylated state [Mass spectrometry analysis allowed identification of numerous phosphorylation sites in cytoplasmic fragment of BRI1, located throughout the juxtamembrane, kinase and C-terminal domains ,39. Phosed state . It has ed state . There aed state ,46.Binding of BR molecule by BRI1 receptor domain encompassing the 70-amino acid island and the downstream LRR induces conformational alterations in the receptor (preformed homodimer or BRI1 homodimerization), causing autophosphorylation within the kinase domain activation loop, what results in stimulation of the kinase activity, what brings about the association and phosphorylation of the second component of receptor complex, what renders it active ,44. BR bIt was shown that heterodimerization of BRI with four members of the Somatic Embryogenesis Receptor Kinase (SERK) subfamily of LRR-RLKs is required for full activation of the signaling pathway. Initially activated by ligand binding BRI1 can not activate downstream signaling components without previous transactivation with SERK proteins ,52. The Auto- and transphosphorylations cause exposition of the sites of interaction with other components of the signaling pathway, what enables formation of the receptor complex and initiates the signal transduction . The BRISimilarly to BRI1, BAK1/SERK3 also functions as a dual-specificity kinase and phosphorylates on tyrosine residues, what influences a subset of BAK1/SERK3 activities ,57. PhosThe transmembrane BRI1-BAK1 heterodimer functioning as BR co-receptor is asymmetric—BAK1/SERK3 is shorter than BRI1 because contains only five LRRs and does not include the pair of cysteines and 70-amino acid island, therefore BRI1 protein is the only component of the receptor complex that binds BR ligands ,32,58. IRecently another mechanism of regulation of the BRI1 receptor kinase activity has been reported. Dephosphorylation of BRI1 by protein phosphatase 2A (PP2A), whose distinct function in BR signaling will be described later, causes down-regulation of BRI1 activity and signaling pathway. This process is suggested to be regulated by BRs through stimulation of the Suppressor of bri1 (SBI1) leucine carboxy-methyltransferase, which methylates the PP2A phosphatase facilitating its interaction with BRI1, what results in dephosphorylation of the receptor kinase ,61.elongated (elg) mutant, which carries a mutation in the extracellular region of BAK1 protein, may be caused by reduced MSBP1-BAK1 interactions, what results in attenuated suppression of BAK1 activity [On the other hand, activity of BAK1 kinase is regulated by interaction with the Membrane Steroid-Binding Protein1 (MSBP1). MSBP1 is capable of binding the BR ligands, however with lower affinity than BRI1, and interacts specifically with the extracellular LRR domain of BAK1 in BR-independent manner. It is suggested that this interaction suppresses BR signaling. MSBP1 enhances BAK1 endocytosis, thus shifts the equilibrium to endosomes. Function of MSBP1 is based on preventing the interactions between BRI1 and BAK1, therefore MSBP1 acts as a negative regulator of the early events during BR signal transduction. Enhanced BAK1 endocytosis triggered by association with MSBP1 causes reduction in BAK1-stimulated BRI1 endocytosis, what results in BR signaling inhibition. It is suggested that BR-hypersensitive phenotype of the activity .It has also been shown that BAK1/SERK3 kinase interacts with other LRR-RLKs to induce their functions and initiate other signaling pathways that regulate plant defense responses –66. ThisOther members of the SERK family including SERK1, SERK2 and SERK4/BKK1 (BAK1-like) also interact directly with BRI1 receptor and redundantly promote BR signaling, however SERK2 plays a minor role in this process . Apart fSeveral new proteins participating in the assembling of the BR receptor complex have recently been identified. One of these components that regulates the BR signaling is BKI1 (BRI1 Kinase Inhibitor1)—negative regulator, which in the absence of BR is a membrane-bound protein anchored in the plasma membrane by N-terminal lysine-arginine-rich motif, whereas its C-terminal domain binds specifically to the cytoplasmic C-terminal domain of BRI1 receptor ,74. It iA proteomic analysis led to the identification of other components of the BR receptor complex—BR-Signaling Kinases (BSKs) belonging to the subfamily of the Receptor-like Cytoplasmic Kinases (RLCK-XII) and functioning as positive regulators of BR signaling. The members of BSK family transmit the signal between membrane-bound receptor complex and cytoplasmic regulators of BR signaling . It was Two homologous proteins—Constitutive Differential Growth1 (CDG1) and CDG-like1 (CDL1), both members of the RLCK-VIIc subfamily of cytoplasmic kinases, have been identified as positive regulators of BR signaling and the substrates of BRI1 kinase domain. Upon ligand binding BRI1 phosphorylates CDG1 on Ser-234 activating its kinase domain. The activated CDG1 phosphorylates BSU1 on Ser-764, located in its C-terminal phosphatase domain, what stimulates its activity and ultimately leads to BSU1-mediated BIN2 dephosphorylation ,80. BothTwo other polypeptides: Transthyretin-like protein (TTL) and TGFβ-Receptor Interacting Protein1 (TRIP1) which are the substrates of the BRI1 kinase have been identified. TTL is a tetrameric, bifunctional protein with decarboxylase and hydrolase activity , which ibri1 Suppressor1 (BSU1) phosphatase plays a crucial role in positive regulation of BR signaling by repressing the activity of BIN2 kinase. BSU1 contains N-terminal Kelch-repeat domain and C-terminal phosphatase domain and shows basal level of BIN2-binding and dephosphorylation. Activated BSU1 interacts with BIN2 kinase and inactivates it through dephosphorylation of Tyr-200, which is crucial residue for BIN2 activity [Cytosolic activity ,79. It wactivity . On the activity . Severalactivity ,79.Drosophila melanogaster Shaggy kinase and mammalian Glycogen Synthase Kinase3, which often are negative regulators of signaling pathways, regulating different aspects of metabolism [BIN2 belongs to a multi-gene family encoding GSK3-like kinases, regulating broad spectrum of physiological processes, including development of generative organs and responses to salinity stress and wounding [Arabidopsis Shaggy-like Kinases (ASKs). Several of them, i.e., BIN2 (ASKη), ASKι and ASKζ, which belong to the subgroup II are redundantly involved in the BR signal transduction through phosphorylation of transcription factors regulating BR-dependent gene expression [A crucial role in BR signaling is played by the serine-threonine kinase Brassinosteroid-Insensitive2 (BIN2), which is another negative regulator of BR signaling, phosphorylating and thus inhibiting transcription factors regulating expression of target genes. BIN2 protein sequence share similarity with tabolism . The Arawounding ,92. BIN2pression –95. A noIn the absence of BR BIN2 autophosphorylates on Tyr-200 residue, which is required for its kinase activity . BIN2 kiCPD gene by heterodimerization with the close homologue of CESTA, BRI1 Enhanced Expression1 (BEE1). BIN2-mediated phosphorylation of CESTA is assumed to regulate the nuclear localization of this transcription factor [BIN2 gene expression level [BIN2 inactivates two closely related transcription factors - Brassinazole-Resistant1 (BZR1) and BRI1-EMS-Supressor1/Brassinazole-Resistant2 (BES1/BZR2) that bind to the promoters of BR-regulated genes ,101. Then factor . Based oon level .The phosphorylation of the BZR1 and BES1 transcription factors by BIN2 in the nucleus reduces their affinity to DNA, therefore attenuating binding to the promoters of target genes. Additionally, phosphorylated BZR1 and BES1 are also prevented from dimerization with other transcription factors . It was The phosphorylation of BZR1 and BES1 mediated by BIN2 kinase located in the cytoplasm results mainly in cytoplasmic retention of these transcription factors, which is caused by binding the 14-3-3 proteins ,106,107.BR perception by the BRI1-BAK1/SERK3 receptor complex causes accumulation of de-phosphorylated and vanishing of phosphorylated forms of BZR1 and BES1/BZR2, which is caused by the inactivation of the BIN2 kinase. Dephosphorylation of BZR1 and BES1/BZR2 transcription factors leads to dissociation of the 14-3-3 proteins and migration of these transcription factors to the nucleus ,106. TheT/CG) but lower affinity for the E-box element (CANNTG). The E-box elements are present in the promoters of both BR-induced and BR-repressed genes, whereas BRREs are more abundant in the promoters of BR-repressed genes [BRI1 gene based on the feedback loop. Additionally, BES1 down-regulates expression of its two homologs–BEH1 and BEH2 [BIN2 gene and enhancing expression of the BSU1 gene [DWARF4 gene, involved in BR biosynthesis, is antagonized by positive effect of the TCP1 transcription factor belonging to the broad family of proteins containing the bHLH domain. BR treatment significantly stimulates the TCP1 gene expression. However, TCP1 does not bind BZR1 promoter [The active, dephosphorylated BZR1 and BES1 regulate the expression of hundreds of BR target genes ,111. Theed genes . Both trand BEH2 . On the SU1 gene . Negativpromoter ,116.MYB30 gene, which is a direct target of BES1 transcription factor and itself regulates a subset of BR-responsive genes [BES1/BZR2 activates transcription of BR-responsive genes together with the group of three BES-Interacting Myc-like proteins (BIM1-3), which interact with BES1 by binding to its HLH dimerization domain ,101,114.ve genes . MYB30 ive genes .As far as the extent of BR-dependent regulation of gene expression is concerned, it was reported that expression of about 1,200 genes is influenced by BRs. Out of this number about 950 genes proved to be direct targets of BZR1 transcription factor, 450 of them were up-, whereas another 462 genes were down-regulated . About 2MYBL2 gene results in a typical phenotype of mutant plant with defects in BR metabolism, what suggests that suppression of BR-repressed gene expression is crucial for optimal BR response. Interestingly, MYBL2 is a substrate of BIN2 kinase. However, unlike BIN2 phosphorylation of the BZR1 and BES1 transcription factors, which renders these factors inactive and directs them to proteasomal degradation, it was shown that BIN2 phosphorylation stabilizes MYBL2 [Recently, a new key factor regulating BR-dependent gene expression has been identified. It has been reported that BES1 interacts with the Myeloblastosis family transcription factor-like 2 (MYBL2), which is a transcription repressor, inhibiting expression of BR-repressed genes. The loss-of-function mutation of the es MYBL2 . Hence, BRI1 and several BR-biosynthesis genes, however the activation of expression of the BR-synthesis genes mediated by this transcription factor may be suppressed by BZR1. RAV1 transcription factor contains B3 DNA-binding domain showing different affinity for E-box elements in the promoters of the regulated genes. Surprisingly, RAV1 is not subject to BR feedback regulation, however is involved in maintaining the expression of the regulated genes implicated in BR signaling and synthesis at the basal