In the present review, we summarize recent findings in signaling underlying MAMP-induced stomatal movement by comparing with other signalings.Stomata, formed by pairs of guard cells in the epidermis of terrestrial plants, regulate gas exchange, thus playing a critical role in plant growth and stress responses. As natural openings, stomata are exploited by microbes as an entry route. Recent studies reveal that plants close stomata upon guard cell perception of molecular signatures from microbes, microbe associated molecular patterns (MAMPs), to prevent microbe invasion. The perception of MAMPs induces signal transduction including recruitment of second messengers, such as Ca As natural openings, stomata are exploited as a major entry route by a wide range of microbes including bacteria, oomycetes, and fungi  and phosphorylation events, which are critical for plant innate immunity (Pseudomonas syringae pv tomato (Pst) strain DC3000, Pst DC3118  are molecular signatures that are highly conserved in whole classes of microbes but are absent from the host, such as chitin for fungi and flagellin for bacteria . Recogniimmunity . Recent immunity . Furthert DC3118 . As a reis topic . In the Arabidopsis guard cells. For details on this topic, we refer readers to excellent reviews , and salicylic acid (SA) are believed to induce stomatal closure in various plants . Particu reviews .2+-independent protein kinase and an essential positive regulator in ABA signaling in Arabidopsis guard cells. In resting condition, OST1 kinase activity is inhibited by clade A Type 2C protein phosphatases (PP2Cs). Upon ABA perception, the interaction of ABA receptors, PYR/PYL/RCAR, and PP2Cs releases the inhibition of OST1, resulting in increment of OST1 kinase activity  KAT1 , CPK3, CPK4, CPK5, CPK6, CPK10, CPK11, and CPK23  KAT1 , K+ upta 6 KUP6; , vacuola 6 KUP6;  and Plas 6 KUP6; . Downstrnd CPK23 , and mitnd CPK23 . The regA wide range of MAMPs from bacteria, fungi and oomycetes have been identified and characterized . New typP. syringae pv. tabaci 6605, and induces stomatal closure and inhibits light-trigged stomatal opening in Arabidopsis , Flagellin-sensitive 2 , which induces stomatal closure in tomato , which is also an LRR-RLK expressed in guard cells  family, among which Brassinosteroid insensitive1-associated kinase1/SERK3 (BAK1/SERK3) plays a dominant role . StructuP. aeruginosa. The LPS induces stomatal closure in Arabidopsis and tomato  that can have variable composition, length, and branching of its carbohydrate subunits. Due to variety in structure and easy contamination during preparation, LPS used in many of the plant researches can be consider a mixture of MAMPs and is derived from human pathogen d tomato . Lipopolbidopsis . The PRRbidopsis . Recent f plants . Interest manner . These rN-acetylglucosamineone and one of the major components of fungal cell wall. Chitosan is the deacetylated form of chitin. Caution has to be paid that commercially available chitosan can be only partially deacetylated. For example, some chitosan from Sigma is 75 to 85% deacetylated. Chitin induces stomatal closure in Arabidopsis (Arabidopsis (Arabidopsis involves three lysin motif type RLKs (LysM-RLKs), Lysin motif receptor kinase 5 (LYK5), LYK4 and chitin elicitor receptor kinase 1 (CERK1)  . It has  (CERK1) . These r (CERK1) . For the (CERK1) . Chitosa (CERK1) . It is f (CERK1) .Mixtures of β-1→3-linked glucans with different DP have been shown to induce stomatal closure and inhibits stomatal opening in grapevine and tobacco . The strArabidopsis (Elicitors from baker’s yeast (YEL) extracted by ethanol precipitation mainly contains fungal cell wall fraction including mannan, β-1→3-linked glucans, chitin, and glycopeptides, and has been widely used as a fungal MAMP to induce plant immune response including stomatal responses . YEL indbidopsis , 2015.2O2, NO, and Ca2+, phosphorylation events mediated by CDPKs, OST1, and MAPKs, and changes of transporter activity. In this section, we review the main signaling events upon perception of MAMPs with focus on findings from Arabidopsis guard cells.Guard cell signaling induced by MAMPs involves in recruitment of second messengers, such as H2O2, are important second messengers in guard cell signaling induced by abiotic and biotic factors H oxidases, RBOHD and RBOHF. NAD(P)H oxidases transfer electrons from cytosolic NAD(P)H to apoplastic oxygen, leading to superoxide production. The superoxide can be converted to H2O2 through dismutation by unknown superoxide dismutase . Regarding the activation of NAD(P)H oxidases, studies have shown that elevation of free cytosolic Ca2+ concentration ([Ca2+]cyt) and phosphorylation by CDPKs are important for flg22- and elf18-induced H2O2 production in leaf disks H oxidases. In ABA signaling, the Ca2+-independent protein kinase, OST1, phosphorylates RBOHF at Ser13 and Ser174 and interacts with RBOHD . Both internal factors, such as SA -sensitive PRXs but not RBOHD or RBOHF are essential for stomatal closure and H2O2 production in guard cells induced by SA, ITCs, methylglyoxal, chitosan and YEL , and flg22 , 2011. Frd cells . It is loduction . Pharmacchitosan ,b. Howevoduction . Enzymatoduction . Pharmacoduction . Howeveroduction . Two Ara closure . Further2+ has long been considered as an important second messenger in guard cell signaling induced by various internal and external stimuli , which are activated by hyperpolarization  are involved in elevation of [Ca2+]cyt in seedlings induced by flg22, elf18, and chitin , other proteins containing EF-hand are CDPKs, CaM-like proteins (CML) and calcineurin B-like protein (CBL). CBLs can form complexes with CBL-interacting protein kinases (CIPKs) to convert the Ca2+ signal to phosphorylation events  represents a Ca2+ sensor of low Ca2+ affinity/high-capacity that does not contain EF-hand motif and is associated with thylakoid membranes . Further research is needed to validate this possibility.To our knowledge, there is no evidence that kinase activity of OST1 is increased by stimuli other than ABA in guard cells. On the other hand, recent studies have shown that flg22 does not increase OST1 kinase in rd cells . OST1 isrd cells . Flg22 drd cells , suggestPst DC3000, in which flg22 is the dominant MAMP sensed by guard cells H-free solution . Further studies reveal that CPK6 is required for activation of ICa channels induced by ABA, MeJA, and YEL , and R-type channels, ALMT12. However, SLAC1 shows the most prominent role in regulation of stomatal closure induced by various stimuli, such as ABA, CO2, darkness and high extracellular Ca2+  as positive regulators and PP2Cs as negative regulators .In channels . These rchannels . Since tgulators . Howevergulators . In casegulators . These rgulators . CPK6 dod by YEL . These rin vitro, which are probably dephosphorylated by PP2Cs  function as the main gate for K+ eﬄux to the apoplast. Both Kin channels and Kout channels are voltage-dependent and do not inactivate with time . On the other hand, YEL and chitosan induced stomatal closure in ABA-deficient mutant , such as 4-hydroxy-2-nonenal and acrolein, is produced by both enzymatic and non-enzymatic mechanisms and regulates guard cell signalings . Recent 2O2 accumulation in guard cells induced by flg22, LPS, elf26 and ABA , LecRK-V.5, and Cysteine-rich receptor-like kinases (CRKs), which are involved in guard cell MAMP signaling. LecRK-V.5 has been reported to negatively regulate stomatal closure and H and ABA . LecRK-V and ABA . Furthercificity . For the2O2 production, sensing of Ca2+, MAPK function and regulation of anion channels. However, these mechanisms do have their own specificity. For example, CPK3 and CPK6 have different Ca2+ sensitivity, with CPK6 activated at lower Ca2+ concentration, but both can phosphorylate SLAC1 . Future research is needed to unravel new Ca2+-dependent mechanisms. Emergent challenge is to elucidate how Ca2+-independent mechanisms, such as the ones mediated by OST1 and MAPKs, are involved in MAMP signaling in guard cells. Signaling specificity provided by RLCKs and CRKs has been observed in MAMP-induced stomatal response. Further elucidation of these specificities is essential for dissecting MAMP signaling in guard cells.Though plasma membrane-localized PRRs in guard cells are widely accepted to function to perceive MAMPs, MAMP signaling also happens in the cell wall. It remains unknown whether the apoplast signaling is regulated by the plasm membrane-localized PRRs or there are PRRs in the apoplasts. Upon binding of PRRs and ligands, co-receptors are immediately recruited to form receptor complexes, which determine the specificity of signalings. The core signaling downstream to induce stomatal closure is that MAMP perception induces CaThe roles of phytohormones in guard cell MAMP signaling are still unclear. Though increase of phytohormone by methods such as direct application can induce stomatal movement, it is less likely that MAMPs increase the level of phytohormone to trigger stomatal movement, as seen in the case that flg22 does not increase ABA content. Therefore, it is important to note that the function of elevating level of phytohormone can be different from that of phytohormone in unstressed plants. An emerging role is that phytohormones, such as ethylene and SA, in unstressed plants regulate the level of PRRs. A possible role is that phytohormones, such as ABA, are important for providing basal activity of important signaling components. The future challenge is to elucidate the roles of endogenous phytohormones in guard cell MAMP signaling.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Deregulated Hedgehog (HH)/GLI signaling plays an etiologic role in the initiation, progression and maintenance of many cancers. Small molecule targeting of HH signaling by inhibiting the essential pathway effector Smoothened (SMO) has proven exceptionally efficient for the treatment of advanced and metastatic basal cell carcinoma. That said, severe side effects, limited response rates, SMO-independent GLI signaling and rapid development of drug resistance limit the therapeutic success of SMO antagonists, urgently calling for the identification of alternative and additional strategies repressing oncogenic HH signaling.In this perspective article we highlight recent findings showing that the Toll-like receptor-7/8 (TLR7/8) agonist imiquimod (IMQ), an immune modulator approved for the treatment of basal cell carcinoma, can also act as a potent cell autonomous inhibitor of oncogenic HH signaling. Surprisingly, IMQ reduces HH signal strength independent of TLR signaling, via adenosine receptor (ADORA)/Adenylate cyclase (AC)/Protein kinase A (PKA) activation. We here highlight the molecular mechanisms of IMQ-mediated repression of HH/GLI and discuss the possible benefits as well as challenges of using ADORA agonists for the treatment of HH-associated cancer. HH/GLI signaling is crucial for proper embryonic development and in adults for tissue maintenance and regeneration by regulating stem cell activation and self-renewal. In line with the requirement of exquisite regulation of signal strength and duration, deregulated HH/GLI signaling can have fatal consequences causing developmental anomalies and cancer (for extensive reviews see -11).HH pathway activation is initiated by binding of HH ligand to Patched (PTCH), a twelve transmembrane domain protein that blocks Smoothened (SMO) in the absence of HH. Ligand binding inhibits the repressive function of PTCH, allowing SMO to enter the primary cilium, an antenna-like