HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) remains a challenging disease. Treatment options are scarce, and their safety and efficacy are currently a matter of concern.We present a case report describing our experience using cyclosporine in a patient with early HAM/TSP who started with a gait disturbance at Vall d’Hebron University Hospital (Barcelona) from August 2012 to October 2013. After 62 weeks of treatment, clinical improvement was observed and proviral load diminished. No safety concerns were observed.Cyclosporine seems to be effective in new-onset HAM/TSP or in chronic HAM/TSP that develops a relapse. However, the duration and safety profile of this steroid-sparing therapy remain unknown and should be further investigated. Approximately, 20 million people are infected with HTLV-1 and 5 million with HTLV-2 worldwide.3 HTLV-1 infection is mainly present in the Sub-Saharan region, Japan, the Caribbean region, and some parts of Latin America, whereas HTLV-2 infection predominates in specific ethnic groups in Africa and America. Additionally, HTLV-2 infection has been found in injection drug users.4 Prevalence in the high endemic Japanese population has been rated at 0.66% in men and 1.02% in women.5 In Spain, the prevalence rate is 0.06% for HTLV-1 and 0.08% for HTLV-2. HTLV-1 was mainly diagnosed in the migrant population.4Human T-lymphotropic virus (HTLV) was the first retrovirus to be described.7The HTLV-1 infection has been associated with 2 life-threatening diseases, T-cell leukemia/lymphoma (TLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Other manifestations, such as eye and skin involvement, have also been related with HTLV-1.8 Symptoms of the disease are insidious and difficult to interpret at the onset of the disease. Paraparesis, spasticity, urinary incontinence, lower back pain, and hyperreflexia are common but not specific findings, hindering both diagnosis and follow-up. A new classification to make monitoring of the therapeutic response easier was developed recently using onset, progression, and activity criteria.9HAM/TSP is an inflammatory disease of the central nervous system. The incidence of HAM/TSP was 5.3 cases per 1000 HTLV-1-seropositive cases per year in a study carried out in Brazil.10Although HAM/TSP has been known for decades and its consequences are devastating, treatment options are still scarce. Initially, antiretroviral therapy was used with contradictory results. Once the immune-mediated theory emerged, immunosuppressive therapy was studied. To date, cyclosporine and interferon-α are the preferred immunosuppressive agents, even though the evidence regarding their efficacy is limited.11 In a recent proof-of-concept study, 7 patients with HAM/TSP in an early/progressive stage of the disease were treated with cyclosporine with encouraging results.10Host–virus interaction is crucial for the HTLV-1 spread and the development of lymphocyte-mediated related diseases. Immunomodulation with cyclosporine after HTLV-1 infection in a rabbit model has shown a decrease of proviral load.10Based on the cyclosporine proof-of-concept study, we relate our experience with cyclosporine in a patient diagnosed with an early HAM/TSP. We utilized the same stage classification and outcome criteria as described by Martin et al.The institutional review board of the Vall d’Hebron University Hospital approved the study. The patient signed a written informed consent for publication of the case report.A 40-year-old woman from the Dominican Republic presented at the emergency room in August 2012 with an 8-month history of gait disturbances and falls. The patient had been living in Spain for the last 14 years and reported no travel history in the last 5 years. Her previous medical status unveiled a treated hyperthyroidism with a normal hormonal study in the follow up, a major depression disorder, and urinary bladder disturbances that had appeared during the previous year and were being studied at another center. She was under treatment with paroxetine and solifenacin succinate.A more accurate medical history revealed blurred vision and muscle spasms. On physical examination on admission, the patient had normal axillary temperature, blood pressure values, and heart rate. Heart, lung, and abdominal examinations were unremarkable. Skin examination showed a pruriginous papular rash in the lower abdomen and scalp. Muscle strength was slightly reduced in the lower limbs, where the deep tendon reflexes were brisk and the reflex area expanded. Moreover, the lower limbs were spastic and vibratory sensation was diminished. The Babinski sign was presented bilaterally. On eye examination, uveitis was diagnosed.3 (10.2%). Biochemical parameters remained within the normal range. Serological tests for hepatitis B and C, human immunodeficiency virus, and Strongyloides stercoralis were negative. Vitamin deficits and autoantibody battery study were also within normal ranges. Feces samples were negative for helminths. The tuberculin skin test was performed twice with negative results.In the blood test, the white cell count only showed a total eosinophil count of 1000 cells/mmA cranial and spinal magnetic resonance imaging demonstrated small demyelinating focal lesions in the subcortical white matter, mainly around the posterior portion of both oval centers, conditioning Wallerian degeneration in the pyramidal tracts bilaterally. The lesions showed no signs of activity.The cerebrospinal fluid showed lymphocytic pleocytosis. An oligoclonal band study was negative and microscopy did not observe any atypical cells.On electromyography examination, a severe involvement of pyramidal and somatosensorial tracts for all 4 limbs was diagnosed, suggesting demyelination. No signs of peripheral neuropathy or motor neuron disease were detected.Staphylococcus aureus.Furthermore, a skin punch examination of the papular rash of the lower abdomen demonstrated an atypical T phenotype lymphoid infiltrate, suggesting an indolent lymphoma form; however, T-cell receptor γ, T-cell receptor β, and immunoglobulin heavy chain reassortment studies were negative. T CD8 lymphocytes in the epidermis also suggested infective dermatitis. Skin cultures were positive for methicillin-sensitive 12 The seropositivity for HTLV-1 was confirmed by line immunoassay . Curiously, the line immunoassay also resulted positive for HTLV-2. The partner of the patient also had antibodies against HTLV-1 and HTLV-2 confirmed by line immunoassay.A definitive HAM/TSP diagnosis was made with the information above, plus a positive serology for HTLV  and a positive HTLV-1 proviral load in peripheral blood.The HTLV-1 proviral load in the peripheral blood before treatment was 1.767/10.000 peripheral blood mononucleated cell. The proviral load from the cerebrospinal fluid was not performed.Clinical status was evaluated using the modified Ashworth spasticity scale, spasticity scale-88 score (SPAST-88), SF-36 health survey scale (SF-36), multiple sclerosis walking scale, Instituto de Pesquisa Clínica Evandro Chagas disability score (Instituto de Pesquisa Clínica Evandro Chagas disability score), the Barthel scale, postvoid residual urine volume, nocturia frequency (per night), and a walking test . These tests were performed before treatment initiation and after 62 weeks of treatment. Results are shown in Table Cyclosporine was started at 25 mg twice a day and titration was done according to plasma cyclosporine levels. Target plasma levels were 80 to 100 ng/mL, and were mainly achieved with 50 mg twice daily.Treatment was discontinued after 62 weeks. During this time, the patient was closely monitored with blood tests and physical examination. No side effects or evidence of TLL were reported.The clinical outcomes and the HTLV-1 proviral load in peripheral blood are depicted in Tables 9Although HAM/TSP was described many years ago, limited efforts have been made to develop new and effective therapies. Different biological approaches have been elaborated to understand the pathogenesis of the disease, and therefore, different treatment options have been tested with inconclusive results, including interferon-α, interferon-γ, antiretroviral therapy, danazol, corticoids, valproic acid, monoclonal antibodies anti-CD25, and others.Despite the fact that available scientific evidence does not support any treatment, the interferon-α treatment strategy is the most widely studied, and there appear to be benefits when applied in the early or progressive stages of the disease. On the contrary, steroids remain a useful resource for punctual use, but their activity seems temporary and treatment potency is lost with continuous use.10 Our baseline and end-of-treatment outcome criteria were compared after 62 weeks of treatment, in contrast with the 48 weeks of treatment and 72 weeks of follow-up in the proof-of-concept study. Despite these differences, we think our experience strengthens the outcomes obtained by Martin et al.10Recently, a proof-of-concept study has supported a potential advantage when using cyclosporine, a steroid-sparing therapy, in the early or progressive phase of the disease.The IPEC and walking tests were the main monitoring criteria used, as they are stable over time and reproducible because they are rarely modified by mood state or psychiatric conditions. Other tests like SF-36 and SPAST-88 may be highly dependent on mood state, so that a state of depression, usually accompanying the disease, may alter the results. Our patient showed a significant improvement in both the walking and IPEC tests.Cyclosporine levels were used to adjust the treatment dose. Three out of the 8 cyclosporine levels were in the range between 80 and 100 ng/mL, 1 was above the upper limit, 2 were between 60 and 65 ng/mL, and 2 determinations were <50 ng/mL. Notwithstanding, both determinations <50 ng/mL were the 2 first determinations (performed in the first month) when there was titration of the dose. We can therefore say that cyclosporine levels were within the normal range or above in 50% of the determinations. Treatment was given under directly observed treatment.HTLV-1 proviral load was only assessed in peripheral blood. Proviral load gradually declined during treatment, as shown in Table Although the patient received immunosuppressive therapy for over a year, no sign of hematological disease appeared. However, a concern exists over whether a longer period of treatment may increase the incidence of TLL. More data regarding this aspect are needed.To summarize, we have reported the case of a patient with an early HAM/TSP treated with cyclosporine, with a satisfactory clinical outcome after 62 weeks of treatment. Cyclosporine seems to be effective in new-onset HAM/TSP or in chronic HAM/TSP that develops a relapse. However, the duration and safety profile of this steroid-sparing therapy remain unknown and should be further investigated.