level. RAV1 expression is suggested to be negatively regulated by auxins through binding of the Auxin Response Factor (ARF) to the RAV1 promoter. This mechanism together with the negative feedback regulation maintains BR homeostasis [The novel transcription factor, Related to ABI3/VP1 (RAV1), has been identified in Arabidopsis and rice, which stimulates expression of both the BR-receptor gene eostasis ,125.PRE1 gene. Activity of PRE1  is based on mechanism, in which dimerization of the transcription factor with classical bHLH protein promotes binding to the E-box motif (CANNTG) in promoter of a target gene, whereas association with atypical, non-DNA binding bHLH factors inhibits DNA binding of both associated transcription factors [PRE1 gene expression regulation, BZR1 exerts negative effect on the expression level of IBH1. Therefore, the antagonistic activity of PRE1 and IBH1 may constitute a molecular mechanism that stimulates growth rate in tissues at early developmental stages, which are known to be more BR-sensitive, whereas in mature organs growth is inhibited [A new group of atypical bHLH transcription factors, comprising six proteins PACLOBUTRAZOL RESISTANT (PRE1–6) is implicated in positive regulation of BR signaling ,126. Exp factors . It is snhibited ,126.ATBS1 and AIF genes is BR-dependent, however these genes show contrary expression profiles—ATBS1 expression is down-regulated, while expression of AIF is induced by BR [ATBS1 is directly regulated by auxin-dependent transcription factor MONOPTEROS. Other PRE factors are implicated in gibberellin and light signaling pathways as well as photomorphogenesis [The same mechanism based on the dimerization of transcription factors, which influences the expression of target genes has been proposed for another representative of the PRE family—PRE3, also named Activation-Tagged BRI1-Suppressor1 (ATBS1), which is a nuclear-localized positive regulator of BR-dependent gene expression. It has been shown that ATBS1 interacts with four paralogous, nuclear-localized atypical bHLH transcription factors ATBS1-Interacting Factor (AIFs), which are known to be negative regulators of BR-dependent gene expression, incapable of binding DNA. It is suggested that AIFs function through interactions with two BEE proteins, but not with the BZR1 or BES1 transcription factors. Positive role of ATBS1 in the regulation of BR signaling is mediated by sequestering the negative regulators—AIFs. This model grew even more complicated by the fact of AIF1 phosphorylation by BIN2 kinase, but the exact effect of this phosphorylation on AIF1 activity is not fully understood ,120. Exped by BR . Moreoveogenesis –130 indiogenesis ,120. It Pathogen attacks pose a major factor limiting seed production . During i.e., flagellin, the elongation factor EF-Tu, the bacterial-cold shock protein and the oomycete elicitor INF1. This molecular responses act as the first line of defence against pathogens [Recent studies clearly indicated, that some of the components of BR signaling pathway act as multifunctional proteins involved in other signaling networks regulating diverse physiological processes, like photomorphogenesis, cell death control, stomatal development, flowering, plant immunity to pathogens and metabolic responses to stress conditions. Regulation of some of these processes is mediated through a crosstalk between BR signaling pathway and signaling cascades of other hormones, including auxin, abscisic acid, ethylene and salicylic acid . One of athogens ,73,140.BAK1 loss-of-function on the EFR-mediated response is less pronounced than on the FLS2-regulated signaling pathway [SERK genes is up-regulated in response to PAMP and pathogen treatment [Nicotiana attenuata plants against its herbivore Manduca sexta. This mechanism is dependent on modulation of the herbivory-induced jasmonic acid accumulation and regulation of activity of defensive secondary metabolites [BAK1/SERK3 is a convergence point that connects growth promoting BR responses with PAMP signalosome ,141. Per pathway ,140,146.reatment , what sureatment ,148. Thureatment . The sigreatment . BR-stimreatment ,150. It abolites .bak1 knock-out mutants have a spreading lesion phenotype upon pathogen infection and premature senescence. This phonotype is further enhanced in double-mutant combinations with its closest paralogue BKK1/SERK4, also mediating BR perception. Interestingly, cell-death phenotype of the double mutant bak1/bkk1 occurs even in a sterile growth conditions. It was also reported that the phenotype is enhanced by light, therefore it is suggested that the double mutant is deprived of the capability to detoxify or restrict the production of phototoxins induced by light. The uncontrolled accumulation of the phototoxins is likely a cause of the observable spontaneous cell death phenotype [BAK1/SERK3 is also implicated in cell-death control, and henotype ,152,153.henotype .bak1-5, demonstrated that the role of BAK1 in various signaling pathways is mechanically separated. The missense mutation in BAK1 kinase domain represses innate immune response, whereas steroid signaling and cell-death control remain unaffected [elongated-D (elg-D), exhibits enhanced BR response through elevated level of BR signaling. This mutant harbours change of the highly conserved Asp-122 in the third LRR of BAK1 co-receptor, which renders the protein constitutively active in the stimulation of BR response. Apart from the increase in BR signaling, elg-D allele causes increased efficiency of infection with Pseudomonas syringae, whereas the allele showed no effect on EFR-mediated signaling. Therefore, it is concluded that this allele of BAK1 leads to the promotion of plant growth, mediated by the elevated level of BR signaling, at the expense of efficiency of the responses to pathogen invasion and salinity stress, which are impaired. It substantiates the general observation that increased growth often impairs the plant resistance to stress conditions [elg-D allele stimulates BR signaling by causing the encoded mutated BAK1 co-receptor to associate preferentially with BRI1 over FLS2, most likely by enhancing the ‘double-lock’ interaction. This affinity difference underlies the opposite gain- and loss-of-function phenotypes in BR and flagellin signaling, respectively [i.e., light, water or nutrients [Identification and characterization of the new BAK1 allele, affected . Anothernditions ,156. It ectively . Therefoutrients .Pseudomonas syringae, AvrPto and AvrPtoB, target early steps in various PAMP pathways, upstream the MAPK cascade important for Pathogen-Triggered Immunity (PTI) [It has been reported that two unrelated effectors from ty (PTI) . Both efty (PTI) . Interesty (PTI) .2+-dependent manner [2+ concentration [2+ to calmodulin induces its conformational changes, which enable interacting with target proteins leading to alteration of their activities [2+-dependent CaM binding to the kinase domain of BRI1 inhibits both auto- and transphosphorylation activity of the receptor kinase. The interaction of calmodulin and BRI1 is suggested to constitute a link between BR and Ca2+ signalosomes [It has been recently reported that the SERK proteins are not the only members of the BR receptor complex that serve as the cross-talk points with other signaling pathways. It has been shown that calmodulin (CaM) interacts with the cytoplasmic kinase domain of BRI1 receptor in Cat manner . It is nntration ,162. Caltivities ,164. It alosomes .It has been recently reported that Brassinosteroid Signaling Kinase5 (BSK5) belonging to the RLCK family, the same to which two paralogous factors, BSK1 and BSK3 mediating BR signaling belong, is up-regulated by BR and ABA. The abiotic stresses, including salinity and drought, also enhance BSK5 expression to various extents. BSK5 kinase is required for salt stress and ABA-mediated drought stress tolerance. BSK5 stimulates salt stress response and suppresses salinity-induced ABA biosynthesis. This kinase regulates process of ABA-dependent stomatal closure under drought stress and negatively influences drought stress response through modulating gene expression .Similarly to the members of the SERK family, several members of the RLCK-VII subfamily, the one to which CDG1 belongs, including plasma membrane-localized protein PBS1, PBS-like proteins (PBLs) and Botrytis-Induced Kinase1 (BIK1) function downstream of multiple immune receptor kinases perceiving pathogen-associated molecular patterns. PBL1 and BIK1 interact directly with the unstimulated flagelling receptor FLS2 and BAK1. Upon binding of flagellin by the FLS2 receptor PBL1 and BIK1 become phosphorylated, and as a result they transphosphorylate FLS2 and BAK1 and dissociate from the receptor complex ,144. ThuRecently it has been reported that BRs are also implicated in the regulation of stomatal development. The regulatory function is based on activation of the MAPK kinase kinase YODA, which is a negative regulator of stomatal development. In the absence of BRs, BIN2 kinase  inhibits YODA activity through phosphorylation of its auto-regulatory domain, leading to suppression of the MAPK signaling ,167. It DWF4 [BIN2 kinase activity is not merely restricted to BR signaling pathway, it also phosphorylates Auxin Response Factor 2 (ARF2), which is a negative regulator of auxin response. The BIN2-mediated phosphorylation inhibits DNA-binding activity of ARF2, hence inducing downstream auxin responses . The dirDWF4 ,169. BINDWF4 , thus BILD and FCA autonomous pathways genes, which suppress the FLC gene expression. However, it is suggested that BR promote flowering also independently of the LD and FCA genes and this pathway is also aimed at BR-induced suppression of the FLC expression [ELF6 and REF6, which are involved in the flowering time control. ELF6 functions in the photoperiodic flowering pathway, whereas REF6 acts as a repressor of the FLC gene expression [Brassinosteroids enhance flowering by stimulating the expression level of the pression . BES1 trpression . It was pression . Apart fpression , what inpression .Another key regulator of BR-dependent gene expression, BZR1 mediates signaling pathways cross-talk between BR and auxin by direct regulation of auxin-responsive genes expression as well as modulation of expression of multiple genes involved in auxin signaling and metabolism. It was also reported that both transcription factors BES1 and BZR1 bind to the promoters of numerous genes involved in the signaling and synthesis pathways of gibberellin (GA), abscisic acid, ethylene, cytokinin and jasmonate, suggesting that BR signaling impacts metabolism of several other plant hormones ,114. It GATA2 gene is mediated by direct binding of BZR1 to the GATA2 promoter, and this mechanisms is crucial to prevent photomorphogenesis in the dark. BR signaling efficiency does not fluctuate significantly under light and dark conditions, however BZR1 binding to GATA2 promoter is reduced in the light, what results in attenuation of the repressive impact of BR on GATA2 expression. Moreover, light stimulates accumulation of GATA2 protein, and on the other hand, inhibits the GATA2 gene transcription in a feedback manner. GATA2 inhibits its own transcription in a