organelle essential for HH signal coordination and transduction -17. CiliOf note, GLI transcription factors can be activated also in a non-canonical SMO independent manner, thereby reversing the therapeutic effect of SMO inhibitors used for targeted inhibition of oncogenic HH signaling -52.The oncogenic role of HH signaling in cancer was first discovered in patients suffering from nevoid basal cell carcinoma syndrome (NBCCS) caused by genetic loss of PTCH function. NBCCS patients are prone to developing multiple basal cell carcinomas (BCC) in response to ligand independent constitutive activation of the HH pathway -56. ClinThe immune modulator imiquimod  represents another FDA approved drug successfully applied for the treatment of superficial BCC, when surgery is less favorable -70.IMQ is a synthetic nucleoside analogue of the imidazoquinoline family . Its antIn a screen for modifiers of HH/GLI signaling that comprised several TLR agonists including IMQ, our group noticed that IMQ has a direct repressive effect on GLI activity in mouse embryonic fibroblasts . In light of the well-documented therapeutic effect on BCC, this led us to hypothesize that IMQ may directly repress oncogenic HH/GLI signaling independent of its immune modulating function.In the study by Wolff et al. , we testTwo previous studies were key to interpret these unexpected and puzzling findings. Schön et al. have shown that IMQ can affect adenylate cyclase (AC) and protein kinase A (PKA) activity via binding to adenosine receptors (ADORAs) independent of TLR7/8 . EquallyIn line with these data, we observed that treatment of BCC cells or human GLI expressing keratinocytes with IMQ induced PKA-mediated GLI phosphorylation, thereby reducing the level of GLI activator and oncogenic HH signal strength, respectively Figure .The study by Wolff et al. therefore identified ADORAs as possible targets for inhibition of HH signaling in BCC, and it is tempting to speculate that other small molecule ADORA agonists currently in clinical or preclinical evaluation may also hold promise for anti-cancer therapy by repressing HH/GLI signaling. In this context it will also be important to address whether ADORA agonists can overcome current limitations of SMO inhibitors for the treatment of acquired or de novo SMO inhibitor resistant malignancies including cancers with SMO-independent GLI activation , 63-65.Although the surprising finding of a direct repressive role of IMQ on HH/GLI via stimulation of ADORA/AC/ PKA signaling revealed a new mode of action of a well-known drug, several questions remain to be addressed before any of these findings may be translated into clinical applications:In Wolff et al.  we invesA number of HH-associated cancer entities such as cancers of the skin, breast, lung and prostate express high levels of different ADORA subtypes , 80. WhiDetailed epistatic mapping of the repressive mechanism of IMQ on HH/GLI signaling will be key to stratify patients with HH-associated cancer into putative responders and non-responders. The data by Wolff et al. support a model where IMQ interferes with HH signaling downstream of SMO Figure , suggestThe identification of IMQ as inhibitor of HH/GLI signaling has strong potential to broaden the spectrum of applications for this drug or derivatives thereof. However, addressing the open questions raised in this article is critical and there is still a significant way to go before optimized ADORA agonists can be clinically evaluated as potential drugs for the treatment of HH/GLI dependent cancers.


Signaling through WD-repeat proteins in plants” consists of two comprehensive reviews, a perspective, and two articles of original research on the field that help understand how some of these WDR proteins from plants interact with other molecules in response to particular signals.Plants are sessile organisms that rely on appropriate signal-transduction responses in order to cope with the challenges imposed by their environment, and must be able to recognize potential damage or benefit to respond accordingly. These response mechanisms are mediated by specific sets of signal receptors, effector proteins interacting through scaffolding assemblies, second messengers, and transcription factors, among other components. The specific responses occur through protein–protein interactions conveyed by particular sequence modules such as the WD-repeat (WDR), which has been evolutionarily conserved in many proteins participating in signaling events. This module consists of a sequence that spans a number of conserved amino acids and ends with tryptophan and aspartate (WD) residues  and Regulator of G-protein Signaling 1 (AtRGS1), mediate D-glucose signaling in A. thaliana. They show signaling crosstalk between these pathways, and that the WDR protein AGB1 is the triggering signaling element after G protein-mediated sugar signaling, followed by the downstream action of HXK1 on AtRGS1 signaling. They also show in planta interaction between RHIP1and both AtRGS1 and AtHXK1-a requirement for some glucose-regulated gene expression, suggesting a role for RHIP1 in glucose signaling. They conclude that glucose signaling is a complex hierarchical relationship specific to the target gene and sugar phenotype.The research article by Sabila et al., addresses the mechanism by which specific tyrosine phosphorylation mediates AtRACK1A interactions. By site-directed mutagenesis in combination with the Yeast Split-Ubiquitin Assay and BiFC Analysis, they determined that phosphorylation on tyrosine 248 is a requisite for AtRACK1A homo-dimerization. They also showed that exposing Arabidopsis plants to heat stress resulted on such Tyr248 phosphorylation. In addition, yeast cells expressing AtRACK1A WT, but not non-dimerizing AtRACK1A-Y248F grew in the Yeast Split-Ubiquitin Assay when exposed to UV-B light. Their results suggested that AtRACK1A homo-dimerization induces UV-B stress resistance and allows contact with partners interacting on the same surface of AtRACK1A, or recruiting novel partners in newly exposed regions, both unreachable in the monomer.Next, the research article by Miller et al. engages in the review of ternary complexes formed by plant proteins containing WD40 repeats and their role on immunity. They specifically focused on Gβ and TRANSPARENT TESTA GLABRA1 (TTG1) proteins, that are constituents of ternary complexes. These proteins contain seven tandem WD40 repeats and are involved in plant innate immune signaling. Gβ is coupled to type-I membrane receptors while TTG1 to transcription factors whereby functioning at opposite ends of a plant immune signaling pathway. When activated, the heterotrimeric G protein releases the Gβγ dimer to regulate the MAPK cascade by interacting directly with a MAPK protein or by recruiting RACK1 as MAPK scaffold. The authors conclude that Gβ and TTG1 represent the interaction hubs in their specific pathways despite the different evolutionary history of their mechanisms and targets.Finally, two comprehensive reviews cover, in one case, the evolution and role of ternary WD40 repeat-containing protein complexes in plant immunity; and in the other, the various described roles of plant RACK1 in plant signaling. The review article by Islas-Flores et al. provide an overview of the most recent findings regarding the various interacting partners and roles of this protein in diverse plant processes. The authors discuss its promiscuous properties emphasizing the spatio-temporal regulation of critical signal-transduction events such as hormonal control, stress responses, development, immune defense, protein translation regulation, miRNA production, photosynthesis, and cell wall biogenesis. They relate these processes from the information of at least 138 reported ligands recently identified using the split-ubiquitin based and conventional yeast two-hybrid assays. The authors conclude from the information acquired that, in spite of the high degree of conservation of its structure, the functions of the plant RACK1 homolog appear to be distinct and diverse from those in yeast, mammals, insects, etc. They also conclude that even though there is significant novel information regarding the many functions in which plant RACK1 has been reported to participate, there is still much to learn about the missing ligand partners.Two decades after the first report of the plant homolog of the Receptor for Activated C Kinase 1 (RACK1) in cultured tobacco BY2 cells (Ishida et al., All authors listed contributed substantially to the work. MV, TI and HU wrote, proofread, and approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The protein–protein interaction interface maps to a patch of highly conserved residues on B′, which when mutated render B′ incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity.To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B′ family of the regulatory subunits. B′-PP2A and HTLV-1 IN display nuclear co-localization, and the B′ subunit stimulates concerted strand transfer activity of δ-retroviral INs  Of the seven retroviral genera , two are known to cause severe and fatal conditions in humans. The lentiviruses human immunodeficiency virus type 1 (HIV-1) and HIV-2 are the aetiological agents of acquired immunodeficiency syndrome. The δ-retrovirus human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T cell leukaemia-lymphoma (ATL) and the neurological disorder HTLV-1 associated myelopathy/tropic spastic paraparesis (HAM/TSP). A range of inflammatory diseases, such as uveitis, infective dermatitis, arthritis and others is also caused by HTLV-1 infection. Approximately 5% of HTLV-1 infected people eventually develop ATL, of whom most die within two years of symptom presentation . Following reverse transcription, IN recognizes and binds to the viral long terminal repeat (LTR) ends of the viral DNA in a sequence specific manner to form the pre-integration complex (PIC) . Within in vitro generated integration sites revealed a strong bias for integration into active regions of the genome  associate specifically and exclusively with INs from the δ-retroviral genus, and the B′ subunits stimulate biologically-relevant concerted integration activity of HTLV-1 and -2 INs. Mapping of the interaction binding site on B′ illustrates that residues critical for binding to and stimulating HTLV-1 and -2 IN are highly conserved.A detailed description of the generation of the DNA constructs and the Methods describing the purification of all recombinant proteins used in this manuscript, the stable HEK293T-derived cell lines generated and conditions for immunoprecipitation and nickel-nitrilotriacetic acid (Ni-NTA) pull-downs can be found in the Supplementary Data.2, 13 mM DTT, 5.8 μM ZnCl2, 0.132 M HEPES pH 7.1 and 300 ng pGEM-9Zf(-) in a total volume of 30 μl and were started by addition of IN to a final concentration of 1 μM to 6 μM. B′γ(11–380) was used at different concentrations as indicated in the figures. Reactions, which spanned 60 or 90 min as indicated in figure legends, were stopped by addition of 0.5% SDS/25 mM EDTA, pH 8.0. Proteins were degraded by incubation with 30 μg proteinase K at 37°C for 1 h. DNA products, concentrated by ethanol precipitation, were separated by electrophoresis through 1.5% agarose and detected by staining with GelRed (VWR). To quantify strand transfer by PCR, donor DNA S20PQ  for HTLV-1 and S24PQ  for HTLV-2 IN were used. Quantification was done as described previously  from extracts of HEK293T cells stably expressing C-terminally Flag-tagged versions of the viral proteins were analysed