Human T-cell Leukemia virus type-1 (HTLV-1) causes several diseases including aggressive Adult T-cell Leukemia (ATL). A major player in the malignant transformation of infected cells is expression of Tax, the virally-encoded oncoprotein. Tax is a potent activator of HTLV-1 transcription. To activate viral transcription, Tax, together with the cellular protein CREB, bind to the viral CRE enhancer elements located in the HTLV-1 promoter. Together, these activators recruit the homologous coactivator proteins CBP to initiate strong transcription of the provirus. Both CBP/p300 are involved in multiple cellular pathways, and deregulation of these proteins is associated with numerous pathologies. Despite extensive work on Tax-stimulated recruitment of CBP/p300 to the HTLV-1 promoter, primarily via their KIX domain, potential interactions between Tax and other domains of CBP/p300 have not been fully investigated. Recently, we have found that Tax interacts with several additional domains of the coactivators, however the significance of these interactions remains incompletely understood. We hypothesize that Tax interaction with various domains of CBP/p300 stimulates the potency of the coactivators and enhanced transcription. Our studies utilize protein-protein and protein-DNA interaction assays using individual domains of the coactivators, together with mutations and deletions that specifically target the Tax-CBP/p300 interface. Competition and activity assays enable us to functionally and biophysically characterize Tax-coactivator interactions. These studies have implications for both viral and cellular mechanisms of gene expression, and expand our understanding of the role of Tax and the cellular coactivators in HTLV-1 transcription and replication.


Nicotiana attenuata. We then discuss means of manipulating jasmonate signaling by creating tissue-specific jasmonate sinks, and the micrografting of different transgenic plants. The metabolic phenotyping of these manipulations provides an integrative understanding of the functional significance of deviations from the canonical model of this hormonal pathway. Additionally, natural variation in jasmonate biosynthesis and signaling both among and within species can explain polymorphisms in resistance to insects in nature. In this respect, insect-guided explorations of population-level variations in jasmonate metabolism have revealed more complexity than previously realized and we discuss how different “omic” techniques can be used to exploit the natural variation that occurs in this important signaling pathway.Plants have evolved sophisticated communication and defense systems with which they interact with insects. Jasmonates are synthesized from the oxylipin pathway and act as pivotal cellular orchestrators of many of the metabolic and physiological processes that mediate these interactions. Many of these jasmonate-dependent responses are tissue-specific and translate from modulations of the canonical jasmonate signaling pathway. Here we provide a short overview of within-plant heterogeneities in jasmonate signaling and dependent responses in the context of plant-insect interactions as illuminated by examples from recent work with the ecological model,  Regulation of plants’ defenses during biotic stresses is a complex phenomenon. In nature, plants are essential nutritional sources for different communities of phytophagous insects. The selection pressure imposed by insect herbivory has likely shaped many of the strategies that plants employ to maximize their fitness in their native habitats. In addition to physical barriers  and constitutive chemical defenses that repel herbivores, plants also rely on more complex defense systems that are specifically produced during insect feeding . These iCOI1 complex via a mechanism in which the F-box protein COI1 (CORONATINE-INSENSITIVE1) binds to JA-Ile and recruits JASMONATE ZIM DOMAIN proteins (JAZs) which are then targeted for ubiquitination and subsequent proteosomal degradation, leading to a release of JAZ-mediated repression of gene expression [Most of the above described anti-herbivore direct and indirect responses are modulated across plant tissues and intrinsically connected with rapid changes in jasmonate pools . Jasmonapression ,32,33.COI1-independent manner [Arabidopsis possess 12 JAZ genes, some of which are subjected to alternative splicing, provides the basis for diversity in small molecule recognition and signaling that may be responsible for additional heterogeneity in jasmonate signaling amongst tissues [N. attenuata, herbivory-induced jasmonate levels greatly vary across geographically-distant accessions and this variation can partly explain differences in levels of secondary metabolite responses [A first dimension of the specialization of the canonical jasmonate signaling lies in the tissue-specific modulation of jasmonates. Other dimensions of variations from the canonical pathway may result from signaling functions of jasmonates other than JA-Ile. 12-oxophytodienoic acid (OPDA) has notably been shown to activate certain physiological responses, such as tendril coiling and carnivorous trap functions and other transcriptional responses in a t manner ,35. The  tissues ,37. An aesponses ,39,40,41N. attenuata. To this end, we provide a three-dimensional roadmap traversing within-plant, population-level to metabolic pathway-specific variations in jasmonate signaling and possible deviations from the canonical perspective. We also discuss means of manipulating jasmonate signaling by creating jasmonate sinks, micrografting procedures and a dexamethasone (DEX) system to disentangle tissue- and age-specificities in jasmonate signaling and their responses. Finally, we discuss the prospect of identifying novel jasmonate-regulated defense metabolites using natural variation in jasmonate signaling. In each of these sections, we highlight how deviations in jasmonate signaling can enhance our understanding of the phenotypic adjustments that are regulated during herbivory by this signaling pathway.In this review, we describe modulations of jasmonate signaling in light of plant-insect interactions as illuminated by examples from recent work with the ecological model, JAZ genes suggests that the diversity is not simply an artifact of gene redundancy. Despite the current consensus that JAZ proteins act as general repressors of jasmonate signaling, little is known about how individual JAZ proteins function. Even in the model plant Arabidopsis, phenotypes of plants with individually silenced JAZ proteins remain incomplete, and their tissue-specific functions remain poorly understood [JAZ genes are differentially expressed in locally elicited leaves, systemic unelicited leaves and roots of N. attenuata when elicited by a standardized method mimicking Manduca sexta herbivory ) [JAZ expression is not fully understood and additional work is needed to spatially dissect the function of this JAZ regulatory network. Recently, JAZ10 was successfully employed as a JA-responsive reporter in Arabidopsis to identify root-specific activators or repressors of wound signaling [Tissue specificity in metabolism in response to herbivory is thought to be mediated, in part, by tissue-specific deviations in jasmonate signaling . Spatialderstood ,44. JAZ  (W+OS)) . Howeverignaling .Nicotiana tabacum, jasmonates regulate the expression of NtPYL4 which encodes an ABA receptor protein affecting the regulation of defensive alkaloids biosynthesis in roots [JAZ proteins [N. attenuata is presented in Interactions with other hormonal signaling are also likely to contribute to these tissue-specific responses. For example, specific sets of genes encoding auxin- and ethylene-related signaling processes show OS-elicited responses in systemic leaf tissues that are distinct from those in locally treated tissues . In Nicoin roots . An antain roots . Gibbereproteins . The balproteins . In contproteins ,52, a crproteins . The kinproteins . Jasmonaproteins . The infproteins ,56; thisproteins . ClearlyGLUTAMATE RECEPTOR-LIKE genes (GLRs) were found to be involved in the propagation of electrical signals and to elicit wound-induced expression of several JAZ genes [When chewing herbivores attack a leaf, a short-distance mobile signal is rapidly triggered, which in turn produces a long-distance mobile signal that moves to unattacked leaves following vascular connections. This short-distance mobile signal travels in particular directions towards the undamaged parts of the attacked leaf where it triggers region-specific jasmonate signaling, while the long-distance signal propagates between leaves to elicit jasmonate signaling and defense responses in distal leaves. Relatively little is known about which signal serves as the initial trigger and how the signaling crosses from wounded leaves to other plant parts. Electric and hydraulic signals are thought to play a central role in this process ,59,60. RAZ genes .Solanum Lycopersicum tomato [de novo in response to transported signal activated by HAMP and DAMP elicitors from damaged leaves [The importance of jasmonate signaling for systemic defensive traits regulation during insect herbivory was first revealed by the seminal work of C.A. Ryan on the induction of digestive proteinase inhibitors in the m tomato ,27. Sevem tomato . During m tomato ,64. Impom tomato . In systd leaves . The lond leaves .Arabidopsis, jasmonate metabolic profiles differ dramatically both in their compositions and accumulations in systemic leaves and midveins of systemic leaves compared with those in local leaves in response to wounding; while within the wounded leaf, certain jasmonate derivatives specifically accumulate in the midvein, distinguishing the jasmonate signatures from those in the lamina [N. attenuata, the conversion efficiency from JA to JA-Ile greatly differs for different positions relative to the wound site [MAPKs (mitogen-activated protein kinases) activation are responsible for the inhomogeneity of the jasmonate accumulation and transcriptional responses in different areas of the leaves following simulated insect herbivory [ArabidopsisLOX6, a lipoxygenase responsible for rapid jasmonate synthesis in tissues distal to wounds, has been shown to be expressed in a group of cells that are tightly associated with xylem vessels. These regions where contact cells are located are highly sensitive to the release of water column tension in response to wounding. The squeeze cell hypothesis elaborated by E.E. Farmer suggests that contact cells that are hydrostatically coupled to the xylem are likely to be squeezed by pressure increases in the xylem after wounding which disperses hydrostatic pressure finally triggers jasmonate synthesis in distal tissues [In many species, compositional variations in jasmonate profiles have been observed both within and between leaves. In e lamina . In N. aund site . Constraerbivory . Most jaerbivory ,69 reinferbivory . Along t tissues . AlthougJAR1, COI1, JIN4, JIN1/MYC2, JAI3 or AXR1. Root-specific molecular aspects of the jasmonate pathway have been recently investigated in depth with the discovery that the organizational principles of the jasmonate signaling machinery differ significantly between the root and the shoot. In this respect, NINJA plays a critical role in repressing jasmonate signaling in roots and thereby maintaining the homeostasis of root growth [MYC2 together with NINJA in constraining jasmonate signaling during normal root cell division and elongation after wounding [JAZ10 reporter was used to screen for mutants affected in the organ-specific activation of jasmonate signaling in A. thaliana seedlings, and highlights the need for tissue-specific manipulations to finely dissect the complexity of jasmonate/JAZ regulatory networks.Root growth inhibition was one of the first observations of a plant’s physiological responses to jasmonates and has been used to screen jasmonate-deficiency and insensitivity which led to the discovery of many important signaling components of the jasmonate pathway such as t growth . A followounding . These sSpodoptera littoralis herbivory of maize leaves exceeded those observed in damaged leaves [M. sexta leaf herbivory in N. attenuata exhibit more pronounced differential responses in roots than in the shoot, suggesting a root-specific contribution to leaf-based immunity [Relatively