feedback manner, and this process serves as the mechanism of desensitization during transition from dark to light. Taken together, this BZR1-mediated repression of the GATA2 gene expression is essential for maintaining complete skotomorphogenesis in the dark [BZS1 was decreased in the light, but induced in the dark. On the other hand, light promotes BZS1 protein accumulation. BZS1 represses BR responses, however positively regulates light signaling pathway. It is suggested that BZS1 interacts with HY5 transcription factor to control expression of a subset of light-responsive genes [BZR1 was reported to repress expression of the GATA2 transcription factor, which is the main component of transcriptional regulation of many genes involved in de-etiolation. GATA is a key transcriptional regulator that mediates cross-talk between BR- and light signaling pathways, being positive regulator of photomorphogenesis. This BR-dependent transcriptional repression of the the dark ,175. It the dark . Recentlve genes .i.e., oxidative stress, drought, salinity, heavy metal stress and thermal stress BRs promote expression and activity of many enzymes, including superoxide dismutases, catalases, ascorbate peroxidises, glutathione reductases and heat-shock proteins, as well as enhance concentration of metabolites, i.e., ascorbic acid, carotenoids, glutathione and phytochelatins. Moreover, in response to biotic stresses, mainly evoked by bacterial, fungal and viral infections BRs stimulate synthesis of abscisic acid, ethylene and salicylic acid [2O2 production, what impacts abiotic and biotic stress tolerance, however the exact mechanism is not fully elucidated [It is now well known that in response to wide variety of abiotic stresses, lic acid . BRs reglic acid –180. BRslic acid . It has lic acid . BR togeucidated .Solanum lycopersicum Eix protein is perceived by two receptors LeEix1 and LeEix2 belonging to the RLK family. However, LeEix2 is the only receptor initiating defence signaling, whereas LeEix1 heterodimerizes with LeEix2 and attenuates the signaling. Moreover, the inhibitory function of LeEix1 depends on association of this receptor with the BAK1/SERK3 kinase. This indicates that Eix-induced defence response mediated by LeEix2 is attenuated by the interaction of BAK1 with the decoy receptor LeEix1. The kinase activity of BAK1 is required for the attenuation of Eix-induced signaling [BRs belong to a vast group of factors influencing ethylene biosynthesis through regulation of activities of key enzymes involved in this process . Recent ignaling .Involvement of BR signaling components in such a wide variety of metabolic pathways regulating diverse physiological processes, including plant responses to biotic and abiotic stresses, indicates that the members of BR signalosome may also serve in the future as target for genetic engineering approaches aimed at improved responses to biotic and abiotic stress factors. In fact, overexpression of the BAK1 homologue (OsSERK1) in rice led to increase in the resistance of transgenic rice plants to blast fungus . Therefo


Human T-Lymphotropic Virus Type 1 (HTLV-1) infection causes lethal adult T-cell leukemia (ATL) and severely debilitating HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in up to 5% of infected adults. HTLV-1 is endemic in parts of Africa and the highest prevalence in West Africa (5%) has been reported in Caio, a rural area in the North-West of Guinea-Bissau. It is not known which HTLV-1 variants are present in this community. Sequence data can provide insights in the molecular epidemiology and help to understand the origin and spread of HTLV-1.To gain insight into the molecular diversity of HTLV-1 in West Africa.HTLV-1 infected individuals were identified in community surveys between 1990–2007. The complete Long Terminal Repeat (LTR) and p24 coding region of HTLV-1 was sequenced from infected subjects. Socio-demographic data were obtained from community census and from interviews performed by fieldworkers. Phylogenetic analyses were performed to characterize the relationship between the Caio HTLV-1 and HTLV-1 from other parts of the world.LTR and p24 sequences were obtained from 72 individuals . Consistent with the low evolutionary change of HTLV-1, many of the sequences from unrelated individuals showed 100% nucleotide identity. Most (45 of 46) of the LTR sequences clustered with the Cosmopolitan HTLV-1 subtype 1a, subgroup D (1aD). LTR and p24 sequences from two subjects were divergent and formed a significant cluster with HTLV-1 subtype 1g, and with the most divergent African Simian T-cell Lymphotropic Virus, Tan90.The Cosmopolitan HTLV-1 1aD predominates in this rural West African community. However, HTLV-1 subtype 1g is also present. This subtype has not been described before in West Africa and may be more widespread than previously thought. These data are in line with the hypothesis that multiple monkey-to-man zoonotic events are contributing to HTLV-1 diversity. Human T-Lymphotropic Virus type 1 (HTLV-1) affects millions of people worldwide. It is very similar to Simian T-Lymphotropic Virus, a virus that circulates in monkeys. HTLV-1 causes a lethal form of leukemia (Adult T-cell Leukemia) and a debilitating neurological syndrome  in approximately 5% of infected people. Based on sequence variation, HTLV-1 can be divided into 7 subtypes (1a–1g) with the Cosmopolitan subtype 1a further subdivided into subgroups (A–E). We examined HTLV-1 diversity in a rural area in Guinea-Bissau, a country in West Africa with a high HTLV-1 prevalence (5%). We found that most viruses belong to the Cosmopolitan subtype 1a, subgroup D, but 2 viruses belonged to subtype 1g. This subtype had thus far only been found in monkey hunters in Cameroon, who were probably recently infected by monkeys. Our findings indicate that this subtype has spread beyond Central Africa. An important, unresolved question is whether persons with this subtype were infected by monkeys or through human-to-human transmission. The retrovirus Human T-Lymphotropic Virus type 1 (HTLV-1) was first discovered in 1979 HTLV-1 is an ancient retrovirus with a very low evolutionary rate  After an initial dispersion in Asia and Africa, the Cosmopolitan subtype 1a spread worldwide and is thought to have been introduced into the Americas during the post-Columbian slave trade The study area of the current analysis is a rural community in Guinea-Bissau and has the highest reported HTLV-1 prevalence in West Africa (5% in 2007) Informed consent was obtained from all the study participants. From 2003 onwards, written informed consent was obtained as required by the Gambian Ethics Committee. In studies prior to the 2003 study, written informed consent was not required by the Gambia Government/MRC MRC Laboratories Joint Ethics Committee nor by the Ministry of Health of Guinea-Bissau and thus verbal consent was obtained. The documentation of the informed consent was done in a two-step process. After information on the study was given by the field worker, and after having responded to any questions that the prospective participant might have, the field worker asked whether the participant was willing to participate in the study. The field worker noted down the answer by ticking one of two boxes (Accepting to participate in study or not); a separate question was asked about providing a blood sample and the answer to this question was noted separately.. All studies, (both prior to and after 2003), were approved by the Gambia Government/MRC MRC Laboratories Joint Ethics Committee and by the Ministry of Health of Guinea-Bissau.The study area, Caio, is a settlement of 10 small villages with 10,000 inhabitants in North-Western Guinea-Bissau and has been described in detail In the studies conducted in 2003, 2006 and 2007, DNA was extracted from blood samples , and stored for molecular analyses. Because these samples have been used in different studies, not each HTLV-1 infected individual had a stored sample; some samples had been depleted during other studies or could not be traced or storage had failed (e.g. freezer breakdowns). The available samples were used for HTLV-1 sequencing for the current study.Samples for LTR sequencing were selected in 2010 from stored DNA samples. All complete LTR sequences obtained from mother-child pairs from the vertical transmission study in 2004 were also used in the current analysis (accession numbers JN655856 to JN655872). Sau 3A (which cuts only HTLV-1) to distinguish products from the two viruses. In this study all PCR-positive samples were confirmed to be HTLV-1 by Sau 3A digestion. The HTLV-1 testing in the 2007 study (2 ELISAs and PCR confirmation) and the HIV testing of previous studies have been described in detail In the 1997, 2003 and 2004 studies, plasma samples were tested for the presence of antibodies to HTLV-1 and HTLV-2 using a Murex HTLV-1+2 ELISA . Reactive samples were retested by the same assay. HTLV-1 infection status was further determined by PCR using primers targeted to the tax/rex gene Amplification of the HTLV-1 proviral DNA was performed with nested PCR. PCR primers were optimized for a melting temperature (Tm) between 57 and 60°C and are listed in www.macrogen.com). Complete LTR and p24 sequences were assembled and ambiguities were resolved using the sequence alignment editor BioEdit. PCR products were purified using Qiaquick Gel Extraction (Qiagen) and sequenced using the inner PCR primers from both directions by Macrogen , 2 siblings (Caio4757 & Caio4758) and 6 mother-child pairs . Within each of these 8 family pairs, the viruses showed 100% identical sequences.First, LTR sequences were analyzed to identify the HTLV-1 subtypes present in the village . IdenticA phylogenetic tree was constructed with all nonidentical LTR sequences (5 cells) and HIV-2  infection in 1990. She had not travelled outside of Guinea-Bissau and had not been bitten by a monkey or hunted monkeys (information obtained in the 1997 survey). Her husband was HTLV-1 negative in the most recent survey (2007).One virus LTR sequence, Caio4064, was very divergent (6%) from the major group of Caio subtype 1a isolates. Caio4064 clustered significantly with an STLV-1 sequence obtained from a wild African green monkey in the Central African Republic 5 cells) and HIV-2  in 1997. He was not married and had no epidemiological link with the patient providing Caio4064. No samples were available from the mothers of subjects providing Caio4064 or Caio65552 to examine vertical transmission.Further evidence for the presence of subtype 1g was obtained by examining a set of p24 sequences obtained from 36 individuals  Remarkable genetic stability in 1aD sequences was observed and in particular, 10 identical LTR sequences and 16 identical p24 sequences were found in people with no identified epidemiological links. These infections differed in time of diagnosis, route of infection  and the length of the infection (a few years vs. >14 years). This observation is consistent with the very low evolutionary rate of HTLV-1  HTLV-1 subtypes Table S1Characteristics of 72 individuals from whom HTLV-1 LTR and/or p24 sequences were obtained, Caió, Guinea-Bissau.(DOC)Click here for additional data file.