by tandem mass spectrometry. Several transcription factors, chromatin remodellers, enzymes involved in DNA damage repair as well as subunits of the cohesin and condensin complexes were isolated in these experiments (Supplementary Tables S2 and S3). Strikingly, subunits of the heterotrimeric phosphatase PP2A co-purified with BLV IN in close to stoichiometric quantities Figure ,25. In cTo investigate whether PP2A is a binding partner of retroviral INs in general, or specifically associates with δ-retroviral INs, IPs were done with extracts from cells that stably express C-terminally Flag-tagged versions of HIV-1 (lentivirus), feline leukaemia virus , murine leukaemia virus , prototype foamy virus , Mason Pfizer monkey virus , BLV and HTLV-1 IN (δ-retrovirus) versus the parental HEK293T cell line. B′-PP2A co-purified specifically and exclusively with IN proteins from the δ-retroviral genus Figure . Immunoshuntingtin-elongation A-subunit-TOR) repeat proteins, the B′ proteins are referred to as pseudo-HEAT repeat proteins  or very low levels (HTLV-1 IN) of concerted integration products are produced in the absence of the PP2A regulatory subunit, strand transfer products made in the presence of B′γ were cloned and sequenced as described previously ,22,23. FTo further map the interaction of IN binding to B′γ, a set of deletion mutants in which pseudo-HEAT repeats were removed from the N- or C-terminal end were designed, expressed and purified from bacteria Figure  and B. B6, whilst mutant 8 and 10 retained some residual binding to HTLV-1 and -2 IN  less able to stimulate strand transfer activity of these δ-retroviral INs Figure –E. Of inmologues   or none (HTLV-1 IN) were identified for the catalytic subunit. This is most likely due to the fact that the catalytic subunit co-migrates with BLV and HTLV-1 IN in SDS-PAGE gels and the bands corresponding to the IN proteins were not analysed by tandem mass spectrometry. Nevertheless, independent Flag-IPs followed by western blot detection illustrates that both delta-retroviral INs specifically co-precipitate the PP2A holoenzyme containing B′ regulatory subunit Figure . A highlA Figure . HoweverA Figure , suggestIt is presently unclear what role B′-PP2A plays in δ-retroviral infection. It is likely that binding of HTLV-1 IN to B′-PP2A is beneficial to the virus for more than one reason. HTLV IN is not the first viral protein known to associate with PP2A. Simian virus 40 small T antigen and polyoma virus middle T antigen disrupt the PP2A holoenzyme to replace the regulatory subunit, hereby inhibiting PP2A activity which contributes to cellular transformation ,19. AlsoIt will be important to determine if the interaction between HTLV-1 IN and B′-PP2A can influence cell cycle checkpoints and/or cellular transformation at the very early stages of infection and whether PP2A is inactivated in malignant ATL clones.Following 3′ processing, the PIC needs to engage host chromatin to catalyse strand transfer. Lentiviruses and mouse mammary tumour viruses are unique in usurping the endogenous nuclear import machinery, whereas all other retroviruses depend on nuclear envelope breakdown during mitosis to bring the PIC into contact with chromatin ,46. By iB′-PP2A dephosphorylates transcription factors such as p53, STAT1 and HDACs ,33, of wHTLV-1 is a notoriously difficult virus to work with; cell-to-cell transmission occurs through the virological synapse , and celB′-PP2A is a functional δ-retroviral IN binding partner that displays some key characteristics of a PIC targeting factor: the interaction between B′-PP2A and IN is genus-specific, both IN and B′-PP2A co-localize in the nucleus of the cell, and B′ significantly stimulates concerted integration of δ-retroviral INs. Current investigations are focussed on determining whether PP2A targets the HTLV-1 PIC to its site of integration. It appears that HTLV INs when present in a 2-fold molar excess do not influence the activity of B′-PP2A on a phospho-Thr peptide. However, more work is needed to verify whether certain PP2A substrate(s) are affected by the association of B′-PP2A with this viral enzyme. Site directed mutagenesis carried out in this work has identified three highly conserved B′ residues critical for the interaction with and stimulation of HTLV-1 and -2 IN activity. Further characterization of the B′-PP2A : HTLV-1 IN interaction interface is ongoing with the goal of identifying the HTLV-1 IN residues involved in binding to B′. This information can then be used to further identify the role(s) of PP2A in δ-retroviral infection.Supplementary Data are available at NAR Online.


Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell lymphoproliferation caused by human T-cell lymphotropic virus type-1 (HTLV-1). This oncogenic human retrovirus can be acquired by mother-to-child transmission through prolonged breast-feeding, sexual transmission, or from transfused infected blood cells or intravenous drug abuse. HTLV-1 infects approximately 5–10 million individuals worldwide and, among them, 1–5% will develop ATLL during their lifetime. Four major geographic molecular subtypes (genotypes) have been reported including the cosmopolitan a-subtype, the Central African b-subtype, the Central African/Pygmies d-subtype and the Australo-Melanesian c-subtype. The results of several studies showed that most cases of ATLL develop in individuals who have been infected with HTLV-1 as young children via their mothers’ breast milk. The very rare ATLL cases observed following transfusion or sexual transmission are still being debated. Here, we report on a Caucasian French patient, with HTLV-1–seronegative parents, who developed ATLL, characterized by a clonal T cell skin proliferation of CD4+ and CD25+ cells, 18 years after highly probable sexual transmission of HTLV-1 through repeated unprotected sexual intercourse with a Cameroonian woman. Indeed, genotyping of the patient's virus revealed infection with an HTLV-1 b-subtype strain, typically of Central African origin, especially Cameroon. This case definitively confirms the hypothesis that ATLL can develop, albeit rarely, after infection during adulthood, outside breast-feeding.


A recent study by Niepel and colleagues describes a novel approach to predicting response to targeted anti-cancer therapies. The authors used biochemical profiling of signaling activity in basal and ligand-stimulated states for a panel of receptor and intracellular kinases to develop predictive models of drug sensitivity. In some cases, the response to ligand stimulation predicted drug response better than did target abundance or genomic alterations in the targeted pathway. Furthermore, combining biochemical profiles with genomic information was better at predicting drug response. This work suggests that incorporating biochemical signaling profiles with genomic alterations should provide powerful predictors of response to molecularly targeted therapies. Significant improvements in cancer therapy have come from identifying and targeting oncogenic drivers in specific tumor types. In breast cancer, targeting estrogen receptor-positive tumors with anti-estrogens and human epidermal growth factor receptor (HER)2-positive cancers with HER2 inhibitors represent significant advances. Recent efforts to find additional oncogenic drivers on a larger scale have focused primarily on high-throughput DNA sequencing. These studies have identified somatic alterations  in a number of genes encoding signaling proteins ,2. ThereCancer Cell Line Encyclopedia have leveraged large cell line panels to identify genomic predictors of response to treatment [Moreover, although many drugs targeting signaling pathways are available, clinical responses are generally seen in only a minority of tumors with alterations in the targeted aberrant pathway. Many studies have focused on understanding what determines and how to predict the response to a molecularly targeted drug. Recent efforts such as the reatment . DespiteRecent work by Niepel and colleagues  takes a PIK3CA) mutations, were key predictors of response. They also showed that HER2 and HER3 receptor abundance together, perhaps acting as a surrogate for heregulin-stimulated Erk, predicted sensitivity to PI3K pathway inhibitors. Combining genomic information with the signaling profiles significantly improved the predictive power of the models. Using signaling profiles alone, predictive models could be developed for 6 of 11 PI3K pathway inhibitors, but these profiles together with PIK3CA or PTEN mutational status predicted the activity of 10 of 11 inhibitors.Using phosphoinositide 3-kinase (PI3K)-Akt inhibitors as an example, Niepel and colleagues showed how their models can be used to identify which signaling parameters are most important to determining response. For PI3K inhibitors, signaling through Erk in response to the HER3 ligand heregulin and HER3 abundance, but not phosphoinositide 3-kinase catalytic subunit alpha (Despite its novelty, this approach has limitations for its applicability to clinical investigation. It uses cell lines in culture, depends upon the ability to measure multiple parameters after perturbations in a controlled system, and focuses mainly on ErbB-PI3K pathway signaling. However, the findings are significant for several reasons. First, they serve as a proof of principle that biochemical profiling on a relatively large scale can predict drug responses. Importantly, modeling of these profiles can identify a few nodes that reflect the activity of larger signaling networks. Second, this work augments the predictive power of genomic and expression profiling and shows how combining biochemical phenotypic data with genomic data can improve the predictive power of both. Third, the incorporation of ligand-stimulated signaling to the modeling adds at least a rudimentary approximation of what is likely occurring in patient tumors exposed to multiple ligands in the tumor microenvironment. Finally, biochemical profiling of patient tumors is currently not feasible on a large scale, but could potentially be performed for those few species predicted to reflect the larger signaling program. A number of issues complicate the measurement of dynamic signaling events in primary tumors, particularly given that ligands cannot be administered to patients. However, presurgical or neoadjuvant studies might be used for measuring not activation but interruption of signaling after treatment with a particular inhibitor, as has been shown for epidermal growth factor receptor inhibitors and anti-estrogens, for example -9.PIK3CA mutations with response to PI3K inhibitors could be extended to other breast tumor subtypes and driver oncogenes. This work also demonstrates that ‘hybrid’ biomarkers combining specific genomic alterations with markers of signaling pathway activation are likely to be required to optimally predict response to targeted therapies.Another promising approach is to use signaling network profiles, such as those described in this work, to refine therapeutic genomic predictors. Studies such as The Cancer Genome Atlas have given us important information on genomic alterations in signaling molecules and pathways, but investigation into how these genotypes affect the signaling phenotypes of breast cancer cells remains limited. The paradigm in this work exemplified by combining HER: Human epidermal growth factor receptor; PI3K: Phosphoinositide 3-kinase; PIK3CA: Phosphoinositide 3-kinase catalytic subunit alpha; RTK: Receptor tyrosine kinase.The authors declare that they have no competing interests.