little work has been conducted on the role of jasmonate signaling in the regulation of defensive metabolism in roots. The systemic responses in roots during leaf herbivory can exceed the amplitude of some of the best systemic responses detected in aboveground tissues. For instance, the transcriptomic changes activated in roots after d leaves . Consistimmunity . Jasmonaimmunity ,74.N. attenuata, a long-distance signal travels from damaged leaves to roots and initiates the transcriptional up-regulation of nicotine biosynthetic genes in the roots [When leaves of tobacco plants are mechanically wounded or attacked by a shoot herbivore, the plants activate synthesis of nicotine in the roots which is then mobilized to the shoot . In N. ahe roots . Surprishe roots .This resN. attanuata which may enable plants to support the synthesis of plant defensive metabolites belowground to attain full defense deployment. Jasmonate signaling plays a key, but not exclusive, function in the regulation of shoot and root carbohydrate turnover.Many plants exhibit an increase in carbon (C) transport from locally damaged leaves as well undamaged systemic leaves to roots in response to herbivore elicitation ,80,81,82N. attenuata is attacked by the larvae of the pith-feeding weevil Trichobaris mucorea, it activates a separate spatial defensive strategy in which JA and JA-Ile levels specifically increase in the damaged pith region but not in connected leaves (manuscript in preparation) [N. attenuata, a phenomenon which is elicited by M. sexta leaf herbivory and requires jasmonate signaling. The larvae of M. sexta, the important night pollinator hawkmoth of N. attenuata flowers, are important herbivores and nectaring during pollination increases oviposition rates. This creates a dilemma for N. attenuata in how to attract pollinators without increasing herbivory rates. When N. attenuata is attacked by M. sexta larvae, the phenology of flowers shifts from night-opening and benzyl acetone scenting flowers to morning-opening flowers that do not scent at night and these flowers attract day-active hummingbirds. This herbivory-elicited shift in floral phenology requires jasmonate signaling [Insect herbivory to plant stems results in distinct jasmonate patterns compared to roots and leaves. When aration) . Anotherignaling . Althougignaling , and theignaling .odorless-2 shows altered morphology, density and metabolites in their glandular trichomes, and as a consequence is susceptible to increased attack from both M. sexta and Colorado potato beetle larvae in the field [Arabidopsis, endogenous jasmonates regulate trichome initiation via the COI1 receptor complex to activate the trichome-specific transcription factors MYB75 and GLABRA 3 (GL3) leading to development of metabolically active trichomes [COI1-dependent jasmonate signaling as aos and coi1, two mutants that completely lack endogenous jasmonate function show reduced trichome numbers after wounding. Intriguingly, the jar1 mutant which is deficient in the generation of the most bioactive jasmonate compound JA-Ile [COI1, other active ligands may be responsible for trichome induction [Another tissue-specific jasmonate-dependent developmental response related to defense against insects is induced trichome formation. Trichomes often play a role in resistance due to the physical barrier they present to herbivores and for being sites of biosynthesis of various secondary metabolites such as terpenoids, flavonoids and alkaloids that function as direct defenses ,88 as wehe field . In Arabrichomes ,92. Wound JA-Ile  is unaffnduction .AtJMT gene in N. attenuata redirects jasmonate flux towards MeJA which competes with the production of the bioactive form JA-Ile and thereby down-regulates dependent defensive responses [Arabidospsis, overexpression of AtJMT results in elevated levels of MeJA and normal levels of JA but constitutively enhanced expression of JA biosynthesis related genes and plant resistance to stresses [N. attenuata plants ectopically expressing AtJMT do not differ from wild type plants in morphology at the rosette stage of growth. When plants reach flowering stage, the overexpression of JMT in N. attenuata severely alters floral organ maturation, corolla limb opening and volatile emissions which is consistent with the key function of JA-Ile in regulating these processes [The most commonly used means of studying hormonal function involves, silencing or knockout mutations of key genes in hormonal biosynthesis pathways . Substraesponses ,102. Thiesponses . With thesponses . In Arabstresses . It is nrocesses . ManipulArabidopsis to dissect systemic jasmonate signaling [N. attenuata and applied to the spatiotemporal modulation of cytokinin signaling during interactions with native herbivore Tupiocoris notatus in both laboratory and field studies [JMT/JME jasmonate sink expression system in N. attenuata plants, will provide an ideal means of disentangling tissue-specific regulation of jasmonate signaling for some of the plant-insect interaction-dependent responses described above. A new frontier will consist in the combination of this DEX-transactivating system with tissue- or cell type-specific promoters when relevant ones are identified in N. attenuata.Given the spatiotemporal specificity of jasmonate signaling and the similar specificity in the activity patterns of interacting organisms, it would be ideal to have sufficient control over gene expression to manipulate these dynamic interactions in real time in different tissues and ideally also under field conditions. ,105,106 ignaling  and is oignaling . Recentl studies ,108. ThiArabidopsis has been developed to manipulate below- and above-ground parts in N. attenuata for the study of shoot-root signaling in plant-insect interactions [N. attenuata lines have been used to reveal that the transportation of nicotine from roots to shoots requires jasmonate synthesis and JA-Ile perception in both shoot and root compartments [AOC. This result highlights the importance of the hormonal crosstalk between JA and ABA in the shoot-root-shoot signaling interplay established during leaf herbivory and induced nicotine mobilization to the shoot.Additionally, (micro)grafting techniques have provided significant insights into the study of systemic signaling. Grafting experiments with tomato mutants in jasmonate biosynthesis and signaling were first employed to reveal that local jasmonate signaling was required to mount systemic responses in distal leaves . Recentlractions . The mairactions . For exaartments . AdditioArabidopsis, MeJA induced gene expressions and glucosinolate accumulation are highly variable among different accessions [Considerable amounts of natural variation in jasmonates and dependent responses have been identified in many species ,111,112.cessions . Modulatcessions .N. attenuata populations exhibit a high genetic diversity within populations, which can be as great as that seen among populations [N. attenuata genotypes accumulating low amounts of jasmonate are more severely damaged by folivores such as grasshoppers, leafhoppers, lepidopteran larvae, beetles and stem-boring weevils in the field [ulations  and thisulations ,117. Thiulations ,39,40 anhe field ,118.Helicoverpa zea can respond to jasmonate signals to upregulate the expression of genes important in the detoxification of host plant defenses [Empoasca leafhoppers have also evolved mechanisms to select host plants in nature by eavesdropping on their JA-mediated signaling capacities. This probe-feeding insect preferentially targets jasmonate deficient plants [Empoasca leafhoppers on untransformed and jasmonate signaling-deficient transgenic plants under field conditions and the measurement of jasmonate-regulated direct and indirect defense compounds. On the other hand, the special fire-synchronized germination behavior of N. attenuata exposes these ephemeral host plants to herbivores from various guilds that colonize the environment after fire, but also to an intense selection for rapid growth with conspecifics and intense competition for resources. Plant’s growth-defense trade-offs involving jasmonate regulation might account for the maintenance of low jasmonate-producing genotypes in native populations, providing them with competitive advantages, despite the clear disadvantage of being defenseless and frequently targeted [Some phytophagous insects appear to directly sense the jasmonate signaling and/or dependent responses of their host plants to evaluate their suitability as food. For example, the corn earworm defenses . Empoasct plants , but thitargeted .M. sexta elicitation among different natural N. attenuata populations and across populations, herbivory induced jasmonate production negatively correlates with flower production of regrowing shoots [N. attenuata accessions, and that individuals even within small populations emit significantly different HIPVs following simulated herbivore elicitation [N. attenuata Arizona (AZ) and Utah (UT) accessions, the AZ accession was shown to exhibit similar capabilities in producing HIPVs despite having jasmonate accumulations that are half those observed in the UT accession [Jasmonate signaling plays a decisive role in modulating regrowth responses after g shoots . Recent citation  which inccession ,122.N. attenuata [Natural variation has been extensively used to discover the function of genes. With current advances in sequencing and with the use of multi-parent advanced generation inter-cross (MAGIC) and recombinant inbred lines (RILs) combined with analytical approaches such as liquid chromatography-mass spectrometry (LC-MS) and NMR for targeted metabolic analysis, the exploration of natural variations in both model and crop species can now be used to identify the genetic fundamentals of metabolic traits via quantitative traits locus mapping ,124,125.ttenuata . The nonttenuata . SignifiNon-targeted MS/MS profiling has the potential to identify unknown metabolites that could be involved in anti-herbivore defenses. The problem is that stress-responsive secondary metabolites are extremely structurally diverse and most plant metabolite databases are too sparsely populated to provide useful hints on these unknown structures. Innovative approaches have therefore to be implemented to accelerate the annotation of MS/MS data. One of the successful approaches pioneered by the Dorrestein laboratory involves the creation of MS/MS molecular networks. This approach involves the generation of networks in which mass spectra (nodes) from one data-set are clustered using edges whose length is inversely proportional to the pairwise similarity between spectra. This molecular networking facilitates the mining of unknown metabolites clustered adjacent to known ones. The network view of large metabolomics data-sets also allows for the combined visualization of other biological information linked to a given metabolite (MS/MS spectrum). This possibility was used to visualize statistical descriptors of natural variation for each MS/MS spectrum (nodes in the network) , and co-More than 50 years of research in jasmonate metabolism and regulation have established the importance of this signaling pathway for a plant’s ecological interactions, particularly those with insects. An increasing number of studies demonstrate that tissue-specific responses to herbivory are also associated with tissue-specific regulation in jasmonate signaling. The development of more sensitive and comprehensive analytical tools becomes increasingly important to improve the analysis of temporal modulations across multiple tissues and of the metabolites that are regulated downstream and function as defenses during herbivory. The acquisition of MS/MS metabolite data and the generation of structurally rich MS/MS similarity networks will facilitate the exploration of the multiple metabolic layers that make up plants’ defense responses. The analysis of natural variation in metabolic responses harbored in native plant populations will greatly enrich our understanding of the diversity in metabolic outcomes that jasmonate signaling can trigger and provide a healthy antidote to the typological thinking that emerges from focusing too much on models raised in the rarefied environments of growth chambers with little of the environmental complexity that sculpted the diversity of specialized metabolism.