HTLV-1 infection increases susceptibility and severity of tuberculosis. Despite the documentation of a decreased in vitro and in vivo T cells response to mycobacterial antigens, the reasons of the HTLV-1 infection increase the susceptibility and make worse the clinic course of tuberculosis are not understood. For instance, it is not known if HTLV-1 infection increases bacillary load and if severity of tuberculosis is due to the inability of these patients to control bacterial growth. The aim of the present study was to evaluate how HTLV-1 might influence the clinic, bacteriologic and immunologic features of tuberculosis.A prospective cohort studies enrolled 13 cases of tuberculosis associated with HTLV-1 (cases) and 25 patients with tuberculosis without HTLV-1 (controls). Clinical findings, bacillary load in the sputum, x-rays, immunological response and death were compared in the two groups.There was no difference in the demographic features, clinical manifestations and in vivo response to PPD. IFN-γ were higher in unstimulated cultures of mononuclear cells of HTLV-1 patients with tuberculosis than in controls (P<0.01). The enhancement in TNF-α and IFN-γ responses after stimulation with PPD was similar in cases and controls. There was no difference in the bacillary load among the groups but baciloscopy became negative faster in cases than in controls. Death only occurred in two co-infected patients.Severity of tuberculosis in patients co-infected with tuberculosis may be due to the enhancement in the Th1 inflammatory response, rather than a decreased ability to control bacteria growth.


Human T-cell Leukemia Virus type I (HTLV-I) infects an estimated 15-20 million persons worldwide. A number of diseases have been associated with this virus including adult T-cell leukemia/lymphoma (ATLL), HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), HLTV-I uveitis, and HTLV-I–associated infective dermatitis. The screening process for HTLV-I among blood banks includes detection by an enzyme immunoassay (EIA) followed by a confirmatory western blot (WB). Seropositive results are defined by the presence of all core bands  as well as the band specific for recombinant HTLV-I Env glycoprotein. However, numerous cases have been documented in which a positive response was detected by ELISA, but WB displayed an incomplete banding pattern. These samples were then categorized as seroindeterminates. Using the LIPS assay, 60% of the 53 seroindeterminate samples previously screened by ELISA and WB displayed anti-HTLV-I Gag, Env or Tax antibody responses . The LIPS assay was also able to detect anti-HTLV-I antibody responses in 6% of the 167 samples determined to be HTLV-I ELISA negative. A number of these samples were subsequently found to have an indeterminate WB pattern. Although the significance of these HTLV-I/II seroindeterminates is unclear, it may suggest a much higher prevalence of exposure to HTLV-I/II than previously estimated, as seroindeterminate samples may indicate exposure to the virus. The LIPS assay is a sensitive and high throughput antibody detection assay which may prove to be a useful tool in HTLV-I related clinical investigations.


In this review, we summarize the major functions of the BMP, FGF, Nodal and Wnt signaling pathways, which have well-established roles in vertebrate embryogenesis. Then, we highlight recent studies in pluripotent stem cells that have revealed the stage-specific roles of BMP,FGF and Nodal pathways during neural differentiation. These findings enhance our understanding of the stepwise regulation of embryo patterning by particular signaling pathways and provide new insight into the mechanisms underlying early embryonic development.Early embryonic development is a multi-step process that is intensively regulated by various signaling pathways. Because of the complexity of the embryo and the interactions between the germ layers, it is very difficult to fully understand how these signals regulate embryo patterning. Recently, pluripotent stem cell lines derived from different developmental stages have provided an  During early development in the mouse, the zygote first develops into a blastocyst containing the inner cell mass (ICM) inside the trophectoderm. At embryonic day (E) 4.5, the blastocyst implants in the uterus and the ICM differentiates to form the primitive endoderm and the early epiblast . At E5.5Xenopus, zebrafish and chick embryos are widely used, as they are easily raised and can be manipulated with various assays. Compared with these species, mice as mammals more closely resemble humans in developmental processes, but mouse embryos are difficult to manipulate, especially after implantation in the uterus. In past years, genetic, molecular and cell transplantation experiments in these animal models have established that the BMP, FGF, Nodal and Wnt signaling pathways play crucial roles in early embryonic patterning. However, there have been inconsistent results obtained from Xenopus, zebrafish and chick, probably caused by species-specific differences or by interference from complex germ-layer interactions. In the mouse embryo, knowledge of the molecular mechanisms governing early embryogenesis is limited due to the small size, complexity and inaccessibility of the early post-implantation embryo. Thus, mechanistic studies require an amenable system with embryonic properties but with the absence of the complexity that exists in vivo. Under these circumstances, pluripotent stem cells derived from different developmental stages have become an in vitro system for investigating the detailed molecular mechanisms through which signaling pathways regulate cell fate decisions. Both human and mouse embryonic stem cells (ESCs) are pluripotent cell lines derived from blastocyst-stage embryos [in vivo [in vitro equivalent of the late epiblast, form a novel system for studying the mechanisms of early embryonic development, especially for mechanistic studies at different developmental stages. In this review, we briefly summarize the key functions of the BMP, FGF, Nodal and Wnt signaling pathways in early embryogenesis, and then we discuss recent findings obtained from studies in ESCs and EpiSCs that reveal the stage-specific functions of BMP, FGF and Nodal signals. These findings begin to elucidate the mechanisms underlying different stages of early embryonic development.For studies of early development,  embryos –7. Under[in vivo –11. Rece[in vivo –14. ESCs2.Xenopus blastula-stage embryos, researchers have shown that activation of the BMP pathway in ectoderm leads to the acquisition of an epidermal fate, whereas inhibition of BMP signaling by antagonists that are secreted by the Spemann organizer leads to a neural fate [Sox2 and late Sox3), but it does not affect the pre-neural marker (early expression of Sox3). Therefore, BMP inhibition is probably required only as a late step during neural induction [The bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF-β) cytokine superfamily. BMP signaling has been shown to play a central role in ectodermal cell fate decisions. Using ectodermal explants  from ral fate ,16. Thesral fate . Since tral fate . Howeverral fate . Furthernduction .chordin and noggin) mouse mutants show relatively little change in the initial size of the neural plate [Bmpr1a) knockout mice. Bmpr1a is the only type I BMP receptor expressed in the epiblast of implanted mouse embryos [Bmpr1a in mouse embryos, which completely inhibits BMP activity, was found to lead to premature neural differentiation of the epiblast accompanied by suppression of mesodermal fate [Other data relevant to the default model come from experiments showing that BMP antagonists are unable to induce neural character in the epidermal or extra-embryonic ectoderm of chick embryos –22. Moreal plate –25. Thes embryos . Knockoumal fate . Therefomal fate .in vivo studies. Recently, some findings in pluripotent stem cells have shed light on these issues. In mouse ESCs, it was confirmed that BMP4 significantly inhibits neural differentiation, as it does in vivo [Id) genes to specifically block neural differentiation [The discrepancies of the data obtained from chick and mouse raise important questions, including whether there is a time point during which the BMP signal inhibits neural induction and what mechanisms are involved in this process. These questions are difficult to answer using only  in vivo . Moreove in vivo . Therefontiation . In addintiation –35. The ntiation . We firsIds, the direct downstream targets of BMP, were found to inhibit the conversion of ESCs into ESD-EpiSCs during the first stage, and to reduce ESD-EpiSC self-renewal, inhibit neural specification and promote mesodermal and trophectodermal differentiation during the second stage. FGF-Erk signaling was also found to be involved in the functions of BMP signaling during different stages. In ESCs, FGF-Erk activity was found to be reduced by short-term treatment with BMP4, whereas in ESD-EpiSCs, FGF-Erk activity increased during long-term treatment with BMP4. Therefore, BMP might perform its stage-specific roles by interfering with the FGF-Erk pathway.Based on this model, the molecular basis of the distinct