Hordeum chilense. Fertility restoration of the CMS phenotype was first associated with the disomic addition of the short arm of chromosome 6H from H. chilense. In further studies it was observed that chromosome arm 1HchS was also implicated, and the combination of genes in both chromosome arms restored fertility more efficiently. In this work we aim to dissect the effect of each chromosome in fertility restoration when combined in different genomic backgrounds and under different environmental conditions. We propose a model to explain how restoration behaves in the msH1 system and generate valuable information necessary to develop an efficient system for hybrid wheat production.Exploiting hybrid wheat heterosis has been long pursued to increase crop yield, stability and uniformity. Cytoplasmic male sterility (CMS) systems based in the nuclear-cytoplasmic incompatible interactions are a classic way for hybrid seed production, but to date, no definitive system is available in wheat. The msH1 CMS system results from the incompatibility between the nuclear genome of wheat and the cytoplasmic genome of the wild barley  The discovery of heterosis or hybrid vigor and its exploitation in modern breeding programs is one of the most important advances in plant breeding in the last century. Hybrid varieties have led to a large increase in yield and uniformity in many crop species . CytoplaTriticum aestivum L.) by [Hordeum chilense Roem. et Schultz. , a diploid wild barley native to Chile and Argentina which possesses some traits potentially useful for wheat breeding [Triticeae tribe [H. chilense cytoplasm was first associated with the addition of the short arm of chromosome 6Hchfrom H. chilense [chac, capable of restoring male fertility even more efficiently than 6HchS was observed while testing this system in other wheat backgrounds [chS which only fully restored fertility when present in homozygosis. Further studies of this acrocentric chromosome with molecular markers and cytological techniques, demonstrated that itcomprises fragments of the short arms of chromosomes 6Hch and 1Hch [The new CMS system msH1 was first reported in bread wheat (m L.) by . This sybreeding –8. Moreoae tribe , 10. Ferchilense . An extrkgrounds . The acrand 1Hch .Fertility restoration is genetically very complex as, though mainly controlled by restorer genes, it is also affected by the genomic background and the environmental conditions. Factors such as temperature, photoperiod and light intensity are thought to be of primary importance for the development of fertility. In fact, the influence of environment on the expression of CMS and its fertility restoration has been observed in a large number of crops species , 13. PhochS and 1HchS chromosomes, and that it is the combination of both, which completely restores male fertility [To date we know that restorer genes in the msH1 system are located in the 6Hertility , 16. ThechS)and 6 (6HchS) of H. chilense in an alloplasmic wheat background with H. chilense cytoplasm. The genetic constitution of each parental line is that described by their authors: lines T21A1H1S and T552 were kindly provided by Steve Reader, JIC, Norwich, UK; lines T218 and T593 are described in [v/v) mixture and stained by the conventional Feulgen technique.Crosses using lines shown in ribed in , 16, resAt least three F1 plants of each cross were sown in pots in November 2013, in Córdoba, in order to assess the effects of the genetic constitution and the environment on fertility restoration. Each of the different alloplasmic F1 individuals was split when they were three weeks old to generate two clones per plant. One of the clones was grown in a greenhouse and the other was transplanted into the field The experimental design was a completely randomized design replicated in two environmental conditions, ‘field’ and ‘greenhouse’.Seed set after anthesis was used as the criterion for assessing male fertility and sterility. Seed set of all plants was evaluated by two methods: (1) by counting the number of grains per lateral flower in 20 flowers located in the middle of every spike; and (2) by counting total number of grains per total number of flowers in the spike. Fertility indices for both estimators were calculated as the ratio of the number of grains per flowers (number of grain/number of flowers), where a value of 1 means total fertility and 0 means total sterility. In both cases an average number of five flowers per spikelet was considered. Along with the seed set of all the spikes in every plant, flowering date, day length, and minimum (Tmin), mean (Tmean) and maximum (Tmax) temperatures at anthesis were recorded.All analyses were conducted with the statistical software R version 3.0.2 . In ordelm and factors effects were checked by an analysis of variance with the function anova. The normality and heteroscedasticity assumptions were tested by plotting the residuals versus the predicted values and Q-Q plots.Fertility data scored with the first method were adjusted to linear models including the following explanatory variables: Location (‘field’/‘greenhouse’), flowering date, day length and minimum, mean and maximum temperatures. Data were adjusted to a linear model with the function The linear model that best explained the observed variation was selected. Briefly, in a first step all the variables and factors were included (saturated model) with all two-way interactions between them. The saturated model was then reduced by the elimination of factors and covariates with non significant effect.glht, package multcomp [p-values adjustment type “free” (see package multcomp for more information). The least-squares (LS) means were calculated with the package lsmeans [The differences between alloplasmic lines were assessed using post hoc multiple-comparison test . As shown in 2 = 0.9296). As also observed in The relationship between the two indices used for evaluating the fertility 1) count of the number of grains produced by the 20 central flowers in the spike, and 2) count of the total number of grains per spike was analyzed. A strong correlation between the two fertility estimators was found when using total data . Within the fertile genotypes differences in robustness were detected, with genotype T218T21A1H ‘field’ .1S and T650T552 genotypes. These differences in fertility patterns with respect to temperature and genotype cause the significant interaction between factors ‘Genotype’, ‘Tmean’ and ‘Location’.Another factor with significant effect on fertility was ‘Tmean’. In general, fertility increased with temperature but in some cases this relationship was unclear due to interactions with factors ‘Genotype’ and ‘Location’ . For thachS and 6HchS chromosomes on fertility, the LS means from the model lm1r1C were calculated with confidence intervals at a significance level of 0.05  monotelosomic addition 1HS (Te1H), b) monotelosomic addition 6HS (Te6H), c) translocation 1HS⋅1BL (Tr1HB) and, d) translocation 6HS⋅6DL (Tr6HD). The model formula is: ‘Fertility ∼ Location + Tmean + Te1H + Te6H + Tr1HB + Tr6HD + Te1H:Te6H + Te6H:Tr1HB + Tr1HB:Tr6HD + Location:Tmean + Location:Te1H + Location:Te6H + Location:Tr1HB + Location:Tr6HD + Tmean:Te1H + Tmean:Te6H + Tmean:Tr1HB + Tmean:Tr6HD’. To compare the effect of each genetic combination involving 1H of 0.05 . The monH. chilense chromosome arm responsible for fertility restoration. The involvement of chromosomes 1HchS and 6HchS in fertility restoration has been reported in previous works of the group [Since the msH1 CMS system was first described, great effort has gone into identifying the he group , 12, 16.Concurrently, two different methods of fertility estimation were investigated, the number of grains produced by 20 central flowers of the spike and the total number of grains per spike. The correlation between them was very high when it is present. Because there are no large differences between the fertility estimation by both methods, we propose the method of the 20 flowers for its speed and ease of evaluation.chS, as expected based on our previous knowledge of the system. Genotypes with the monosomic telocentric addition of either chromosome 1HchS or 6HchS do not show an increase in fertility. However when both chromosome arms are present as telocentric additions, the level of fertility is significantly increased. Higher fertility levels are shown by those genotypes harbouring the 6HchS⋅6DL translocation. Conversely lines with the translocation 1HchS⋅1BL do not show increase in fertility. However, lines that present both translocations 6HchS⋅6DL and 1HchS⋅1BL show a much larger increase.The results presented in this work show that the most important factor controlling fertility restoration in the msH1 CMS system is the genotype, in particular the chromosome arm 6HchS itself does not increase fertility, it enhances the positive effect of chromosome 6HchS when is present translocated with a wheat chromosome. This implies that the major gene/s controlling fertility restoration are located on the short arm of chromosome 6Hch but that an enhancer gene/s are also present in the 1HchS chromosome. These results are consistent with previous observations on the restoration ability obtained with the Hchac chromosome. It has been recently shown that Hchac is a kind of zebra-like chromosome conformed by fragments of the 6HchS and 1HchS arms, indicating again that restoration is enhanced by the interaction of genes in both chromosomes [Although the region of chromosome 1Homosomes .The location where plants are grown is the second factor affecting fertility restoration. In this work, plants grown in the greenhouse have higher and more stable fertility values  than those grown in the field. Plants grown in the field are exposed to more extreme environmental conditions, which may negatively affect fertility. However, in the greenhouse, environmental conditions are damper and more stable, which might promote a more stable fertility as well. The factor ‘Location’ could then be masking the effect of other factors; therefore a deeper examination of the environmental conditions is needed to find other abiotic elements affecting the fertility restoration in the msH1 system, apart from those studied in this work.chS⋅6DL. Plants with this translocation have a positive response to temperature in both environments showing male sterility at low temperatures and restored fertility at high temperatures. Furthermore, the increase of fertility by degree of average temperature is greater than in the rest of the chromosomal combinations studied in this work. This could be explained by the existance of a gene/s located on chromosome 6DS with a negative effect on fertility, and that is absent in the translocation event (6HchS⋅6DL). Thermo-sensitive cytoplasmic male sterility lines have been frequently observed in other CMS systems. Indeed, this is the basis for the development of ‘two line hybrid systems’ in which temperature at anthesis determines fertility phenotype [Lastly, the average temperature at anthesis is significantly associated with fertility. Overall fertility of plants grown in the field increased with an increase in temperature. On the other hand, in the greenhouse, fertility does not change with temperature, or it even decreases as temperature increases. An exception is plants with the translocation 6Hhenotype , 21.H. chilense cytoplasm with and without nuclear genes restorers are needed.We have observed some association between photoperiod and plant location; however, our results demonstrated that fertility was not influenced by day-length as in other CMS systems . For thichS⋅6DL and 1HchS⋅1BL translocations is the best one for fertility restoration considering all the situations tested. In any case, the presence of genes from both 6HchS and 1HchS is necessary to achieve full fertility restoration. It has been reported that the expression of partial restorer alleles is more strongly affected by environmental conditions than the expression of fully restoring and non-restoring alleles [In conclusion, the main factor explaining fertility restoration in the msH1 system is the genotype. The combination of both 6H alleles . Thus fu