Mean T/S ratio was 1.02 ± 0.16 in HTLV-1, 1.03 ± 0.17 in HTLV-2 and 0.99 ± 0.18 in HTLV seronegative subjects (p = 0.322). TL was not associated with HTLV-1 or -2 proviral load. Shorter TL was significantly associated with impaired vibration sense in the HTLV-2 positive group only. Overall, we found no evidence that telomere length was affected by chronic HTLV-1 and HTLV-2 infection. That TL was only associated with peripheral neuropathy in the HTLV-2-positive group is intriguing, but should be interpreted cautiously. Studies with larger sample size and telomere length measurement in lymphocyte subsets may clarify the relationship between TL and HTLV-infection.Short or damaged telomeres have been implicated in degenerative conditions. We hypothesized that analysis of telomere length (TL) in human T-cell lymphotropic virus (HTLV) infection and HTLV-associated neuropathy might provide clues to the etiology of HTLV-associated disease and viral dynamics. A subset of 45 human T-cell lymphotropic virus type 1 (HTLV-1), 45 human T-cell lymphotropic virus type 2 (HTLV-2), and 45 seronegative subjects was selected from the larger HTLV Outcomes Study (HOST) cohort, matched on age, sex and race/ethnicity. Telomere-to-single-copy gene (T/S) ratio (a measure of TL) and HTLV-1 and HTLV-2 proviral loads were measured in peripheral blood mononuclear cells (PBMCs) using quantitative PCR (qPCR). Vibration sensation measured by tuning fork during neurologic examinations performed as part of the HOST study allowed for an assessment of peripheral neuropathy. TL was compared between groups using  Telomeres, the repetitive nucleoprotein complexes that protect chromosome ends from damage, can shorten with each cell replication. If telomere length decreases beyond a critical threshold, normal cells typically enter a senescent state or can be directed to an apoptotic pathway. Hence, telomeres have been termed “clocks” that count down with each cell cycle, marking progress through the replicative lifespan of individual cells, unless they are rebuilt, usually by the telomere DNA synthesizing enzyme telomerase.Human T-cell lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) infection has been associated with increased all-cause mortality ,4 and inBecause HTLV proviral DNA integrates into the host chromosome and promotes cell division as part of a replication strategy, TL in infected cells could be affected as a result: either rapidly shortening TL or altering TL regulation to promote survival of HTLV-infected cells, similar to cancer cells where reactivation of telomerase maintains cells with shorter telomeres, thus contributing to genomic instability and cellular malfunction) ,17. HTLVGiven the associations between TL, interferon-related gene expression , oxidatiFrom the HTLV outcomes study  ,24. Thes6 cell aliquots in liquid nitrogen. Each PBMC aliquot that was analyzed for the current study was selected from the final HOST study visit, thawed and split, with one-half used for proviral load measurement and one-half used for telomere-to-single-copy gene (T/S) ratio (a relative measure of TL) measurement.Fresh whole blood samples from HOST subjects, collected at study visits, were centrifuged to separate peripheral blood mononuclear cells (PBMC’s), which were washed and stored in 5 × 10Telomere measurement was by quantitative PCR (qPCR)  at the BProviral loads were measured using real-time qPCR amplification with primers common to both HTLV-1 and HTLV-2 and detection by SYBR Green , which allowed direct comparison of proviral loads between types 1 and 2. Type-specific TaqMan probes  were used to distinguish HTLV-1 from HTLV-2 provirus. The specific method has been previously published .10 transformed. Seropositive cases with no detectable virus were imputed with a proviral load of 1 per 106 cells (which is the lower limit of detection) in order to allow for log transformation). Unpaired t-tests  were performed to compare HTLV-1 and HTLV-2 cases and controls in terms of demographic characteristics, smoking and alcohol history, and telomere length (mean T/S ratio).Univariate analysis of subjects’ demographic characteristics, smoking and alcohol-use history, and laboratory measurements was performed to describe the study population groups. Proviral load was loge T/S ratio) on proviral load (log10 transformed). Potential confounders, including age, sex, body mass index (BMI), smoking (cumulative pack-years), and alcohol use (drinks/week in previous two years) were evaluated for possible inclusion in an adjusted model, using a p-value < 0.10 as a threshold for consideration.Unadjusted linear regression was used for an initial assessment of the dependence of telomere length . Potential confounders were included in the final model based on biological plausibility and association with telomere length in bivariate analysis at significance of p < 0.10. Multivariable logistic regression was also performed to assess whether PBMC telomere length was predictive of vibration-sense impairment. Potential confounders identified in bivariate analysis (p < 0.10) were included in the adjusted model based on likelihood ratio tests and biological plausibility. Statistical analysis was performed using Stata .Multivariable linear regression was used to assess the independent association of telomere length on proviral load . In unadjusted regression analysis by HTLV-status , telomere length (loge T/S ratio) was significantly associated with age (inversely) only in the HTLV-negative control group (p = 0.007). Cumulative smoking (lifetime pack-years) was inversely associated with telomere length in the HTLV-1 cases (p = 0.009), but not associated within HTLV-2 cases or controls. Log10 proviral load had a borderline-significant association (p = 0.069) with telomere length only in the HTLV-1-positive group. Within the HTLV-2 cases, TL in subjects with HAM was shorter (mean T/S ratio 0.78 ± 0.15) than in those without (mean T/S ratio 1.04 ± 0.17), although the paucity of HAM cases precluded formal assessment of this difference.No significant differences in mean telomere length were observed, comparing either HTLV-1 or HTLV-2 cases to controls, or combined HTLV cases to controls . Mean T/p = 0.003), smoking (p = 0.015) and sex (p = 0.097) reached the threshold (p < 0.10) for consideration as candidates in an adjusted regression model of loge T/S ratio. Higher age and smoking and female sex were negatively associated with TL. In a multivariate model that adjusted for age, pack-years of smoking and sex, log10 proviral load was not associated with telomere length in either HTLV-1 or HTLV-2 cases. In the control group only, higher age remained significantly associated with lower loge TL (p = 0.006) after adjustment for smoking and sex. In the HTLV-1 positive group, cumulative smoking history had a borderline significant inverse association with loge T/S ratio (p = 0.052). There were no significant predictors of T/S ratio in adjusted HTLV-2-specific analysis.In an analysis combining HTLV-1 and -2 cases and controls , age (p p = 0.025), greater cumulative smoking (p = 0.027) and lower loge T/S ratio . Shorter telomere length, the primary independent variable of interest with respect to neurologic outcomes, was associated with increased odds of tuning fork sensation impairment in the HTLV-2 cases (p = 0.014), but not in the HTLV-1 cases or controls. While the data are suggestive of seronegative subjects with impaired sensation demonstrating shorter TL when compared with seronegatives with normal sensation, the large variability results in lack of statistical difference.e T/S ratio, the pattern of significant predictors of vibration sense impairment was markedly different among HTLV-1 and -2 cases and controls. In the control group, age did not retain its significant association with the odds of impaired vibration sensation after adjustment. Smaller loge T/S ratio was significantly associated (p = 0.026) with increased odds of vibration-sensation impairment only in the HTLV-2-positive group.In a logistic regression model that adjusted for age, smoking and logOur main