functions of BMP4 was further investigated . Ids, thXenopus. However, the ectoderm stage is usually overlooked during early embryonic development in chick and mouse because of its difficult accessibility and lack of markers. To confirm the default model of neural induction from ectoderm and the function of BMP during this process, an in vitro model of ectodermal cells needs to be established. We have recently identified ectoderm-like cells that form during mouse EpiSC neural and epidermal differentiation [Due to the complexity of embryogenesis, the BMP signal may have divergent roles at other time points during the multi-step developmental process. For instance, BMP specifically induces an epidermal fate in ectoderm, which has been demonstrated in ntiation , and the3.Xenopus, it has been demonstrated that in addition to inhibition of BMP, pre-gastrula FGF signaling is also required in the ectoderm for the emergence of neural fates [Xenopus [The studies reviewed above indicate a central role for BMP signaling in both the maintenance of mouse ESC pluripotency and neural inhibition. Data from different species show that the signaling of fibroblast growth factors (FGFs) also plays an important role during early embryogenesis through both BMP-dependent and -independent mechanisms. In al fates . In chical fates . Moreoveal fates ,21,39. Aal fates ,41. FGFs[Xenopus ,43, zebr[Xenopus ,45 and m[Xenopus ,47. ThusFgf4 or Fgfr2 results in peri-implantation lethality [Fgf8 and Fgfr1 are also expressed in the blastocyst, but they appear to function later. Both Fgf8 mutants and Fgfr1 mutants die at late gastrulation with impaired axis formation and mesoderm specification [Fgf8−/− embryos, patterning of the prospective neuroectoderm is greatly perturbed and the range of anterior neuroectoderm markers is widely expanded [Hesx1 [However, an important role for FGF signaling in neural induction has not been conclusively demonstrated in mice. Mutation of ethality ,49, whicfication ,51. In Fexpanded . Anotherd [Hesx1 . TherefoXenopus and chick. Recently, the early roles of FGF signaling have become increasingly understood using mouse pluripotent stem cells. In 2007, Stavridis et al. found that inhibition of FGF/Erk during mouse ESC differentiation abolished neuronal induction with no reduction in levels of the pluripotency marker Nanog [et al. further showed that mouse ESCs lacking Fgf4 or Erk2 and those treated with FGFR inhibitors not only resist neural induction, but also fail to undergo mesodermal differentiation even when BMP4 is added. Moreover, Erk2-null ESCs retain their expression of the pluripotency markers Oct4, Nanog and Rex1 when differentiated in adherent culture [Thus, the function of FGF signaling in the pre-implantation mouse embryo needs to be elaborated. The answer to this question may explain the neural induction effect of FGF observed in er Nanog . Kunath  culture . Therefoet al. found that FGF-Erk inhibitors, in combination with the inhibition of glycogen synthase kinase 3 , keep mouse ESCs in an undifferentiated state called the “ground state” [Subsequently, Ying d state” . Based od state” . Followid state” ,57. Reced state” . These dd state” . These rd state” .Pax6 expression [In addition to the early roles of FGF, the subsequent functions of FGF signaling starting from the late epiblast stage are being investigated in EpiSCs. First, as in human ESCs, FGF2 is required to maintain EpiSC pluripotency ,13. Howepression . Blockagpression . These rpression ,51,61,62In summary, FGF signaling plays distinct roles during different developmental stages. FGF signaling is crucial for priming ESCs to differentiate into the late epiblast state. It then acts to inhibit the subsequent transition to neuroectoderm. This conclusion is distinct from the notion that neural differentiation is promoted by FGF signaling. In the future, it will be interesting to determine the exact functions of divergent FGF ligands during commitment to the three germ layers from the epiblast and to elucidate the molecular mechanisms underlying these processes using EpiSCs.4.Nodal, a member of the TGF-β family, plays central roles in early embryo patterning during the induction of mesoderm and endoderm and the specification of left-right asymmetry. In some cases, Nodal signaling is also referred to as “Activin/Nodal” signaling because another TGF-β family member, Activin, binds to the same receptors as Nodal (with the exception of the coreceptor Cripto) and triggers similar intracellular events .Xenopus, Activin and Xnrs  function as mesoderm inducers in whole embryos and explanted animal caps [Cyclops and Squint, the orthologs of Nodal, lack head and trunk mesoderm and fail to form the germ-ring, an organizer analogous to the mouse primitive streak [The roles of Nodal signaling are evolutionarily conserved and have been well established using molecular genetic studies in various animal models. In mal caps –66. Inhimal caps –69. Genee streak . Similare streak . In addie streak . AVE proe streak . AlthougIn vitro studies have also confirmed that Nodal signaling is required for the maintenance of undifferentiated human ESCs and mouse EpiSCs [NANOG promoter and thereby activates NANOG gene transcription in human ESCs [in vitro studies have revealed the corresponding molecular mechanism underlying this function.Nodal signals may also act as anti-differentiation signals to maintain epiblast proliferation. Analysis of Nodal mutants and embryo explants suggests that Nodal signaling within the epiblast is essential for maintaining pluripotency determinants such as Oct4 and Nanog and for preventing precocious neuroectoderm differentiation ,75. In ve EpiSCs ,76. Actiman ESCs ,78 and mman ESCs . These ret al. provided an explanation for how Activin/Nodal signaling maintains pluripotency without inducing mesendoderm in human ESCs and mouse EpiSCs [SIP1 in human ESCs, thereby limiting the neuroectoderm promoting effects of SIP1. Therefore, SIP1 limits the mesendoderm inducing effects of Activin/Nodal signaling without inhibiting the pluripotency maintaining effects exerted by SMAD2/3. This conclusion was confirmed in mouse EpiSCs, implying that these mechanisms are conserved in different species and may operate in vivo during mammalian development [In mouse ESCs, EpiSCs and human ESCs, Activin/Nodal signaling is also known to drive differentiation toward mesendoderm ,79–81. Te EpiSCs . They foelopment . Overall5.Xenopus and chick embryos, Wnt signaling represses neural fate and induces epidermal fate by attenuating the responsiveness of epiblast cells to FGF signaling [Xenopus found that Wnt signaling promotes neural development in ectoderm through inhibition of BMP4 expression [Xiro1, a downstream target of the Wnt pathway, was shown to repress BMP4 expression [Wnt signaling is mediated by blocking the activity of glycogen synthase kinase 3β (GSK3β), which promotes β-catenin degradation. Inhibition of Gsk3β activity thus stabilizes β-catenin, which activates the Wnt pathway. Wnt signaling plays multiple roles during early embryonic development. In both ignaling –86. In aignaling ,88. Convpression . Furtherpression . These cpression . Howeverpression .in vivo, Wnt signaling is required for mesoderm differentiation in mouse ESCs. It has been shown that inhibition of endogenous Wnt signals in mouse ESCs prevents the expression of primitive streak-, endoderm- and mesoderm-associated genes and abrogates the functional development of mature mesodermal lineages [The functions and mechanisms of Wnt signaling have also been investigated in mouse ESCs. In agreement with observations lineages . Howeverlineages . In summet al. showed that Wnt activation stimulated by 6-bromoindirubin-3′-oxime (BIO), a specific pharmacological inhibitor of GSK3, sustains the expression of the pluripotent state-specific transcription factors Oct4, Rex1 and Nanog in mouse and human ESCs [et al. reported that overexpression of stabilized β-catenin in mouse ESCs inhibits neuronal differentiation and delays loss of pluripotency; moreover, β-catenin forms a complex with Oct4 and enhances Oct4 activity [et al. showed that blockage of GSK3 in mouse ESCs enhances growth capacity and suppresses neural differentiation, but it also promotes non-neural differentiation. To block differentiation of ESCs into the cells that make up the three germ lineages, the combination of a GSK3 inhibitor and an FGF-Erk inhibitor is necessary [Recently, Sato man ESCs . In addiactivity . Therefoecessary .At present, no published data fully elucidate the functions of Wnt signaling in EpiSC differentiation. Considering the multiple roles of Wnt signaling in early embryonic development, we speculate that Wnt signaling may play distinct roles at different stages of development, similarly to the BMP, FGF and Nodal pathways. Further investigation into the functions of Wnt and its crosstalk with other signaling pathways is needed, especially in EpiSCs.6.in vitro systems for investigating the complex mechanisms through which signaling pathways play distinct roles during different developmental stages. Recently, published data [in vitro tool to study the mechanisms involved in ectoderm commitment and in neural and epithelial differentiation during embryonic development. Studies in pluripotent stem cells that correspond to different developmental stages would provide a foundation for efforts to guide the differentiation of pluripotent stem cells along selected developmental pathways for potential therapeutic use.The BMP, FGF, Nodal and Wnt signaling pathways play important roles during embryogenesis. However, the mechanisms underlying cell fate decisions during vertebrate embryogenesis are complex. ESCs and EpiSCs provide hed data  and our