A biological signal is transmitted by interactions between signaling molecules in the cell. To date, there have been extensive studies regarding signaling pathways using numerical simulation of kinetic equations that are based on equations of continuity and Fick’s law. To obtain a mathematical formulation of cell signaling, we propose a stability kinetic model of cell biological signaling of a simple two-parameter model based on the kinetics of the diffusion-limiting step. In the present model, the signaling is regulated by the binding of a cofactor, such as ATP. Non-linearity of the kinetics is given by the diffusion fluctuation in the interaction between signaling molecules, which is different from previous works that hypothesized autocatalytic reactions. Numerical simulations showed the presence of a critical concentration of the cofactor beyond which the cell signaling molecule concentration is altered in a chaos-like oscillation with frequency, which is similar to a discontinuous phase transition in physics. Notably, we found that the frequency is given by the logarithm function of the difference of the outside cofactor concentration from the critical concentration. This implies that the outside alteration of the cofactor concentration is transformed into the oscillatory alteration of cell inner signaling. Further, mathematical stability kinetic analysis predicted a discontinuous dynamic phase transition in the critical state at which the cofactor concentration is equivalent to the critical concentration. In conclusion, the present model illustrates a unique feature of cell signaling, and the stability analysis may provide an analytical framework of the cell signaling system and a novel formulation of biological signaling. Protein interaction is essential for cellular activities such as cytoskeleton formation Systems biology can describe the kinetics of a signal pathway usingsimultaneousequations of a complex reaction network. On the other hand, there are other types of models consisting of many simultaneous reaction rate equations, including more than ten variables ic  including chemical reaction items can be described using diffusion coefficients iD, kinetic coefficients ik, and concentrations of individual compounds ic as follows:In the current study, to understand biological signaling pathways, we used the following three novel perspectives (A)–(C) based on a non-linear and non-equilibrium kinetic model, which included only two concentrations of the signaling molecule to describe the biological signaling pathway. (A) An equation of the continuity of the chemical concentration of In the above formula, the diffusion rate is hypothesized to obey Fick’s law. In general, the diffusion items and chemical reaction items are thus separately described. On the other hand, because the biological signaling pathway network, including protein interactions, is limited by the slow diffusion rate of the signaling molecular proteins, kinetic coefficients generally depend on the diffusion coefficient. Therefore, diffusion items and reaction items cannot necessarily be separated without validation in the biological reaction. In the current model, we therefore described kinetic coefficients in a diffusion-coefficient-dependent manner.(B) A feedback process due to non-linear self-catalytic reactions was not assumed in the current model, but instead, interactions between signaling molecules in their diffusion was assumed to give non-linearity to the model.(C) A model system far from equilibrium due to a continuous supply of chemicals from the outside was hypothesized. The main issue is how minimal extracellular changes can be transformed into intracellular environmental changes. We aimed to evaluate the behavior of the model around the critical state by perturbation expansion using a minimal change of the supplied molecule concentration. By this mathematical evaluation, we aimed to illustrate the dynamic continuous oscillatory concentration change of signaling molecules from a static state.A.1). Following the protein interaction, one of the signaling molecules is phosphorylated or is bound to a cofactor such as GTP or ATP. In the kinetics of the protein interaction, fluctuation analysis was not fully performed in spite of the greater fluctuation in concentration relative to the solution of small molecules. Furthermore, we systematically analyzed the roles of the fluctuation in cellular signaling.In the current study, we constructed a novel model and aimed to evaluate the general intrinsic properties underlying cellular signaling based on signaling molecule interaction kinetics. Previous systems biology models have not necessarily focused on the diffusion process of proteins. Given the non-linearity during diffusion, we assumed kinetic instability of the signaling molecule interaction, and the sensitivity of the cell signaling in response to the environmental change was evaluated. The model system consists of several steps as follows: (i) the signaling molecule achieves an interaction active state by reversibly binding a cofactor that provides the signaling molecule with interaction activity; (ii) the signaling molecule has the ability to hydrolyze the cofactor; (iii) the signaling molecule interaction activity becomes lower when binding a hydrolyzed inactive cofactor compared to the signaling molecule binding an active cofactor; (iv) the signaling molecule has the ability to exchange the inactive cofactor with an active one; (v) active cofactors are supplied continuously from the outside. Thus, we set the interaction activity to be self-limiting, causing dynamic instability of the signaling molecule interaction. In the present model, we assumed that a signaling protein diffuses relatively slowly in the cytoplasm, and the whole signal transduction is a diffusion-limited reaction .X), and an inactive cofactor-binding signaling molecule (Z). An active cofactor is non-hydrolyzed, and the inactive cofactor is the hydrolyzed type. X has the higher interactive activity and Z has the lower interactive activity. First, X can associate with oligomeric enzyme complex R consisting of X and Z, which transforms the active form X into the inactive form Z by hydrolysis of the binding cofactor and is to be released as Z irreversibly:The model scheme is shown in −1k) of an encountering pair X and Z is significantly slower than the hydrolysis rate of the active cofactor changing into inactive cofactor (relating to 2k) . On a simple consideration of the diffusion limited step, when the kinetic rate can be described according to Fick’s law using diffusion coefficients XD and ZD of X and Z, respectively:In the above formula, we assumed that signaling proteins diffuse relatively slowly in the cytoplasm and that the dissociation rate Signaling molecules have the potential to hydrolyze the cofactor by interacting with identical species:In (2.4) and (2.5), likewise in (1.2):A.1). In this case, the whole reaction system can be regarded as diffusion-limited Here, kinetic equations were set according to the above simple reaction cascade. The equations consist of protein interactive items and an item of small-molecule-cofactor exchange. When the concentration of the protein is sufficiently small, the dependency of the diffusion coefficient on the concentration is linear 3k ’ PZ. Further,Here, the exchange rate of the cofactor is expressed by the item Because kinetic coefficients depend on diffusion coefficients, we set:Using (2.10), (2.8) and (2.9) are rewritten as follows:de novo asssembly is considered to be much slower than monomer interaction at the steady state :Here we set the oligomer concentration as constant because Here, a small letter affixed signifies values at the steady state.This assumption is based upon on the steady state in the protein assembly Setting the right hand sides of Eqs. (2.11) and (2.12) gives:Subsequently, let us consider the fluctuation of participant proteins. We set:R interacts with other proteins, X and Z, in the course of diffusion  to the mix solution of two macromolecules, X and Z:Xv and Zv are the partial specific volumes of X and Z with molecular weights XM and ZM, respectively. X AX’, AX’’, AZA, Z’A, and Z’’A are the second virial coefficients. In actuality, X and Z are the same molecules except with bound ATP or ADP. The fluctuation of the diffusion coefficient is given by:Further, we hypothesized that X contributes to a decrease in diffusion coefficient 1D in thefluctuation item, ax (a>0), because of the higher interaction activity that reducesdiffusion; in contrast, increased Z contributes to increases in the diffusion coefficient 1D in the fluctuation item bz (b>0).Here, an increase in Likewise in (4.5),Using (3.5) and (3.6), Eqs. (2.5) and (2.7) give the fluctuation kinetic equations:Further, using matrix formulation,Here,a, b, c, d, e, and f, of 1D, 4D, and 5D are of the same order of magnitude, 102 (100∼856). The concentrations of X and Z at the steady state are given by Eq. (2.14). On the basis of A.3, 1D, the diffusion coefficient of the assembling rate of X to R, is significantly smaller than 4D and 5,D which are diffusion coefficients of the assembling rate between X and X, and X and Z. Applying the above conditions, the simulation results are shown in p increases to the values that satisfy:The time-course of the signaling molecule concentrations was simulated via the substitution of appropriate numerical values into (3.9). A numerical calculation was performed over a sufficiently long period to evaluate the trend of signaling protein behavior. In the current simulation, the fluctuation coefficients t = 50, around when the oscillation initiates, to t = 1000, the mean amplitude was calculated as the division of the sum of each size of the peak by the number of peaks. In addition, the frequency was estimated by dividing the number of the peak by Δt = 950.cp−p  is given by Lc in (3.10):For mathematical analysis of stability around the critical point, Eq. (3.9) (4.10) was formulated. When L, , coordinate transformation is performed as follows:Using the eigenvectors of The above parameters are subsequently set as:u is given by:Further Therefore, usingia in (6.5) are given  in (6.4), we can obtain the kinetic stability equation of fluctuation ν using coefficients in  as follows  has two real number solutions of v other than zero, indicating the bi-stability of the fluctuation v; when ε≥7.0×10−4 (6.9), it has only the zero solution of the fluctuation (See ε is predicted (v is simply given by the linear equation (6.3), this directly demonstrates the amplitude bifurcation of x and z.As a result, when redicted . BecauseHere, we presented a model of cell signaling systems and performed mathematical analysis on the model in addition to numerical simulations. An increase in the supply of the cofactor near the critical concentration induces a ‘phase transition’ of the system, indicating that the model system has the ability to transform information on the concentration change of a cofactor outside the system into inside information, i.e., the amplitude or frequency of the concentration oscillation of the signaling molecule. This term, ‘phase transition’, is a metaphor implying that the model system nearly discontinuously acquires the ability to dynamically transform outside information into inside information.The introduced non-linear kinetic equations include only two independent parameters, the active or inactive cofactor binding protein. The observed oscillation of signaling molecules in the simulation is not a chaotic behavior that requires more than two parameters in vivo. Still, the theoretical basis of cell signaling systems for quantitative evaluation requires further formulation on the basis of experimental study.The simulation allowed us to define the formula (5.3) mentioned above. The formula using the logarithmic function brings to light an important idea. The present simple model illustrates an essential property in which a system is relatively stable to the outside environment, because a minimal increase in the concentration of the cofactor is transformed into the logarithm of the concentration change inside the cell system. These features may be crucial in responding to transformations of the outside environment while minimizing the inside influence caused by outside alteration. Further, the present simple formulation is reminiscent of the definition of entropy in informatics. In conclusion, our model indicates that the non-linearity of a protein interaction theoretically gives an interesting cohesive behavior, such as an oscillation, of the signaling system leading to self-organization Appendix S1(DOCX)Click here for additional data file.