hypothesis was not supported as telomere length did not differ by HTLV infection status and neither HTLV-1 or HTLV-2 proviral load was predictive of TL. However, we did observe that age effect was weaker in HTLV-1 and -2 cases compared to seronegative controls. Interestingly, shorter TL was associated with impaired vibration sense among HTLV-2 subjects only.The failure to detect any significant differences in mean T/S ratio between cases and controls could have several explanations. First, our hypothesis that there is an underlying difference in the mean telomere lengths of the target groups may have been wrong. Telomere length is under several complex controls  and in HConsistent with previous studies and underlying theory age was inversely associated with telomere length in all subjects combined. But when subjects were separated by infection status, age retained this association only in the control group. Although most age-related telomere shortening occurs early in life , past reThat proviral load was not associated with telomere length in multivariable analysis may reflect the true lack of an association, compensatory mechanisms and in vivo cellular selection (as discussed previously), small sample size, a relatively low percentage of infected PMBCs in these subjects, or unaccounted confounding. There are several reasons to expect proviral load to be associated with telomere length in the PBMCs of HTLV-infected subjects. First, HTLV-provirus spread largely results from clonal expansion of infected cells, and telomeres shorten with each cell division. Therefore, it would be expected that higher proviral load would be associated with shorter telomeres. Recent research suggests that in HTLV-1-infected subjects, even the provirus-free T-cells have altered telomerase activity . Hence, p = 0.052) after adjustment. Evidence from previous research suggests that smoking is indeed a risk factor for telomere attrition [It is interesting that only in the HTLV-1 group did proviral load and smoking show some evidence of association with TL in bivariate analysis, although only smoking remained as a borderline significant predictor -induced damage in impaired tuning fork sensation in this study population. Neurologic dysfunction due to HTLV infection has been associated with inflammation and altered cytokine expression ,33. TherThis study benefited from a well-characterized HTLV cohort with good collection of medical information. It utilized a telomere length assay performed in the laboratory of the discoverer of telomerase and a proviral load assay performed in the laboratory of the creator of the HTLV-specific qPCR protocol. While the number of male subjects is small , the cases and controls were group-matched on age, sex, and self-reported race/ethnicity, and all subjects in this analysis were living in or near major population centers, which may have also helped to evenly distribute unknown confounders between the groups.The major weaknesses of this study were the relatively small sample size, the lack of longitudinal telomere length measurements, and measurement of TL in unsorted PBMC rather than lymphocyte subsets. The sample size for the study was moderate and, in addition, a small number of subjects had moved out of the area since enrollment in the larger HOST cohort, further reducing the sample size for the peripheral neuropathy analysis. While the correlation between impaired tuning fork sensation and progression to HAM in HTLV-infected persons has not been demonstrated, Saeidi et al. reported that 30% of patients with HAM/TSP also experienced peripheral neuropathy . Due to In conclusion, this study did not find any differences in mean TL between HTLV cases and controls but there was some evidence that the relationship between neuropathy and TL is different among HTLV-1-positive, HTLV-2 positive, and seronegative individuals. Since only a small proportion of HTLV-infected individuals progress to clinical disease, it would be interesting from an etiologic perspective, and possibly useful as a prognostic tool, to determine whether telomere length plays a role in the progression to neurologic disease. A study with larger sample size, CD4/CD8 subset sorting, longitudinal telomere length measurements, and measurement of additional potential confounders, such as diet and antioxidant intake, may better able to unravel any possible relationships between telomere length, HTLV-infection, and HTLV neurologic disease.


The pathogenesis of HTLV-1-associated diseases involves complex interactions between HTLV-1 and the infected host, and is dependent on viral replication and gene expression. Previous studies from our laboratory have shown that T cell receptor- and phorbol ester (PMA)-mediated stimulation of chronically infected CD4 T-cells increases the expression of integrated HTLV-1 proviruses. Detailed analysis of HTLV-1 RNA and protein species following PMA treatment of the FS and SP HTLV-1-infected T cells demonstrated rapid induction of tax/rex mRNA peaking prior to other HTLV-1 RNA species. This rapid increase in tax/rex mRNA was associated with markedly enhanced tax/rex mRNA stability. (from 3.5 hr to >24hr in treated cells), while the stability of unspliced or singly spliced HTLV1 RNAs did not increase. PMA treatment also increased RNA transcription. These data contrast with a delayed increase in HTLV-1 RNA transcription following treatment the HDAC inhibitor, SAHA. To further identify the elements and mechanisms responsible for enhanced tax/rex RNA stability, we adapted a luciferase reporter assay in which tax/rex cDNA sequences were linked to the 3’ end of luciferase gene. Increased luciferase activity was observed with this reporter plasmid upon PMA treatment, as compared with a reporter plasmid containing tax antisense sequences. Our data support a model whereby T cell activation leads to increased HTLV-1 gene expression through increased tax/rex mRNA stability, which in turn results in induction of Tax protein expression, and further activation of HTLV-1 gene expression, and associated changes in cellular gene expression, contributing to increased T cell survival and proliferation and disease.


The Human T-Lymphotropic Virus 1 (HTLV-1) is endemic to central Australia. No attempt has been made to control transmission among Indigenous Australians and no epidemiological studies are available that could direct any intervention. Here we report the epidemiological associations of HTLV-1 infection in a hospital cohort of socially disadvantaged Indigenous adults. Results of HTLV-1 serology were obtained from the Alice Springs Hospital (ASH) pathology database for Indigenous adults admitted 1st January 2000 to 31st December 2013. HTLV-1 proviral loads were determined for 109 asymptomatic adults. Among 1950 Indigenous adults screened, HTLV-1 western blots were positive for 621 (31.8%) and indeterminate for 51 patients (2.6%). Two of 17 (11.8%) children from western desert communities were HTLV-1 infected. Seropositivity rates increased with age and were highest for older men. In a multivariable model, increasing age, male gender, residence in the south or west and having positive syphilis serology were associated with HTLV-1 infection. Three seroconversions were recorded among 351 adults who were tested more than once during the study period. The annual incidence rate of HTLV-1 infection in this sub-group was 0.85 per 100 person-years. HTLV-1 proviral loads were <0.01 per 100 PBMC in 53 (48.6%), and exceeded 1% PBMC in 23 (19.7%) asymptomatic adults. In a hospital-based cohort of Indigenous Australians, we found childhood infections suggestive of mother-to-child transmission, document horizontal transmission and reveal an association with positive syphilis serology in a multivariable model. Cultural practices may have further amplified infection rates among older men. Multiple modes of transmission are therefore likely to contribute to high rates of HTLV-1c infection among the Indigenous population of central Australia. Most asymptomatic adults who were HTLV-1 infected appeared to have good control of HTLV-1 replication.