Bacillus subtilis, where it activates the σB transcription factor in response to a variety of sharp environmental challenges, including acid, ethanol, heat or salt stress. However, details of the signaling mechanism within the stressosome remain uncertain. The core of the complex comprises one or more members of the RsbR co-antagonist family together with the RsbS antagonist protein, which binds the RsbT kinase in the absence of stress. As part of the response, RsbT first phosphorylates the RsbRA co-antagonist on T171 and then RsbS on S59; this latter event correlates with the stress-induced release of RsbT to activate downstream signaling. Here we examine the in vivo consequence of S59 phosphorylation in a model strain whose stressosome core is formed solely with the RsbRA co-antagonist and RsbS. A phosphorylation-deficient S59A substitution in RsbS blocked response to mild stress but had declining impact as stress increased: with strong ethanol challenge response with S59A was 60% as robust as with wild type RsbS. Genetic analysis narrowed this S59-independent activation to the stressosome and established that significant signaling still occurred in a strain bearing both the T171A and S59A substitutions. We infer that S59 phosphorylation increases signaling efficiency but is not essential, and that a second (or underlying) mechanism of signal transduction prevails in its absence. This interpretation nullifies models in which stressosome signaling is solely mediated by control of RsbT kinase activity toward S59.The stressosome is a 1.8 MDa cytoplasmic complex that controls diverse bacterial signaling pathways. Its role is best understood in  Bacillus subtilis, where the stressosome controls activation of the general stress factor σB in response to rapidly increasing physical or chemical signals in the environment Particular bacterial sensory modules often regulate activity of a variety of output domains to accomplish different signaling tasks. The 1.8 MDa stressosome complex appears widespread among the eubacteria, and its components are encoded in different genome contexts, implying it provides sensory function for diverse signaling pathways B. subtilis stressosome is formed from three different types of proteins: one or more members of the partially redundant RsbR co-antagonist family, the RsbS antagonist, and the RsbT serine-threonine kinase sulfate transporter/anti-sigma factor antagonist) domains, whereas the smaller RsbS antagonist comprises only a STAS domain Genetic and biochemical analysis indicates that the rsbRAMore is known about downstream signaling events. Stressosome output is represented by release of the RsbT kinase, which then binds and activates the RsbU environmental phosphatase by direct protein-protein interaction B. subtilis and other bacteria – resets the system by removing the phosphate from S59 and T205 According to the model shown in Thus the RsbR co-antagonists and the RsbS antagonist have both negative and positive signaling roles, and these roles can be differentially affected by mutations within their structural genes. The known negative role of the RsbR co-antagonists is their required cooperation with RsbS to sequester RsbT in unstressed cells B in an S59A mutant. A further investigation of the influence of S59A on stress signaling therefore seemed warranted. We report here that the S59A substitution decreases signaling efficiency but does not prevent the stress response. Moreover, mutant analysis locates the S59-independent signaling mode to the environmental branch of the network, leading us to propose that it reflects the fundamental mechanism of signal transduction within the stressosome.Perhaps due to this uncertainty, prevailing models of stressosome signaling focus on how the phosphorylation state of S59 can be modulated by controlling RsbT kinase activity B equally well in response to ethanol stress, but their stressosomes exert different degrees of control over the unstressed output of the signaling network, with the wild type complex having the lowest basal output and thus the greatest control, RsbRB slightly less control, and RsbRA less still For our initial experiments investigating the effect of RsbS S59A on stress signaling, we used three strains whose stressosomes differed in their complement of RsbR family co-antagonists: one had the wild-type set of all four co-antagonists whereas the others had only one of the two principal co-antagonists, either RsbRA alone or RsbRB alone, together with the RsbS antagonist. Such strains activate σrsbU allele and thus lacking the RsbU environmental phosphatase: the rsbS-S59A rsbU double mutant had same low activity as the rsbU null control . We were therefore unable to achieve responses of the magnitude necessary for full comparison to the ethanol data. However, the 0.3 M NaCl experiment did yield a potentially interesting result: the maximum response of the parental strain was 472 units  whereas that of the S59A mutant was 28 units , for a fractional response of 0.06. When this additional point was plotted as the open symbol in Most existing models of stressosome operation have focused on phosphorylation of RsbS S59 as the key signaling event within the stressosome complex In strong support of this view, the genetic results presented here speak against the prevailing phosphorylation models in their simplest forms. Stressosomes composed solely of mutant RsbRA and RsbS proteins, with phosphorylation-deficient T171A and S59A substitutions, were nonetheless capable of transmitting environmental stress signals in vivo . In vitrB. subtilis and several other bacteria Our demonstration of a phosphorylation-independent signaling mechanism chiefly relied on a strain encoding a widely-used model stressosome in which RsbRA was the sole co-antagonist. This strain serves as an archetype for those bacterial species whose genomes encode only a single RsbR co-antagonist The existence of an RsbS-independent signaling role for RsbRA was initially suggested by the genetic epistasis test of Kim et al. Based on the results reported here, we conclude that the stressosome manifests two mechanisms of signal transduction in response to acute environmental stress. We further suggest that these two mechanisms share an underlying commonality: a hypothetical signal-induced shift in conformational equilibria within the stressosome core B. subtilis strains shown in ctc-lacZ transcriptional fusion provided an indirect measure of σB activity −2 s−1). This illumination saturated the blue light-sensing positive regulator YtvA to establish a constant effect on assay results A420×1,000 min−1 mg protein−1 . Stress activation was calculated as maximal post-stress activity minus basal activity just prior to stress addition.Assays were conducted as described previously


Human T lymphotropic virus type I (HTLV-I) infection is largely latent in infected persons. How HTLV-1 establishes latency and reactivates is unclear. Here we show that most HTLV-1-infected HeLa cells become senescent. By contrast, when NF-κB activity is blocked, senescence is averted, and infected cells continue to divide and chronically produce viral proteins. A small population of infected NF-κB-normal HeLa cells expresses low but detectable levels of Tax and Rex, albeit not Gag or Env. In these “latently” infected cells, HTLV-1 LTR trans-activation by Tax persists, but NF-κB trans-activation is attenuated due to inhibition by HBZ, the HTLV-1 antisense protein. Furthermore, Gag-Pol mRNA localizes primarily in the nuclei of these cells. Importantly, HBZ was found to inhibit Rex-mediated export of intron-containing mRNAs. Over-expression of Rex or shRNA-mediated silencing of HBZ led to viral reactivation. Importantly, strong NF-κB inhibition also reactivates HTLV-1. Hence, during HTLV-1 infection, when Tax/Rex expression is robust and dominant over HBZ, productive infection ensues with expression of structural proteins and NF-κB hyper-activation, which induces senescence. When Tax/Rex expression is muted and HBZ is dominant, latent infection is established with expression of regulatory (Tax/Rex/HBZ) but not structural proteins. HBZ maintains viral latency by down-regulating Tax-induced NF-κB activation and senescence, and by inhibiting Rex-mediated expression of viral structural proteins. Most HTLV-1-infected individuals are asymptomatic. It is thought that the proviral DNA is transcriptionally inert and HTLV-1 replicates through mitotic expansion of host cells. The evolving provirus integration patterns in HTLV-1 carriers, however, suggest new infection occurs continuously. Whether or how HTLV-1 establishes latency and reactivates