This research topic highlights the great interest and cautious optimism surrounding our constantly expanding understanding of the roles played by non-coding RNA (ncRNA) molecules in fundamental biology as well as in human health, aging, and disease.Caenorhabditis elegans, a simple and elegant model organism that continues to be at the forefront of ncRNA research (Youngman and Claycomb). She also discusses how this worm provides a tractable genetic system in which researchers continue to study aging, epigenetic inheritance, and the role of ncRNA in such processes. Continuing with contributions from genetic model organisms to the field, Mekhail then discusses how yeast, fly, mouse, and human systems have helped uncover roles of ncRNA molecules in neural function and neurological disorders across the lifespan . In particular, this article discusses the role of different classes of ncRNA including miRNA and lncRNA in healthy neural function and aging before highlighting how deleterious processes can also be triggered by ncRNA and/or its regulators in neurodegenerative diseases especially as we age. Suh then summarizes the rich connections between a great number of disease-linked miRNAs and the insulin growth factor 1 (IGF-1) signaling pathway, which regulates diverse processes from development to aging (Jung and Suh). Discussed connections between miRNAs and diseases including cancers, diabetes, and aging are astounding. This certainly highlights the therapeutic potential of miRNAs and miRNA-targeting nucleotides. Fish then discusses how networks of miRNAs and lncRNAs modulate NFkB signaling, a major driver of blood vessel inflammation . Such inflammation can start at a younger age but lead to heart attack and stroke later in life highlighting the therapeutic potential of ncRNA modulation to treat vascular inflammation and its associated complications across the lifespan. Mhlanga discusses how ncRNAs can be manipulated by pathogens such as the human immunodeficiency virus (HIV) . This article reveals how ncRNA molecules are at the center of a power struggle between virus and host fighting for the regulatory potential of ncRNAs. Finally, Palazzo helps us take a step back and carefully assess the possibility that most ncRNA is junk transcriptional noise . Contrary to the first impression that this article is anti-ncRNA, this hypothesis and theory article should help the field better distinguish functional from junk ncRNA.In this topic, five review articles and one hypothesis/theory paper cover in breadth and depth the established, emerging, and controversial aspects of ncRNA biology. Claycomb discusses how we owe, at least in large part, today's broad interest in this area of biology to the discovery and characterization of RNA interference in the tiny soil worm In conclusion, I thank the authors and my co-guest editor for their contributions to this research topic. I also extend my most sincere thanks to the tireless researchers in the ncRNA field for their dedication to decipher what continues to be one of the most fascinating areas of biology. In addition to revealing secrets of human health, disease, and aging, this field is helping us better understand the evolution of processes that are so vital to our existence as a human species. By uncovering these processes, we learn more about who we are in an effort to extend our health span.The author confirms being the sole contributor of this work and approved it for publication.KM is supported by the Canadian Institutes of Health Research Open Operating Grant Program and the Ontario Early Researcher Award. KM holds the Canada Research Chair in Spatial Genome Organization.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evaluation of the effects of betamethasone on patients with intrauterine growth restriction in couple with preeclampsia is not well studied. This study was designed to assess and compare the changes of Doppler flow in maternal, fetal and placental arteries in singleton pregnancies complicated by IUGR and preeclampsia which are at 24-34 weeks of gestation after betamethasone therapy. This prospective, longitudinal and multicenter study was conducted in 2013 on the 40 singleton pregnant women with IUGR fetuses and concerned over maternal or fetal well-being. Three Doppler measurements including absolutely before betamethasone, one day after betamethasone and 5 days after betamethasone administration were performed. Flow velocity waveforms were obtained from uterine arteries (UA), Umbilical (UM), and middle cerebral artery (MCA). The Systolic/Diastolic ratio (S/D), Resistance Index (RI), and Pulsatility Index (PI) were determined for waveforms. Comparison of baseline mean scores between IUGR with and without preeclampsia showed no statistically significant differences. The mean scores of UA, MCA-UM-RI, UM-S/D, UM-PI, and UM-RI did not differ statistically significant between three time points when compared trend between negative and positive preeclampsia subjects. However, UM-RI had close P value to the margin of statistical significance . In other words, in our study, UM-RI had a clear tendency to be significance. We can conclude that preeclampsia alone could not be major prognostic factor in pregnancies with IUGR. While, other prognostic factors such as gestational age, fetal weight, and fetal vascular Doppler flow may are more important for decision making about termination of preeclampsia. Preeclampsia usually presents as high blood pressure and proteinuria after 20 weeks of gestation . IntrautAbnormal implantation along with the syndrome of preeclampsia and IUGR is the major risk factor for maternal and perinatal morbidity and mortality. Early onset preeclampsia contributes greatly to IUGR, abnormal umbilical and uterine artery blood flow, and adverse outcomes for both mother and fetus. It is suggested that vascular disorders may lead to abnormal implantation and consequently vascular diseases in the mother. The evaluation of arterial stiffness by Pulsed Wave Analysis (PWA) showed more arterial stiffness in women with early onset preeclampsia or with IUGR but normal blood pressure compared with women with late onset preeclampsia or healthy women . In addiDoppler ultrasonography is well-known as a non-invasive procedure for evaluating surveillance of the fetus and as well as vascular blood flow velocity such as umbilical artery, umbilical vein, aorta, middle cerebral artery. For example, a meta-analysis of randomized controlled trials of the high risk pregnancies showed that umbilical artery Doppler investigation is an effective test for reducing morbidity and mortality of fetus . CompariThis study was designed to assess and compare changes of Doppler flow in maternal, fetal and placental arteries in singleton pregnancies complicated by IUGR and preeclampsia which are at 24-34 weeks of gestation after betamethasone therapy.th percentile for gestational age). In other words, they had high probability of preterm delivery and termination of pregnancy.This prospective, longitudinal and multicenter study was conducted in 2013 on the 40 singleton pregnant women with IUGR fetuses and concerned over maternal or fetal well-being. Women of our study were complicated with intrauterine growth-restriction  for fetal lung maturation and fulfilled the following inclusion criteria: (1) gestational age at 24 to 34 weeks  (2) singleton pregnancies in which no fetal anomalies had been detected by ultrasonography, (3) the subsequent administration of a course of betamethasone, and (4) fetuses with confirmed IUGR.Exclusion criteria were (1) structural, congenital anomalies, or chromosomal disorders (2) patients who could not complete the study due to Intra-Uterine Fetal Death (IUFD) or need to emergency terminate the pregnancy and (3) starting betamethasone therapy course before enrolling in the study.Participants’ informed consent was gained; voluntary participation and confidentiality were guaranteed. We had not any missing data. The study was approved by the Ethics Committee of the department of Obstetrics and Gynecology .Three Doppler measurements absolutely before betamethasone, one day after betamethasone and 5 days after betamethasone administration were performed. For all cases two doses of 12 mg betamethasone given intramuscularly repeated at 24-hour apart. All Doppler studies were performed by ultrasound examinations. Doppler Ultrasonography was performed using A Acuson Antares Ultrasound System .Characteristics data included maternal age, gestational age at diagnosis, BMI (Body Mass Index), maternal blood pressure (BP), maternal diabetes, preeclampsia and parity. Preeclampsia was defined as systolic BP>= 140 mmhg or diastolic BP >= 90 mmhg and proteinuria >= 300 mg/day after 20-week of gestation. Flow velocity waveforms were obtained from the left and right maternal uterine arteries (UA), fetal umbilical artery (UmA), fetal middle cerebral artery (MCA), and MCA-RI/UmA-RI ratios. All recording were taken during fetal inactivity to ensure steady signals in the venous channel. The systolic/diastolic ratio (S/D), resistance index (RI), and pulsatility index (PI) were determined for waveforms.The analysis was carried out with SPSS software program version 17. Continuous variables were described with mean ± standard deviation, and qualitative variables were expressed as percentage value. Analysis of variance for repeated measures was used to study the Doppler velocity waveform patterns of UA, UmA, MCA, and MCA-RI/UmA-RI longitudinally over three time points. Independent t-test was used to compare baseline mean of blood flow velocity between preeclampsia and non preeclampsia groups. Significance level  of 0.05 was deemed to indicate the statistically significant difference for all tests.Among 40 singleton pregnant women with IUGR, 16 (40%) had preeclampsia. For all patients, mean ± SD score for maternal age was 29.8 ± 7.7 years old, gestational age was 29.7 ± 2.8 weeks, and BMI was 26.2 ± 3.8 kg.Independent t-test results for patients’ characteristics between positive and negative preeclampsia groups are given in Women with intrauterine growth restriction and early-onset preeclampsia (n=16) and intrauterine growth restriction without preeclampsia (n=24) were studied at 24 to 34 weeks of gestation. Measurements at three time points were conducted to study and compare effects of betamtethasone usage on vascular dysfunction in both groups. We found that betamethasone therapy had significant effects on UA-PI, UM-PI, UM-RI, UM-S/D, MCA-RI, and MCA-UM-RI over 5-day follow up time period in each group  .In some studies, no sever adverse effects after betamethasone administration have been reported during second half of pregnancy. However, some other investigations found adverse neonatal effects such as decreased fetal movement, respiratory activation, and beat to beat variability after betamethasone usage . These sBetamethasone administration improves end diastolic blood flow of umbilical artery in fetuses with IUGR or placental insufficiency symptoms . There iFetal ductus venosus blood flow is valuable in taking an accurate decision about timing of pregnancy termination in the intrauterine growth restriction syndrome . Our finEvidences about fetuses at risk of preterm delivery or placental insufficiency showed contrast results. It had been shown that betamethasone does not change umbilical artery pulsatility index in perinatal period .Another studies reported decreased MCA-PI after maternal betamethasone therapy . In a stIn a study by Yinon and et al., they compared vascular dysfunction between four groups including early-onset preeclampsia, late-onset preeclampsia, and intrauterine growth restriction without preeclampsia, and prior normal pregnancy at postpartum months. They concluded that risk of future vascular disease was higher only in women with a history of early-onset preeclampsia or intrauterine growth restriction without preeclampsia . Their fFinally, we found that betamethasone therapy had similar effects on maternal, placental and fetal arteries blood flow velocity between intrauterine growth restriction with and without preeclampsia within 5-day follow-up based on Doppler ultrasonography studies.We can conclude that preeclampsia alone could not be major prognostic factor in pregnancies with IUGR. While other prognostic factors such as gestational age, fetal weight, and fetal vascular Doppler flow may are more important for decision making about termination of preeclampsia. As our study showed, the response of IUGR with and without preeclampsia to betamethasone therapy was the same within 5 days follow up. Therefore, it is more probable that placental vascular dysfunction may lead to intrauterine growth restriction. At the end, we suggest that perinatologists should be more careful in interpreting Doppler ultrasonography reports of IUGR fetuses. Also, it is better to take more attention to other prognostic factors for termination of pregnancy.