Hesperomyces virescens is a fungal ectoparasite  that infects adult ladybirds. Research has recently focused on this parasite due to the discovery of its prevalence on the globally invasive harlequin ladybird Harmonia axyridis and for its potential use in studies of co-evolution and pathogen spread. We collected adults from ten species of ladybirds in the Western Cape Province, South Africa, and screened for the presence of H. virescens. Infections with H. virescens were found in the samples of two species, H. axyridis and the native Cheilomenes propinqua. This marks the first record of H. virescens on H. axyridis from the African continent and the first record on Cheilomenes worldwide. Hesperomyces virescens Thaxt.  [H. virescens reportedly infects several ladybird species from around the world. It was described in 1891 by Thaxter [Chilocorus stigma  [as C. bivulnerus] from California, USA, and in 2002 it was found for the first time to parasitize the invasive harlequin ladybird, Harmonia axyridis in Ohio, USA [H. virescens-H. axyridis is currently widespread in the eastern United States and Western Europe, and has also been reported from the host’s native range (PR China) [H. axyridis has H. virescens captured the attention of scientists. Although known for over 100 years, this fungus has been historically overlooked despite the biological and ecological importance it may have for studies of co-evolution of host and parasite.eniales)  is an obeniales) . Althoug Thaxter  on ChiloR China) . Only sithalli are formed directly from ascospores . These logy see , but gerlogy see . The stithe host . Non-ranthe host , 22, 27.H. axyridis, which overwinters in large and dense aggregations, H. virescens is also socially transmitted. In the course of winter, the infection rates typically increase, suggesting that transmission through direct physical contact in overwintering aggregations is an important mechanism of spread of this fungus [H. axyridis can reach high levels, but can vary widely between locations and from season to season [In s fungus , 20, 22.s fungus , 22. Funo season , 19.Harmonia axyridis has received considerable attention because it is a striking example of a biological introduction that did not turn out as planned. Being a globally invasive alien species (IAS), its impacts are considered “immense, insidious, and usually irreversible” [Harmonia axyridis is highly competitive with native ladybird species, and its strong dispersal capacities allow rapid range expansion into new ecosystems, hence its reputation as a devastating invader [H. axyridis. Many regional reports have been published about parasites, parasitoids, and pathogens of H. axyridis both within the native and invaded range but much more could be revealed from a systematic approach [ersible” . Harmoni invader . Therefoapproach .H. virescens in the Western Cape Province, a region with the earliest records of established H. axyridis in South Africa dating from 2001 [In this study, we collected and screened adult individuals of ten species of ladybirds for the presence of rom 2001 .Both native and invasive ladybirds were collected from 2013 to 2015 by hand in Stellenbosch, South Africa  from rose bushes planted along vineyards, oak trees, and gardens. Only adult ladybirds were collected, and specimens that had visible ectoparasitic fungal infections were not discounted during collection.Thallus observations were made using a Leica M125 stereomicroscope, and photographs were taken using a Leica DFC320 camera and Leica Application Suite 4.0 software.H. virescens were identified based on morphology [Voucher specimens of rphology  and are rphology  from 8 trphology .H. axyridis collected in Stellenbosch were infected by H. virescens while none of the other alien species showed signs of infection . We also generated SSU and LSU rDNA sequences of H. virescens isolated from C. propinqua . All sequences match the already known sequences of H. virescens in GenBank with 96–100% similarity, which confirms our morphological identification of the fungus.We generated SSU, ITS, and LSU rDNA sequences of H. virescens on H. axyridis from the African continent, and the first record on C. propinqua worldwide. Hesperomyces virescens has a wide host range spanning 15 ladybird genera  and is reported from all continents except Antarctica and Australia  [Hesperomyces virescens has been found on H. axyridis in China, the USA, Belgium, the Netherlands, Germany, United Kingdom, Czech Republic [H. virescens were known in Africa, on Chilocorus bipustulatus  from Morocco [Exochomus laeviusculus Weise, 1909 from La Réunion [This report marks the first record of ollowing , given tipelago) , 26. HesRepublic , Croatia Morocco  and on EH. axyridis likely facilitates the transmission of H. virescens. This is a feature shared with Adalia bipunctata , C. bipustulatus, and C. propinqua . However, H. axyridis combines this life-style feature with a number of other characteristics that make it the most suitable host species for H. virescens. Harmonia axyridis is highly promiscuous and overwinters in dense aggregations, supporting many more inter-generational contacts between infected and uninfected ladybirds. These characteristics provide opportunities for transmission of spores and promote the fungus to grow in large densities on individual hosts [Harmonia axyridis has become the “main host” species at least in some geographic areas, such as Belgium, the Netherlands, and parts of the eastern United States, with locally very high parasite prevalences (Ceryngier and Twardowska  noted thvalences .H. virescens is spreading around the world, possibly following H. axyridis as its main host. Interestingly, we also found the fungus on a native species in South Africa, C. propinqua, which raises the question of whether a host shift event might have taken place from the native to the invasive host following an ecological opportunity or vice versa [This report supports the hypothesis that ce versa . These h


Current technologies have changed biology into a data-intensive field and significantly increased our understanding of signal transduction pathways in plants. However, global defense signaling networks in plants have not been established yet. Considering the apparent intricate nature of signaling mechanisms in plants (due to their sessile nature), studying the points at which different signaling pathways converge, rather than the branches, represents a good start to unravel global plant signaling networks. In this regard, growing evidence shows that the generation of reactive oxygen species (ROS) is one of the most common plant responses to different stresses, representing a point at which various signaling pathways come together. In this review, the complex nature of plant stress signaling networks will be discussed. An emphasis on different signaling players with a specific attention to ROS as the primary source of the signaling battery in plants will be presented. The interactions between ROS and other signaling components, e.g., calcium, redox homeostasis, membranes, G-proteins, MAPKs, plant hormones, and transcription factors will be assessed. A better understanding of the vital roles ROS are playing in plant signaling would help innovate new strategies to improve plant productivity under the circumstances of the increasing severity of environmental conditions and the high demand of food and energy worldwide. Plants are increasingly subjected to a variety of environmental stresses which diminish the productivity of various economically important crops. Every year, the world loses a huge amount of crop production through scarcity of water, extreme temperatures, high soil salinity, herbivore attack, and pathogen infection.The sessile nature of plants has resulted in the evolution of complicated protection mechanisms to survive different environmental challenges. One of the stress tolerance mechanisms is the ability to sense complex stress factors and respond appropriately. Activation of complex signaling pathways helps plants to achieve this. To better understand plant signaling pathways would enable us to modify plants to improve their adaptability. However, this requires reducing the complexity associated with signaling pathways. Focusing on the points at which different signaling pathways converge, rather than studying the branches of these pathways, would be helpful as a starting point. The rapid generation of reactive oxygen species (ROS) represents a common plant response to different biotic and abiotic stresses  and thusRecent genomic technologies, especially global gene expression tools, have not only produced new details about plant signaling pathways but also raised many historical questions including the followings: Is there a specific linear signaling pathway for each stress? If so, what about the observed cross-talk? Is there a big common signaling network from which many branches arise for specificity? If so, what about the different receptors? What do represent the points at which different branches of signaling pathways converge? If ROS are at the points of integrating signaling outputs from different signaling pathways, then what are the ROS receptors? What are the upstream and downstream signaling components of ROS? How do ROS set signaling specificity? What about the photosynthetic machinery that generates ROS; does it and its ROS and redox system represent a primary source of the plant signaling battery? As discussed throughout this review, most of these questions have been answered (or are being answered) while we will be in a better shape in providing more definite answers to the remaining ones in the near future.This review presents a discussion about these historical questions by considering the so complex nature of plant stress signaling networks. A special attention will be given on reviewing signaling players and events such as receptors/sensors, secondary messengers, specificity, cross-talk, redundancy, feedback regulations, alternative promoter usage, alternative splicing, nucleo-cytoplasmic trafficking, and epigenetics. Here, an attempt will also be made to indicate the kinds of studies required to fill in the gaps. A specific thought to photosynthetic activities and ROS as the primary source of the signaling battery in plants will be presented. As ROS production represents a common plant response to almost all environmental challenges, a special emphasis will be devoted here to ROS production, scavenging, damaging effects, signaling roles and how they work upstream and downstream of other signaling components, e.g., calcium, redox homeostasis, membranes, G-proteins, MAPKs, plant hormones  and transcription factors (TFs). We hope to present a holistic summary of various signaling components and concepts that are important for a plant biologist to take into consideration when analyzing signaling events involved in plant response to the ever changing environment. This understanding would help construct comprehensive signaling networks which in turn innovate new strategies to improve plant productivity under the increasing severity of environmental stress conditions and the high global demand for food and energy.Recently, our knowledge about signaling mechanisms in plants starting from stimulus sensing to final response has increased. It is obvious that there is a large number of components underlying