is unclear. We show that HTLV-1 infection in culture can lead to two alternative outcomes — productive infection accompanied by senescence or latent infection followed by clonal expansion — based on the relative expression of regulatory proteins: Tax, Rex, and HBZ. HTLV-1 latency is established by HBZ, and reactivation is achieved by Rex through regulating nuclear export of viral mRNAs. Elucidating mechanisms underlying HTLV-1 latency and reactivation can facilitate virus control to prevent progression to disease. Human T-lymphotropic virus type 1 (HTLV-1) is a complex human retrovirus that infect approximately 10–20 million people worldwide de novo infection through error-prone reverse transcription. However, longitudinal studies of HTLV-1 carriers indicate that the patterns of proviral DNA integration in PBMCs continue to evolve over time de novo infection of naïve cells occurs constantly in virus carriers  in the region that spans nucleotides 5318-5784 of the HTLV-1 genome  gene under the transcriptional control of 18 copies of the Tax-responsive 21-base-pair enhancer element ine, MT2 . Prolifeine, MT2 . While aAs indicated by immunoblotting, most if not all HeLa-G/ΔN-IκBα/HTLV-1 cells were productively infected by HTLV-1 and abundantly expressed Gag , Env (gp46), Tax, and Rex . Togetheenv mutations detected by PCR (We next examined in depth the 3 cell lines derived from the minor population of proliferating HTLV-1-infected HeLa-G (HeLa-G/HTLV-1) cells whose NF-κB activity was unaltered. Interestingly, all three expressed Tax and Rex, albeit at lower levels . The absd by PCR . Since oActivation of viral transcription by Tax is intact in LICs and PICs  as revealed by significant luciferase activities after transfection with an LTR-Luc reporter . This suenv mutations (While the Tax/Rex mRNA (pXIII) levels in LICs were lower than those in PICs as indicated by mRNA quantitation . We thinutations . As mentSince the level of Rex is lower in LICs, we tested the possibility that their lack of Gag expression might be caused by a block in the nuclear export of unspliced viral mRNAs. Nuclear and cytoplasmic RNAs were fractionated for the LIC clones 1–3 and PIC clones 1 and 2, and subjected to qRT-PCR to quantify the nuclear and cytoplasmic levels of Gag-Pol, pX-III, and the control β-actin mRNAs. As indicated in We next asked if p24 expression could be reactivated by Tax or Rex in the LICs. Contrary to conventional wisdom, over-expression of Tax in LICs via transfection of an expression vector, Bc12-Tax, had very little impact on inducing p24 expression except in LIC clone 3 . These rTo determine if the activity of Rex was blocked or defective in LICs, we transfected both LICs and PICs with an HTLV-1 Rex reporter plasmid, pRxRE1-RLuc . This reWe next asked if the lack of Rex activity in LICs might be due to inhibition by a trans-acting viral factor. Of all viral proteins, we thought HBZ to be the most likely to have a role in inhibiting the nuclear export function of Rex. This is because low or no NF-κB activation was detected in LICs despite Tax expression, suggesting that HBZ was expressed in LICs and was inhibiting NF-κB activation and senescence induction as previously proposed To test if HBZ could block nuclear export of unspliced mRNA by Rex, we titrated Rex and pRxRE1-RLuc plasmids to determine the minimal amount of Rex DNA needed to achieve maximal reporter activity  with HTLV-1-producing MT2 cells (3×106) that have been mitotically inactivated by mitomycin C (MMC) treatment (10 µg/ml for 2 hours). The co-culture was carried out in the presence of polybrene (8 µg/ml) for 16 hours. MT2 cells were then removed by washing with phosphate buffered saline (PBS). Fresh media was added and cells were grown for an additional 24 hours, and then harvested. GFP-positive cells were isolated using a cell sorter (BD FACSAria) housed in a lamella flow hood under aerosol-protection condition. Sorted cells were plated at low density on a 15-cm dish. Individual proliferating colonies were picked after a week into 96-well plates and further screened for the integrated HTLV-1 genome by PCR.HTLV-1 infections were performed in a 10 cm dish by co-culturing HeLa-G or HeLa-G/ΔN-IκBα cells  Nuclei were counterstained using 4′,6′-diamidino-2-phenylindole (DAPI). The slides were then mounted in a fluorescence mounting medium (Dako). Images were captured using a Zeiss Pascal inverted confocal microscope.Standard procedures were used for immunoblotting. Typically, 30–50 µg of total proteins was used in each sample. The HTLV-1 Tax hybridoma monoclonal antibody 4C5 had been described previously HTLV-1-infected cells generated as above were harvested, washed, and dissolved in lysis buffer . DNA was then precipitated with isopropanol. To screen for the full-length HTLV-1 provirus, sequence-tagged site polymerase chain reaction was carried out as described −ΔCt method Total mRNA, nuclear and cytoplasmic mRNAs from HTLV-1-infected cell clones were isolated using the PARIS kit (Ambion) according to manufacturer's instructions. Contaminating genomic DNA was removed using the turbo DNA-free kit (Ambion). Complementary DNA (cDNA) was synthesized from 500 ng of RNA in a total volume of 10 µl with iScript reverse transcription super mix (Biorad). The cDNA used for HBZ mRNA quantitation was prepared using a gene-specific antisense primer, HBZ-R2 (see supplementary Table S 2), to avoid contamination from HTLV-1 sense strand cDNA. Real-time PCR was performed using 2 µl of the cDNA as template in a 20 µl reaction, using gene-specific primers (see supplementary 5) were seeded into a 24-well plate overnight. After 16 hours, DNA transfections were performed using Fugene HD reagent (Promega). Two hundred nanograms of each reporter plasmid HTLV-1 LTR-Luc, E-selectin-Luc, or RxRE-RLuc were used in the respective luciferase reporter assay. The RxRE1-RLuc contains the HTLV-1 Rex-response element downstream of the Renilla luciferase reporter (RLuc) gene located within an intron (kindly provided by Dr. Jaqueline Dudley). The amounts of Rex or HBZ expression plasmid used range from 0 to 200 ng. All transfections were performed in duplicates. The total DNA amount (500 ng) was kept constant in all transfections using an empty vector plasmid, pcDNA3.1. Twenty nanograms per well of control luciferase plasmid pGL3-Luc (firefly) or pRL-TK (renilla) were also included in each transfection. After 48 hours, cells are harvested, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Transfection efficiencies were normalized using either TK-renilla or PGL3-Luc. Data are mean ± s.d. from at least three independent experiments.Cells , HeLa-G (negative control), and representative HTLV-1-infected clones of each of the HeLa-G and HeLa-G/ΔN-IκBα groups (G1-3 and ΔN1-2) characterized in (TIF)Click here for additional data file.Figure S2Reactivation of latent HTLV-1 in LIC clone 3 by Tax and Rex individually or in combination. Cells of LIC clone 3 (3×105 cells per well in a 6-well plate) were transfected for 48 hours with expression constructs for Tax (Bc12-Tax) and Rex (pRSV-Rex) either individually or in combination using Fugene reagent (Promega). Whole cell lysates were analyzed by immunoblotting using antibodies against p24, Tax, Rex, and β-actin (Actin).(TIF)Click here for additional data file.Table S1Primers used for sequence-tagged-site (STS) PCR detection of various regions of HTLV-1 genome. The regions of interest (STS), the nucleotide positions correspond to each of the upstream primer sequences in the HTLV-1 genome, the PCR product sizes are indicated.(DOCX)Click here for additional data file.Table S2Nucleotide sequences of primers used for RNA quantitation by real-time qPCR are listed.(DOCX)Click here for additional data file.


The HTLV-1 ORF-1-encoded p12 protein induces T-cell activation and proliferation, while the cleaved p8 protein downregulates TCR signaling. In this study, we investigated whether there is a correlation between genetic variation within ORF-1 and the clinical status or proviral load in HTLV-1 infected individuals. The ORF-1 gene was amplified by PCR from PBMCs of 163 HTLV-1-infected patients  from different geographical regions and a total of 1,640 clones were sequenced. The majority of the patients (73%) carried mutations in ORF-1 that resulted in the expression of more p12 than p8 (50% Carriers and 50% HAM/TSP). The highest genetic variability within ORF-1 was found in the two transmembrane domains of the protein. Of interest, a subclass of mutations was found more frequently in HAM/TSP patients compared to Carriers. While higher proviral loads were found in HAM/TSP patients compared to Carriers (p=0.0001), no correlation between proviral load and the ORF-1 isoform expressed was observed . Currently experiments are aimed at determining the significance of these mutations in ORF-1 function in regard to T-cell activation and proliferation. In addition, mutations will be characterized for their role in viral infectivity and transmission rates ex vivo and in a rhesus macaque model. Determining the effects of ORF-1 mutants on viral replication, spread and latency will provide insight into the pathogenesis of HTLV-1 infection.