As a major regulatory pathway for embryonic development and tissue patterning, hedgehog signaling is not active in most adult tissues, but is reactivated in a number of human cancer types. A major milestone in hedgehog signaling in cancer is the Food and Drug Administration (FDA) approval of a smoothened inhibitor Vismodegib for treatment of basal cell carcinomas. Vismodegib can block ligand-mediated hedgehog signaling, but numerous additional clinical trials have failed to show significant improvements in cancer patients. Amounting evidence indicate that ligand-independent hedgehog signaling plays an essential role in cancer. Ligand-independent hedgehog signaling, also named non-canonical hedgehog signaling, generally is not sensitive to smoothened inhibitors. What we know about non-canonical hedgehog signaling in cancer, and how should we prevent its activation? In this review, we will summarize recent development of non-canonical hedgehog signaling in cancer, and will discuss potential ways to prevent this type of hedgehog signaling. Drosophila in 1980, hedgehog (Hh) signaling was regarded as an essential regulator for tissue polarity during development. It was not until 1996, when two independent research groups discovered somatic inactivating PTCH1 gene mutations in a genetic skin cancer disease, Gorlin syndrome, and subsequent discovery of activating mutations of smoothened (SMO) in skin cancer basal cell carcinomas [For a long time, since its discovery in rcinomas ,3. It isrcinomas ,5, and mrcinomas ,8. PTCH1 (inactivating) or smoothened (SMO) (activating) result in constitutive activation of the Hh pathway. Vismodegib, a specific inhibitor for smoothened, has been approved by FDA to treat locally advanced and metastatic basal cell carcinomas. The current understanding for hedgehog signaling activation in these tumors is described as the following: In normal situation, PTCH1 functions to suppress smoothened signaling but this suppressive effect can be relieved when hedgehog ligands bind PTCH1. In these tumors, somatic mutations of Ptch1 knocked out or with expression of oncogenic SmoM2 develop tumors similar to Gorlin syndrome patients, BCCs and medulloblastomas. These results provide convincing evidence for the driver role of Hh signaling for cancer development. In addition, rhabdomyosarcomas frequently occur in mice after SmoM2 expression or Ptch1 knockout [Mice with knockout ,10,11,12Ptch1 knockout under the control myeloid lineage specific cre LysM results in the development of gastrointestinal stromal-like tumors (GIST) [SmoM2 expression is not sufficient to drive tumor formation, it increases the tumor number. Conversely, mice with Smo knockout have reduced tumor number [Shh expression in the epithelium of esophagus results in elevated Hh signaling in the stroma [SmoM2 has no effects on Kras-induced pancreatic cancer development [SmoM2 does not lead to formation of prostate cancer [In addition to medulloblastomas and rhabdomyosarcomas, there are numerous types of cancer associated with Hh signaling activation. However, in each tumor type, the mechanism underlying the hedgehog signaling activation differs. For example, s (GIST) , supportr number . In a Bae stroma ,16. The elopment . Similare cancer . It needWe propose three major roles of Hh signaling for cancer development: tumor driving effect, tumor promoting role, or regulator for residual cancer cells. Numerous studies have demonstrated a tumor-driving role of activated Hh signaling in BCCs, medulloblastomas, rhabdomyosarcoma, GIST and Barrett’s esophagus ,15,19,20PTCH1, smoothened (SMO) or Su(Fu) (Mode 1). Furthermore, hedgehog signaling can be activated through over-expression of hedgehog ligand, leading to either paracrine Hh signaling  (Mode 2) or autocrine Hh signaling  (Mode 3). In addition, Hh signaling may also be activated via non-canonical signaling, through targeting Gli transcriptional factors  or independent of Gli transcriptional factors . In addition, Hh signaling may synergize with other signaling pathways, including EGF signaling, to regulate certain target genes [As to the hedgehog signaling modes of action, at least three types have been reported in human cancer. First, hedgehog signaling is constitutively activated through somatic gene mutations in genes like gh PTCH1  or regulgh PTCH1 . The beset genes . Since tIn normal situation, the transcription factor Gli is activated through ligand binding of PTCH1 and activation of SMO and this is the canonical Hh signaling pathway. However, in some situations, the Gli transcription factors can be activated by other molecules/signaling independent of ligand and SMO. This type of Gli activation is called non-canonical Hh signaling. Non-canonical Hh signaling is mainly investigated in the context of malignant diseases. The molecules/signaling pathways that can bypass the ligand-receptor signaling axis to activate Hh signaling pathway include Kras signaling, TGFβ, PI3K, PKC and epigenetic regulators. GLI1 expression in cancer cells and had no effects on the multistage development of PDAC tumors [GLI transcription in the neoplastic ductal cells is regulated through alternative manners. Since Kras mutation presents almost universally in PDA and is one of the earliest genetic alterations, it is reasonable to speculate that the RAS/RAF/MEK signaling plays a vital role in the non-canonical activation of Hh signaling in cancer cells. Hedgehog signaling plays a functionally important role in the genesis of Pancreatic ductal adenocarcinoma (PDAC) ,35. HoweC tumors , indicatGLI, as indicated by GLI1 and PTCH1 mRNAs [in vitro. In gastric cancer cells, the KRAS-MEK-ERK cascade has a positive regulatory role in GLI transcriptional activity [GLI transcriptional activity is regulated by the pattern of Gli phosphorylation [Indeed, ectopic expression of oncogenic Kras in normal human pancreatic cell line HPDE-c7 can increase transcription activity of Gli molecules . SimilarH1 mRNAs  in PDAC activity . How RASrylation . In 3T3 rylation , this enrylation ; in anotrylation . Based orylation . In kerarylation   can affect the activity of GLI in a SMO-independent manner or by potentiating the existing Hh Signaling. In human normal keratinocytes, EGFR can transduce signal through RAF/MEK/ERK to cooperate with GLI proteins to selectively regulate some target genes , and synt manner , but in t manner , indicatGLI2 expression and consequently GLI1 expression in various cancer cell lines, normal fibroblasts and in keratinocytes [TGFβ and its receptors are widely expressed in human body, and its signaling plays a major role in human diseases. During the progression of malignant disorders, TGFβ signaling acts both as a tumor suppressor and as a tumor promoter depending on tumor context (see review ). There inocytes . GLI2 induction by TGFβ is independent from the Hh/Ptch/Smo axis and de novo protein synthesis [SMO-deleted pancreatic cancer cell line from mouse, TGFβ treatment leads to marked elevation of GLI1 and GLI3, even when GLI2 expression is undetectable [SMO mRNA is not detectable, and GLI2 expression was not repressed by SMO inhibitor cyclopamine but regulated by TGFβ signaling  is a family of widely distributed phospholipid-dependent serine/threonine kinases, which can be divided into 3 groups: conventional or classic PKC isozymes , novel or non-classic PKC isozymes , and atypical PKC isozymes . As a receptor for the tumor-promoting phorbol esters, the function of PKC in cancer has been intensely investigated in the context of cancer . The croet al. found that PMA, a classical PKC and novel PKC stimulator, can induce GLI-luciferase reporter activity in this cell line through PKCδ [GLI1 expression and PKCα decrease GLI1 expression in 293 T cells independent of MAPK signaling [GLI1 mRNA in NIH/3T3 cells and human Hepatoma Hep3B Cells [aPKCι is also an Hh target gene, and is required for maximal sustained Hh signaling [Here, we mainly discuss the studies about how PKC signaling activates Gli transcription factors without going through SMO. The role of PKC in Gli activity is controversial. By using LIGHT2 cells, as well as NIH/3T3 cells, Riobo ugh PKCδ . This acignaling . Cai and3B Cells . In mous3B Cells . In addiignaling . In summPTEN, a negative regulator of PI3-kinase activity, occur frequently in human cancer and overexpression of IGF I exists in malignant disease, this study indicated that activated AKT/PI3K may facilitate Hh signaling pathway and promote tumor formation. In human PTEN-deficient glioblastomas, Hh signaling is activated [Riobo and colleagues demonstrated that activation of PI3-kinase/Akt increases Shh-induced Gli transcriptional activity through antagonizing PKA-dependent Gli2 inactivation in several experimental systems . Since lctivated . Compensctivated . In mouse melanoma cells, COS7 cells, human prostate cancer LNCaP cells and human glioma U87 cells, the acquisition of oncogenic RAS-RAF-MEK and/or AKT signaling could be responsible for cell-intrinsic enhancement of GLI1 transcriptional activity driven by enhanced nuclear localization and transcriptional activity . In a recent study in esophageal adenocarcinomas, it was revealed that GLI1 activity can be enhanced by TNFα/mTOR pathway independent of SMO activity . TNFα inCancer epigenetics is a rapidly developing field of cancer research since more and more epigenetic regulators are found mutated in malignant diseases and more and more evidence is showing the important role of epigenetic dysregulation in cancer growth and metastasis. Not surprising, many compounds have been reported targeting components of the epigenetic machineries for cancer therapy. in vivo and in vitro [Recently there are some data showing that Hh pathway can be regulated epigenetically during cancer development. SNF5, a tumor suppressor, is core component of the SWI-SNF chromatin remodeling complex. Jagani and coworkers found that SNF5 interacts with GLI1 and is enriched at Gli1-regulated promoter region . Inactivin vitro . AlthougMEN1 is frequently mutated in this disease. Gurung and colleagues found that menin combines with Gli1 promoter, and recruits protein arginine methyltransferase 5 (PRMT5) to promote repressive histone arginine methylation, leading to decreased Gli1 expression independent of the canonical GAS1/PTCH1/SMO-mediated Hh signaling [GLI1 for treating these tumors. Malatesta et al. reported that p300/CREB-binding protein-associated factor (PCAF), a histone acetyltransferase, interacts with GLI1 and is recruited to the promoter of GLI1 responsive gene leading to H3K9 acetylation of Hh target gene promoters and their activation in medulloblastoma and glioblastoma cells, and PCAF downregulation in these cells leads to decreased proliferation and increased apoptosis [GLI1 protein at lysine 518, preventing its nuclear translocation and promoter occupancy [Multiple endocrine neoplasia type 1 (MEN-1 syndrome), a hereditary condition associated with tumors of the endocrine (hormone producing) glands, and the tumor suppressor gene ignaling . This stpoptosis . Howeverccupancy , whereasccupancy .in vitro and in vivo, such as basal cell carcinoma, medulloblastoma and atypical teratoid rhabdoid tumor [BRD4, a conserved member of the bromodomain and extraterminal (BET) family of chromatin readers, plays a