signaling mechanisms, including a high degree of interconnectivity, many spatio-temporal levels, and a complicated tune of signal transduction pathways. For example, the changes at the expression of certain genes under a definite environmental condition are not necessarily translated into metabolic and structural changes where the interactions between various aspects, including post-transcriptional and post-translational modifications, compartmentalization, metabolite stability, substrate availability may lead to an unexpected response .Figure 1). Each sensor controls a branch of the signaling pathway activated by one aspect of the stress condition. For instance, temperature stress is well-known to change the physical state (fluidity) of membranes . Consequently, multiple signaling mechanisms may be activated by one stimulus/stress initiating pathways differ in time, space, and outputs. Using shared signaling intermediates, such as phytohormones, these pathways may interconnect or interact with one another producing an intertwined signaling network  that regulate signaling pathways. In the following sections, these latter aspects will be briefly discussed.The changes in gene expression do not represent end points for signaling pathways. There are many other aspects, including transcriptional, post-transcriptional and post-translational regulation, redundancy, alternative promoter usage, alternative splicing, protein trafficking, non-coding RNA, and epigenetic effects  are a class of small non-coding RNAs that are increasingly being recognized as key modulators of gene expression , 2015. math PCD; . The dowbidopsis . During ignaling .Epigenetics .Molecular oxygen, in its ground state, is relatively unreactive. Nevertheless, during normal metabolic activity, and as a result of various environmental stresses, Oradicals . Reactivgen 1O2;  or from cal OH∙; . The fre2O2 and singlet oxygen, themselves, can be quite toxic to cells although they are non-radicals. Accordingly, the term ROS has been introduced to describe collectively, not only free radicals but also other toxic non-radicals  at PSII and superoxide radicals 2 reduction (electron transfer) at PSI, superoxide radical formation takes place  and Oes place . Then threaction .Reaction (2)2 to water through the respiratory electron transport chain requires four electrons.On the other hand, mitochondria represent a main source for ROS generation in aerobic organisms. It was estimated that from 1 to 5% of the oxygen taken up by isolated mitochondria is used in ROS production . The comReaction (3)2 cannot accept the four electrons at once, but one at a time. As a result, during O2 reduction, stable ROS intermediates such as 2O2, and OH∙ are formed in a stepwise fashion as follow;But, as a consequence of spin restrictions, OReaction (4)2O2 is produced during the process of photorespiration via the action of the enzyme glycolate oxidase , catalases and peroxidases (which remove H2O2). The non-enzymatic mechanisms of ROS removal include antioxidant molecules, such as ascorbic acid, glutathione, carotenoids, and α-tocopherol , chlorophyll movement, suppression of photosynthesis, and photosystems and antenna modulators .2O2 can defuse through the biological membranes through aquaporins , an answer to this question could be revealed.To this end, it is quite evident that ROS operate as intracellular signaling molecules, but how they can set specific signaling duties is still controversial. This controversy arises from what seems to be a paradox between the reactive nature of ROS that renders them indiscriminate and the specificity that is required for signaling . In geneE. coli, the steady-state concentration of -11 M; 2O2. The instability of  limited .2O2H is actually a poor oxidant and reacts mildly with [Fe–S] (rate constant of 102–103 M-1 s-1), loosely bound metals and, very slowly, with glutathione and free cysteine (Cys)  . By contredoxins .∙ is the most highly toxic ROS. It has high indiscriminate reactivity, which limits its diffusion to sites of production  is an excited state molecule. The half-life time of 1O2 is very short (∼100 ns) and it can travel only a very short distance in cells .The perceived ROS signals work upstream as well as downstream from many other second messengers in addition to many feedback and feedforward regulations in an interwoven manner to establish specific responses to different developmental and environmental cues. Currently, a major gap exists in our understanding of how ROS induce large-scale and coordinated expression from many genes. In addition, the big challenge is to identify the upstream sensing and signaling events through which ROS are perceived and delivered to the ROS-induced TFs. Do ROS activate the expression of TFs directly or through another set of signaling intermediates? How could secondary messengers such as G proteins, MAPKs, CaPhotosynthesis represents the most peculiar feature that distinguishes plant and animal systems. In photosynthesis, through intersystem electron transport, the light energy captured by photosynthetic pigments is transformed into chemical energy which pumps reductants (NADPH) and ATP into the Calvin cycle (dark reactions) supplying carbon skeletons (sugars) for all major metabolic processes . In addi1O2 and OH∙; 2O2 from chloroplasts led to the induced expression of many genes coding for membrane-bound receptor proteins and signaling components  processes are under precise control. Information on the redox situation is generated and transferred by various redox components, including various ROS and different antioxidants, that are parts of a robust network that links metabolism with regulation and signaling. Under environmental challenges, the imbalance in the network is sensed, and transformed into redox signals that are transmitted in order to elicit specific responses at various levels of regulation and in different subcellular compartments . Thus, RAs redox homeostasis is greatly influenced by most, if not all, conditions that affect plant growth and development, the changes of intracellular redox determine various signaling events through their interaction with many other secondary messengers, such as protein kinases and phosphatases, phytohormones and calcium. Intensive current research might confirm that ROS-antioxidant interactions act as a metabolic interface between environmental changes and the concomitant signaling responses , 2012. F2O2 responds rapidly to this change, leading to the activation of small heat shock protein synthesis , diacylglycerol, and jasmonates. IP3 and diacylglycerol are secondary messengers that can activate protein kinase and induce Caassembly . In addifluidity . In turnMembrane-bound NADPH oxidases are a group of enzymes that catalyze the production of superoxide radicals  are found in almost all organisms from prokaryotes to humans . G proteignaling .Arabidopsis leaves to ozone, the first biphasic oxidative burst is greatly attenuated or completely absent in mutant plants lacking Gα protein or Gβ protein. This finding suggests that the ROS produced by ozone in the apoplastic fluid do not themselves enter cells to activate intracellular ROS-producing systems. Rather, the extracellular ROS activate the G protein either directly or indirectly , calmodulin-like (CML), calcium-dependent protein kinase (CDPK), and calcineurin B-like protein  gene, the content of CAM proteins and the expression of many antioxidant genes in maize are increased after treatment with ABA or H2O2. Furthermore, pre-treating plants with CAM inhibitors almost completely blocked the upregulation of many antioxidant enzymes  and Arabidopsis roots  attenuated the inducing effect of H2O2 on ERF6, suggesting that Ca2+ is playing a signaling role, which is downstream from ROS, in the induction of this TF by H2O2  is a small, water-, and lipid-soluble free radical gas with well-characterized signaling roles in mammalian systems . Nitric 2O2 in response to pathogen infection was found to mediate defense responses similar to those seen following H2O2 production /sucrose non-fermenting 1 (SNF1)-related protein kinase 2.6 -activated protein kinases (MAPKs) are evolutionary conserved enzymes. In eukaryotes, signaling pathways arbitrated by MAPKs have been considered as a general signal transduction mechanism that links different receptors to their cellular and nuclear targets . The sigArabidopsis genome, 20 MAPK, 10 MAPKK, and 60 MAPKKK encoding genes were identified  was reported to connect protein phosphorylation, Ca2+, and ROS in wound signaling pathways  or in an ABA mutant plants, indicating that ABA is required for H2O2 production in these compartments , such as Botrytis cinerea. On the other hand, the SA signaling pathway is supposed to mediate resistance to biotrophic pathogens (feed on living tissues), such as P. syringae  is well-known to regulate both systemic acquired resistance (SAR) and local disease resistance mechanisms, including host cell death and defense gene expression  mutant (pression . It was pression . One of ed genes . The extbidopsis . It was rd cells . It was ) mutant .Jasmonic acid (JA) and methyl jasmonate (MeJA), are natural products regulating plant development, response to environmental challenges, and gene expression (Arabidopsis inhibited O3-induced H2O2 production and SA accumulation and completely abolished O3 induced cell death (RbohD and RbohF mutant plants with MeJA failed to increase the expression levels of various MYC2 downstream genes (cat2 mutant Arabidopsis plants leads to activating the JA pathway and its related genes with glutathione accumulation as an intermediate (pression . A signapression . Currentll death . It was am genes . A dynamam genes . It was rmediate .2H4)Ethylene  enhanced an oxidative burst which was inhibited in ethylene-insensitive mutants, etr1 and ein2, indicating a requirement of ethylene signaling for ROS production  upregulation of TF expression or increasing both mRNA stability and translation; (2) increasing the stability of the TF by decreasing its association with the protein degrading ubiquitin E3 ligase complex or by inhibiting this complex; (3) nucleo-cytoplasmic traffic by transferring or masking nuclear localization signals, or by releasing the TF from partners or from membrane anchors; and (4) DNA binding and nuclear transactivation by adapting TF affinity toward DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes  and TFs. The recent research implies an early and vital role for photosynthesis in sensing various environmental insults, not only abiotic, but also biotic ones, a concept that needs to be taken into consideration when studying stress signaling pathways in plants. Despite all of these achievements, great efforts are still required to be able to reconstruct larger signaling networks that may include ROS at the convergent points. In this regard, bioinformatics and systems biology approaches are nominated to greatly help in constructing global signaling networks. As a result, these global networks would improve our understanding of plant biology and assist us to develop new strategies for higher plant productivity in the face of increasingly severe environmental conditions and the high demand for food, fiber, and energy crops.All authors wrote and approved the final version of the manuscript.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.