SLC22A12 gene cause renal hypouricemia type 1 (RHUC1), whereas renal hypouricemia type 2 (RHUC2) is caused by mutations in the SLC2A9 gene.Familial renal hypouricemia (RHUC) is a hereditary disease characterized by hypouricemia, high renal fractional excretion of uric acid (FE-UA) and can be complicated by acute kidney failure and nephrolithiasis. Loss-of-function mutations in the SLC2A9 gene: p.Arg380Trp, already identified in heterozygosity, and p.Gly216Arg, previously found in homozygosity or compound heterozygosity in some RHUC2 patients. Compared with previously reported patients harbouring these mutations, our proband showed the highest FE-UA levels, suggesting that the combination of p.Arg380Trp and p.Gly216Arg mutations most severely affects the renal handling of UA.We describe a 24-year-old Pakistani man who was admitted twice to our hospital for severe exercise-induced acute renal failure (EIARF), abdominal pain and fever; he had very low serum UA levels (0.2 mg/dl the first time and 0.09 mg/dl the second time) and high FE-UA (200% and 732% respectively), suggestive of RHUC. Mutational analyses of both urate transporters revealed a new compound heterozygosity for two distinct missense mutations in the The clinical and molecular findings from this patient and a review of the literature provide new insights into the genotype-phenotype correlation of this disorder, supporting the evidence of an autosomal recessive inheritance pattern for RHUC2. Further investigations into the functional properties of GLUT9, URAT1 and other urate transporters are required to assess their potential research and clinical implications. Renal hypouricemia is a heterogeneous genetic disorder characterized by impaired tubular transport, reabsorption insufficiency and/or accelerated secretion of uric acid (UA) accompanied by severe complications, such as exercise-induced acute renal failure (EIARF), chronic kidney disease (CKD) and nephrolithiasis. Diagnosis of renal hypouricemia is based on biochemical markers, i.e., hypouricemia and increased fractional excretion of UA (FE-UA).SLC22A12 gene encoding the transporter URAT1, which leads to a partial UA absorption defect  and higher renal UA excretion (>100%) [normal range 4–14%] [Renal hypouricemia type 1  is a recessive condition caused by mutations in the e 4–14%] ,7.SLC22A12 gene have been described, whereas only a few patients with defects in the SLC2A9 gene have been characterized. SLC22A12 and SLC2A9 are the two genes that most commonly influence the serum level of UA, exerting their effect by modifying renal UA absorption [SLC22A12 gene encodes two transcript variants of the URAT1 transporter, and both variants are specifically expressed on the apical membrane of the proximal tubules in the kidneys [More than 100 patients with mutations in the sorption . The SLCSLC2A9 gene encodes two isoforms of GLUT9, long and short, through the use of alternative promoters. GLUT9 is expressed in human kidney proximal tubule epithelial cells, in particular, GLUT9L might be localized to the basolateral side and GLUT9S to the apical membrane, as suggested by expression studies performed using the Madin-Darby canine kidney cells [The human Dinour and coworkers speculated that UA efflux is mediated solely by GLUT9L on the basolateral side, whereas UA absorption from the tubular lumen is carried out not only by URAT1, but also by GLUT9S and possibly other apical transporters . AccordiSLC2A9[In patients with RHUC2, either heterozygous mutations ,12 or coSLC2A9,10,13,14SLC2A9, a total of 12 mutations  are currently reported in the HGMD professional 2012.4.To date, in SLC2A9 mutations cause hypouricemia mainly via haploinsufficiency [Heterozygous ficiency ,12,15. Ificiency ,10,13,14ficiency .SLC2A9. Mutational analysis of the SLC22A12 and SLC2A9 genes revealed the presence of two previously described mutations in the GLUT9 transporter, one found only in heterozygosis and the other either in compound heterozygosis or homozygosis, allowing RHUC2 diagnosis and providing new insights into the genotype-phenotype correlation in this disorder.Here, we report a young Pakistani man with severe EIARF who required temporary hemodialysis and who presented with very low serum UA levels. The FE-UA was high (>150%), confirming the clinical diagnosis of RHUC. The severe clinical manifestation of low UA and very high FE-UA were suggestive of RHUC2 secondary to compound heterozygous or homozygous mutations in A 24-year-old Pakistani man presented to the emergency room of our hospital suffering from diffuse abdominal and bilateral loin pain, diarrhea, vomiting and fever which began three days after a cricket match. The laboratory tests revealed kidney failure , and the patient was admitted to our department. The patient appeared alert without neurological deficits, his temperature was 38°C, blood pressure was 146/75 mmHg, pulse was 72 beats per minute rhytmic and regular, respiratory rate was 16 breaths per minute. Complete laboratory tests confirmed acute renal failure (serum creatinine 6.3 mg/dl) with metabolic acidosis, hypokalemia and hyperphosphatemia. Urinalysis showed many UA crystals, microscopic hematuria and significant eosinophiluria (2%) without proteinuria. The patient’s serum UA was 2.9 mg/dl, creatine kinase was 1,155 U/l, and Prothrombin Time (PT) was 52% with an International Normalized Ratio (INR) of 1.6. All other laboratory tests were within the normal range .Kidney function gradually improved after the first dialysis session, and over the following nine days, there was complete recovery of renal function along with the disappearance of loin and groin pain and fever. Laboratory tests repeated before discharge were unremarkable, except for a very low plasma UA level (0.2 mg/dl). Steroid treatment was rapidly tapered off, and the patient was discharged on the thirteenth day with a serum creatinine level of 1.6 mg/dl.Five months later, the patient presented again with recurrent loin pain, vomiting, diarrhea and acute renal failure (creatinine 2 mg/dl); two days before he had played a cricket match and the day before he had taken 1 g of paracetamol to treat a fever above 38°C.The physical examination only showed bilateral loin tenderness; there were no signs of fluid overload, blood pressure was 121/68 mm Hg without orthostatic hypotension, and body temperature was 37°C.The chest radiogram was normal, and renal ultrasonography showed normal-sized kidneys with only slightly increased echogenicity. Blood tests Table  showed sSLC22A12 gene  and of both SLC2A9 isoforms  were analyzed by direct sequencing with the ABI3130XL Genetic Analyzer.Genomic DNA was isolated from peripheral blood by standard protocol and all the exons and intron flanking regions of the Sequence descriptions of the mutations were verified using the Alamut software, version 2.2.SLC22A12 did not identify casual mutations; however, in SLC2A9, two distinct missense mutations were identified  can favor either hypovolemia or rhabdomyolysis. The environmental situation , exercise intensity, medications or toxins  and infections can also cause rhabdomyolysis. In addition, genetic diseases should be considered .Our patient displayed neither significant rhabdomyolysis nor identifiable causes. His serum UA concentration was unexpectedly low during acute renal failure and decreased to very low values when renal function recovered.Hypouricemia is arbitrarily defined as a serum UA concentration lower than 2 mg/dl (119 μmol/l). It is due to either decreased production (isolated defect in purine metabolism) or, more commonly, reduced renal tubular reabsorption of UA. The latter can be inherited (through mutation of genes encoding tubular reabsorption transporters), acquired as a result of disease  or as a result of drugs that enhance UA excretion.Differential diagnosis between the different causes of hypouricemia is based on FE-UA. Hypouricemia with a high FE-UA is found in the familiar form of renal hypouricemia, diseases associated with Fanconi syndrome, hypervolemic states and diseases that increase intracranial pressure  .In our patient, a familial form of RHUC2 was considered based on the lack of other causes of hyperuricosuric hypouricemia, very low plasma UA, extremely elevated FE-UA (>150%), clinical presentation, and history of a sister with similar symptoms. EIARF associated with hyperuricosuric familial hypouricemia occurs predominantly in young males , mostly after strenuous exercise, such as a short-distance race. The initial symptoms are nausea, vomiting, loin pain, abdominal pain, general fatigue and low-grade fever.Almost 25% of patients with RHUC2 experience recurrent EIARF at various intervals. Serum UA level is normal or only slightly reduced on presentation  and decreases to less than 1 mg/dl after renal recovery, which usually occurs in all patients.SLC2A9 analysis identified two distinct missense mutations (p.Gly216Arg and p.Arg380Trp); although segregation analysis was not performed, the severe clinical phenotype strongly suggests that they are present in compound heterozygosity. Both missense mutations have already been described in patients with RHUC2, but they have never been identified in combination in a single patient.Our patient represents the most severe end of the spectrum of RHUC2 clinical and biochemical characteristics. In fact, SLC2A9 mutations associated with UA homeostasis. The p.Arg380Trp substitution was one of the first causal mutations identified in the SLC2A9 gene, being found in heterozygosity in a Japanese mother and her son who had serum UA levels of 1.5 and 2.7 mg/dl respectively, and an FE-UA of ~15% [In previously documented patients, the recessive p.Gly216Arg mutation was either homozygous or in compound heterozygous patients , whereas of ~15% . These tvs. 0.1 mg/dl) and the FE-UA was significantly lower (93% and 45.8% vs. >150%) compared with our patient. Therefore, we can speculate that the p.Arg380Trp substitution most severely impairs the GLUT9 function in comparison with the p.Gly216Arg and p.Asn333Ser mutations, giving our compound heterozygous patient an increased risk of EIARF.The p.Gly216Arg substitution was originally described in a 14-year-old boy in compound heterozygosity with the p.Asn333Ser mutation . The patSLC2A9 gene, suggesting an autosomal recessive inheritance pattern for RHUC2 , very high FE-UA (>150%) and a high incidence of EIARF, nephrolithiasis and CKD compared with the asymptomatic heterozygous carriers, who showed only moderately low serum UA levels . Additional factors, such as sequence variation in other UA transporters , age, sex, diet, drugs, physical activity, environment and volume status can influence the degree of UA tubular absorption and the risk of EIARF or nephrolithiasis. These factors could explain the 7/17 (41%) reported asymptomatic patients with homozygous/compound heterozygous SLC2A9 mutations that show very low serum UA levels without clinical complications (Table The data presented here confirm that loss-of-function mutations in The pathogenesis of EIARF is unclear. There are two main hypotheses: one based on UA precipitation and the other on its antioxidant activity. The UA precipitation hypothesis proposes that if patients are exposed to a UA load, they are likely to have exaggerated excretion of UA. Prolonged or severe exercise is associated with an elevation in UA production and urinary excretion. If this condition is associated with hypovolemia, increased urine concentration and low urine pH, it favors the precipitation and crystallization of UA. The occurrence of UA nephrolithiasis in patients with renal hypouricemia and the finding of renal tubular obstruction by UA crystals in one patient with EIARF support a pathogenetic role for UA precipitation . In our The second pathogenetic hypothesis is based on the powerful antioxidant activity of UA ,7. HowevOne could speculate why our patient had only two episodes of EIARF at the age of 24 and not all the times he had anaerobic physical activity. Possibly not all the exercises cause renal failure; other factors like duration and strength of the exercise, environmental temperature, relative humidity, state of normal or reduced hydration of the subject, changes in urine pH due to different foods and use of some drugs could play a role. Due to the severity of renal hypouricemia, our patient was instructed to restrict strenuous anaerobic exercise and take sufficient water after exercise. To prevent recurrence of EIARF prophylaxis with allopurinol, oral supplementation of antioxidants or scavengers, such as glutathione, vitamins C and E, and beta-carotene, are also recommended .SLC2A9 mutations described to date. Further investigations of the functional properties of GLUT9, URAT1 and other urate transporters are required to assess their potential research and clinical implications.In conclusion, the clinical and molecular findings on the present patient provide new insights into this disorder, which is understood to a limited degree owing to the small number of patients with Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.CKD: Chronic kidney disease; EIARF: Exercise-induced acute renal failure; FE-UA: Fractional excretion of uric acid; RHUC: Renal hypouricaemia; UA: Uric acid.The authors declared they have no competing of interests.GJ, NC, MR, MC and GC designed the study. GJ, MG, PM, FV, SP and GC made the clinical diagnosis of the patient and performed the follow-up. MR, NC and SQ performed the molecular analyses. MR, NC, GJ, MC and GC researched the literature, reviewed and prepared the manuscript. MC and GC edited and coordinated the manuscript. All of the authors discussed, read, and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/15/3/prepub