critical role in transcription by binding acetylated histones and recruiting transcriptional complexes . Recentlid tumor ,70. HoweEWS-FLI1 resulting from the chromosomal translocation t. EWS-FLI1 can induce GLI1 transcription by direct binding to the Gli1 promoter, and overexpressed GLI1 is an important mediator in ESFT development [Ewing sarcoma family of tumors (ESFT) is characterized by producing an aberrant fusion gene elopment . Earlierelopment . Severalelopment ,72. How et al. reported that in the situation of insufficient cellular energy, AMPK is activated by accumulated ADP and AMP, and then phosphorylates directly GLI1 at serines 102 and 408 and threonine 1074, leading to suppression of GLI1 transcription activity. In this manner, when energy stores are inadequate, cells or organs slow or postpone developmental steps driven by Hh signaling in order to enhance the chance of survival of a growing animal [Gli1 activity is also regulated by energy sensor, AMP-activated protein kinase (AMPK). Recently, Li g animal . In meduIt is clear that regulation of Gli transcription factors is not only restricted to the canonical Hh/SMO-dependent cascade but also through the convergence of multiple SMO-independent pathways. These findings not only explain the ineffectiveness and failed clinical trials using SMO antagonists, and also encourage developing new strategies to suppress non-canonical Hh signaling. Unlike the canonical pathway, there are different ways by which non-canonical Hh signaling can occur. In some cancer types, more than one type of non-canonical Hh signaling may co-exist. Thus, there is a need of paradigm shift in designing strategies to block hedgehog signaling in cancer. First, canonical Hh signaling in cancer is quite limited to tumors with gene mutations in PTCH1 and SMO, which will be sensitive to SMO signaling inhibitors. In most cancer cells, one or more non-canonical Hh signaling modes of action play a critical role in cancer development. Thus, there is an urgent need to understand the specific non-canonical signaling mechanism in a given tumor type before novel mitigation strategies can be established. Second, due to the co-existence of activation of multiple signaling pathways in a given cancer types, as well as different mechanisms of non-canonical Hh signaling, combined target therapy will be more effective in suppressing tumor progression. Third, despite the existence of multiple aberrant signaling in a given cancer cells, each alteration may affect different stages of cancer development. Thus, physiologically relevant mouse models will be needed to test the role of each aberrant signaling. Taken together, we believe that the unsuccessful clinical trials using SMO signaling inhibitors have revealed novel mechanisms underlying Hh signaling activation in cancer, particularly non-canonical Hh signaling. Thus, inhibition of Hh signaling at the level of GLI transcription factors, rather than the level of SMO, might be a more effective way to combat cancer ,75,76,77


Transgenic expression in Drosophila of the alternative oxidase (AOX) derived from Ciona intestinalis has previously been shown to mitigate the toxicity of respiratory chain inhibitors and to rescue mutant and knockdown phenotypes associated with cytochrome oxidase deficiency. We therefore tested whether AOX expression could compensate the mutant phenotype of 25ttko using the GeneSwitch system to activate expression at different times in development. The developmental delay of 25ttko was not mitigated by expression of AOX throughout development. AOX expression for 1 d after eclosion, or continuously throughout development, had no effect on the bang sensitivity of 25ttko adults, and continued expression in adults older than 30 d also produced no amelioration of the phenotype. In contrast, transgenic expression of the yeast alternative NADH dehydrogenase Ndi1 was synthetically semi-lethal with 25ttko and was lethal when combined with both AOX and 25ttko. We conclude that AOX does not rescue 25ttko and that the mutant phenotype is not solely due to limitations on electron flow in the respiratory chain, but rather to a more complex metabolic defect. The future therapeutic use of AOX in disorders of mitochondrial translation may thus be of limited value.A point mutation [ Drosophila provides a useful animal model for human genetic diseases  point mutation in the gene for mitoribosomal protein S12 , and a set of linked phenotypes that share features with human mitochondrial disease, including hearing impairment and sensitivity to antibiotics that impair mitochondrial protein synthesis  from Ciona intestinalis  and 1sesB (adenine nucleotide translocase) as well as 25ttko display a characteristic seizure pattern  in every case.Bang sensitivity is generally considered to arise from a functional defect of nerve conduction during high-frequency stimulation in the giant fiber pathway  stages  system that contain mitochondrial translation components .In previous studies we found decreased steady-state ATP levels in extracts from 25ttko homozygotes compared with heterozygous controls (25ttko homozygotes was also elevated in every case compared with heterozygous controls (25ttko organismal phenotype is also not directly determined by ROS.We confirmed the previous observation of decreased ATP levels in controls  but founcontrols . This waCiona intestinalis can ameliorate the mutant phenotype of 25ttko, which carries a mutation in mitoribosomal protein S12, resulting in globally decreased OXPHOS capacity. We found that induced AOX expression, whether during development, in freshly eclosed adults, or maintained in adults over a period of 30 d, has no effect on 25ttko, nor does it produce a phenocopy of 25ttko in wild-type flies. In contrast, ubiquitous expression of Ndi1, the alternative NADH dehydrogenase from yeast, was highly deleterious to 25ttko during development and was lethal when combined with both 25ttko and AOX.In this work we set out to determine whether AOX from 25ttko exhibits a functional deficiency of all four OXPHOS complexes containing mitochondrial translation products (25ttko, and that the phenotype is entirely due to cI dysfunction. The second postulates that AOX is unable to rescue 25ttko because, as a nonproton-motive enzyme, it does not support the synthesis of ATP, and ATP deficiency is what underlies the mutant phenotype. A third possibility is that the phenotype is a consequence of one or more processes on which AOX does not impinge, such as elevated ROS production, or proteotoxicity due to the protein synthesis defect. Although none of these can be entirely eliminated, the fact that Ndi1 expression worsens the phenotype, either alone or in combination with AOX, and that changes in ATP level or mitochondrial ROS production do not correlate with it, suggest that the mutant phenotype is determined either by a complex interplay of factors or by other metabolic effects that are as yet unknown. Disrupted redox homeostasis resulting from a cI defect should be rescuable by Ndi1. A combined limitation on electron flow at cI and cIII and/or cIV should be alleviated by combined expression of Ndi1 and AOX. Manifestly, these predictions are inconsistent with our findings.25ttko because the apparent additional decrease in ATP level (25ttko flies (Ndi1 is constitutively active (TP level  was mode5t flies , it was 25ttko phenotype.Proteotoxicity due to imbalance between cytosolic and mitochondrial protein synthesis has been implicated as a longevity mechanism, acting hormetically via the induction of the mitochondrial unfolded protein response . However25ttko reflects a similar finding for a second mutant affecting mitochondrial ATP production, 1sesB (1sesB carries a mutation in the gene encoding the major adult isoform of the adenine nucleotide translocase (25ttko, 1sesB mutant flies show decreased steady-state ATP levels as well as bang sensitivity and developmental delay (The failure of AOX to rescue bang sensitivity and developmental delay in Drosophila and is due to lesions affecting a variety of cellular or physiological pathways, including, in addition to mitochondrial protein synthesis, adenine nucleotide transport and the TCA cycle (25ttko, show only a modestly decreased lifespan and associated neuropathology (25ttko over 30 d of adult life, in contrast to the synergistic and progressive effects on bang sensitivity seen when 25ttko is combined with other bang-sensitive mutants (25ttko is a developmentally determined phenotype, at least in an otherwise wild-type genetic backgroundBang sensitivity is a commonly observed mutant phenotype in CA cycle , they fa25ttko. Despite profound effects on flies exposed to toxins or mutations directly or indirectly affecting cytochrome oxidase (25ttko phenotype.AOX has been proposed as a therapeutic tool relevant to a wide variety of mitochondrial disorders (25ttko has been considered as a model for mitochondrial diseases, exhibiting not only seizures and developmental delay but also hearing impairment (25ttko constitutes a model for such diseases, yet neither AOX nor Ndi1 ameliorated the phenotype, and Ndi1 was even deleterious. As already indicated, Ndi1 and AOX do not restore proton pumping at the respiratory chain segments that they bypass, nor can they alleviate, a priori, all other aspects of mitochondrial dysfunction. Their uses in eventual therapy for disorders of mitochondrial translation therefore may be limited and clearly requires a fuller understanding of the pathophysiological mechanism case by case.


The clonal expansion and malignant transformation of HTLV-1 infected CD4+ T-cells have been linked to the reprogramming effects of HTLV-1 on host transcriptional profile. Coupled to transcription, alternative splicing (AS) is a post-transcriptional process that plays critical role in the complexity of transcriptome and splicing abnormalities frequently occur in cancer. To examine whether AS modifications associate with HTLV-1-associated leukemogenesis, we compared the exon expression profiles of ATL cells with that of CD4+ T-cell clones obtained by limited-dilution cloning of PBMC deriving from HTLV-1 carriers. 3 ATL cells and 12 untransformed infected clones clustering in infected, uninfected, PHA-stimulated or unstimulated CD4+ T cells were compared for exon RNA content using Exon Chip Human microarray. Hierarchical clustering analysis identified 12516 alternative spliced events (3642 genes) that clearly separated ATL samples from the 4 untransformed phenotypes mentioned above. In contrast, the exon content of 1539 genes differed between untransformed infected and uninfected T-CD4+ cells. Overall, less than 5% alternatively spliced genes were found differentially expressed at the transcriptional level. Microarray data were confirmed for 18 AS events using exon specific RT-PCR analysis. Pathway analysis of alternatively spliced genes (3642) in ATL cells revealed new AS-based pathways for p53 signaling, cell cycle and DNA replication while those of untransformed infected CD4+ T-cells were enriched in pathways for cellular movement and DNA repair. These findings unveil a new layer of complexity in the interplay between HTLV-1 and host cell gene expression machinery in which AS might play a central role in tumor initiation and promotion.