In the early 1990's several groups were searching for the molecular basis of the signal transduction triggered by the engagement of plasma membrane receptors. The laboratories of Darnell, Kerr and Stark were first to identify proteins, which acted as intermediaries in interferon (IFN) signaling, known as the signal transducer and activator of transcription (STAT). At the same time, the work of Pellegrini's, Ihle's and Carter-Su's groups demonstrated that non-receptor tyrosine kinases, previously described by John Krolewski and Andrew Wilks, called Janus Kinases (JAK), played a role in cytokine receptor signaling. We have since learned that multiple negative regulators, mainly tyrosine phosphatase (SHP), Protein Inhibitors Against Stats (PIAS), and Suppressor Of Cytokine Signaling (SOCS) proteins, modulate and eventually extinguish JAK-STAT signaling [In invertebrates a single JAK-STAT module controls anti-viral and anti-bacterial responses, leukocyte-like hemocyte generation, cell fate determination, brain development, cardiogenesis, as well as intestinal stem cells. The increase of JAK-STAT pathway components has coincided with the emergence of adaptive immunity and the expansion and diversification of cytokine receptors. As ligands bind to cognate receptors, they trigger the recruitment and activation of JAKs. Activated JAKs can then phosphorylate the receptor favoring the STAT docking and ultimately their activation via tyrosine phosphorylation. Phospho-STAT dimers accumulate in the cell nucleus, bind to enhancer elements and regulate gene expression. In parallel, JAKs may fire other downstream signaling cascades (MAP kinase and PI-3-kinase/AKT pathways), or within the nucleus by phosphorylating DNA regulatory proteins (histone H3 and methyltransferase) modulate gene expression and the epigenetic program of cells .There is comprehensive evidence that abnormal JAK/STAT signals can lead to immunodeficiencies, a spectrum of cytokine mediated inflammatory diseases and cancer. Hyperactivation of STAT signaling is common in hematopoietic disorders through several different mechanisms -3. JAK2 Searching for genomic defects responsible for the transformation and the maintenance of the neoplastic phenotype of ALK-ALCL, our groups have used massive genomic sequencing. These studies demonstrated the presence of recurrent activating mutations of JAK1 and/or STAT3 and novel tyrosine kinase fusions. JAK1 and STAT3 mutants result in a hyperactivated STAT3, which sustains cell transformation, and whose pharmacological ablation produces tumor cell growth inhibition. Interestingly, we found that ~30% of systemic pSTAT3 positive ALK-ALCL carry both JAK1 and STAT3 mutations that work synergistically . FurtherAlthough JAK1/STAT3 mutants are oncogenic, they require lymphokine engagement  and JAK activation, whose inhibition  leads to STAT3 dephosphorylation, cell growth inhibition and lymphoma control in a Patient Derived Tumor Xenografted (PDTX) model. In addition, STAT3 hyperactivation forces the preferential differentiation of T-cells and the secretion of selective lymphokines . Therefore, the inflammatory phenotype associated with ALCL hijacks host elements and favors protumorigenic phenotypes  and/or the emergency recruitment of immunosuppressive T-cells.These data validate JAK and STAT as therapeutic targets. Inhibitors of JAKs were recently introduced and it is anticipated that these new therapies, either alone or in combination, might be beneficial -8. We pr


Connexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown.+/− aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37−/− and Cx40−/− aortas but not in Cx40+/− aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40−/− and Cx40+/− aortas but not in Cx37−/− aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype.Cx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40Cx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40.The online version of this article (doi:10.1186/s12964-015-0110-1) contains supplementary material, which is available to authorized users. Theyesponses . Under sce versa .in vitro [It is now increasingly recognized that connexins do not only function as connexons or gap junction channels; they can also regulate the function of other proteins via protein-protein interactions –7. It hain vitro . Howeverin vitro . Howeverin vitro . It is ten face preparations of WT, Cx37−/− and Cx40−/− aortas. These experiments showed that WT mouse aortic endothelial cells expressed Cx37 and Cx40 at cell-cell interfaces whereas Cx43 was barely detectable  or Gja5 (the gene coding for mouse Cx40) was quantified by real-time PCR performed on total aortic mRNA. As expected, mRNA coding for Cx37 was detectable in WT aortas but not in Cx37−/− aortas  and was comparable to WT levels in Cx40+/− aortas  and was significantly reduced at the level of protein . However, the expression of Cx37 in Cx40+/− aortic endothelial cells was not significantly different from its expression in WT aortic endothelium -induced contractions in aortas isolated from WT, Cx37−/−, Cx40−/− and Cx40+/− mice using wire-myography. Initially, PHE-induced in absence of pharmacological inhibitors were studied. These experiments showed that the amplitude of the PHE-induced contractions was significantly enhanced in Cx37−/− and Cx40−/− aortas as compared to WT aortas  would increase the contractile amplitude. Indeed, the amplitude of PHE-induced contractions was significantly increased in aortas isolated from WT mice in presence of L-NAME . However, Cx37 seems to be necessary for a direct regulation of eNOS function since basal NO release in Cx37−/− aortas, which have normal Cx40 expression levels, was reduced. Exactly how Cx37 regulates the basal release of NO is currently unclear. One possibility would be via direct effects on eNOS expression levels. We therefore studied eNOS expression by western blotting of mouse aortic endothelial cell lysates collected from WT, Cx37−/−, Cx40−/− or Cx40+/− mice. In line with previous findings [−/− endothelium, but expression levels were similar in Cx37−/−, Cx40+/− and WT mice  expressed at cell-cell interfaces and ii) in a close proximity to eNOS. Thus, they suggest that endothelial Cx37 expression may be dependent on proper Cx40 expression. Moreover, these findings imply that Cx37 and/or Cx40 could regulate eNOS-dependent NO production tas Fig. . The amp− aortas , the PHEtas Fig. . InteresAME Fig. , Table 1−/− Fig. , Table 1ice Fig. , Table 1els Fig. , Table 1findings , 18, eNOice Fig. . NO prodiopterin . Theoret−/−, Cx40−/− or Cx40+/− mice were studied. In WT aortas ACh induced relaxations that reached maximal amplitudes at an ACh concentration of 300 nM  but was observed in Cx40−/− aortas (which express low levels of Cx37) and in Cx40+/− aortas (which express Cx37 at WT levels). Furthermore, changes in the expression of Cx37 are most likely also responsible for the changes in the sensitivity for ACh that were observed since these changes were fully normalized in Cx40+/− aortas. However, half of Cx40 expression may also be sufficient for normal endothelial function. Surprisingly, ACh-induced NO-mediated dilatation was not affected by Cx40  in contrast to earlier work from our group [vs non-cumulative CRCs, different timing of agonist application).Basal and activated NO release do not necessarily depend on similar mechanisms. Findings regarding basal NO release can therefore not always be extrapolated to activated NO release. For that reason, CRCs to acetylcholine (ACh) during PHE-induced pre-contractions in aortas isolated from WT, Cx37 nM Fig. , Table 2 nM Fig. , Table 2as Table , Table 2tas Fig. . CRCs fotas Fig. , Table 2tas Fig. , Table 2ur group . The rea−/− and Cx40+/− but not Cx37−/− aortas. Interestingly, it has been suggested that gap junctional communication between endothelial cells or, alternatively, between endothelium and smooth muscle cells could be responsible for the contractile effects of ACh in the aorta of spontaneously-hypertensive rats [+/− and Cx40−/− aortas but not in Cx37−/− aortas, we conclude that Cx37 is necessary to allow the effect of INDO.Various studies have described endothelium-dependent contractile effects of high ACh concentrations –23. Indeive rats . Howeverive rats . As effe−/− and Cx40−/− aortas may be due to i) a changed sensitivity of the aortic endothelial cells for ACh, ii) an altered eNOS expression level (in the Cx40−/− but not the Cx37−/− aortas) or iii) a changed sensitivity of the vascular smooth muscle cells for NO. The latter possibility was tested by generating CRCs to an NO donor ) during PHE-induced precontractions  in WT, Cx37−/−, Cx40−/− or Cx40+/− aortas. These experiments showed that aortas isolated from WT, Cx37−/−, Cx40−/− or Cx40+/− mice were equally sensitive to NO ) but likely reflected endothelial changes. Indeed, involvement of Cx40 (and most likely Cx37) in the endothelial Ca2+ agonist responses, which govern NO release, are agonist specific [I.e. ATP-induced increases in intracellular Ca2+ in mouse endothelium are homogeneous and do not require synchronization by connexins [2+ elevations mediated by ACh are heterogenous and require synchronization by connexins to reach other cells [2+ concentration is unaffected by genotype then a decreased eNOS sensitivity for Ca2+ could explain both the effect observed regarding basal NO release (decreased NO released at fixed Ca2+ concentration) and the apparent effect on sensitivity of ACh . Obviously, this will be the topic of further studies.The altered sensitivity for ACh that was observed in Cx37specific . I.e. ATonnexins . In conter cells . This ober cells )) but do−/− aortas’. Clearly, these questions warrant a specific exploration in further studies.In summary, the current study was conducted to address whether Cx37 or Cx40 is more pronouncedly involved in the regulation of vasomotor function. The data indicate that Cx37 is more markedly involved in basal NO release, release of COX-products and the regulation of the sensitivity for ACh as compared to Cx40. These findings are exciting since they, for the first time, allow for a discrimination of the functional role of Cx37 and Cx40 in mouse aortic endothelium. However, various questions remain e.g. ‘How does Cx37 modulate basal NO release without modulating eNOS expression levels?’, and importantly, ‘Why is the sensitivity for ACh altered in Cx37n = 10), Cx37−/− (n = 10), Cx40−/− (n = 8) and Cx40+/− (n = 8) mice were progeny of the original colony on a C57BL/6 J background and were housed in a 12-hours day-night cycle [The Swiss Federal Veterinary Office approved animal housing and animal experiments. Wild-type  to the repeated sequences of the amplification product. Under a fluorescence microscope, the result was detected as red spots, which were localized by staining the EC nuclei with DAPI. For the proximity ligation assays performed with antibodies targeting Cx40 and Cx37 in the en face aortic strips, a monoclonal antibody specific to mouse Cx40 was used (Invitrogen).Immunostainings and escribed , 11. In RNA isolation, DNAse treatment and reverse transcriptase reactions were performed on whole mouse aortas as previously described . Equal lThe expression of Cx37 and Cx40 was determined as previously described . In brie2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25.0 mM NaHCO3 and 5.5 mM glucose. High K+-KRB solution was prepared by replacing NaCl with KCl. Buffers containing intermediate K+ concentrations were prepared by mixing volumes of KRB and K+-KRB. Acetylcholine (ACh), phenylephrine (PHE), Nω-nitro-l-arginine methyl ester (L-NAME), sodium-nitroprusside (SNP) and indomethacin (INDO) were obtained from Sigma-Aldrich (Germany). All compounds were dissolved in KRB-buffer with the exception of INDO, which was dissolved in ethanol.Krebs-Ringer bicarbonate-buffered physiological salt buffer (KRB-buffer) contained 118.5 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl+ (Kmax) were obtained (Table Mice were sedated (ketamine (100 mg/kg) + xylazine (100 mg/kg), i.p. injection). Subsequently, the left ventricle was punctured with a 23 G needle. Then, the animals were perfused during 3 min under constant flow (flow rate 3.33 mL/min) with a total volume of 10 mL KRB-buffer (pre-heated at 37 °C). After perfusion, aortic segments were isolated and mounted in a multi-myograph . After mounting, the segments were stretched to the diameter at which maximal contractile responses to 40 mM Ked Table . Next, tP-values < 0.05 were considered statistically significant.Results are shown as mean ± SEM. One or two-way ANOVAs followed by Bonferroni’s multiple comparisons tests were performed using Graphpad Prism 6.0 to compare mean values between the groups.