The acquisition of plastids is a landmark event in plant evolution. The proper functionality of these organelles depends on strict and continuous communication between the plastids and the nucleus to precisely adjust gene expression in response to the organelle’s requirements. Signals originating from the plastids impact the expression of a variety of nuclear genes, and this retrograde communication is essential to couple the nuclear expression of plastid-localized products with organelle gene expression and, ultimately, functionality. Major advances have been made in this field over the past few years with the characterization of independent retrograde signaling pathways and the identification of some of their components. One such factor is the nuclear transcriptional factor ABI4 (ABA-INSENTIVE 4). ABI4, together with the plastid PPR GUN1 protein, has been proposed to function as a node of convergence for multiple plastid retrograde signaling pathways. ABI4 is conserved among plants and also plays important roles in various critical developmental and metabolic processes. ABI4 is a versatile regulator that positively and negatively modulates the expression of many genes, including other transcriptional factors. However, its mode of action during plastid retrograde signaling is not fully understood. In this review, we describe the current evidence that supports the participation of ABI4 in different retrograde communication pathways. ABI4 is regulated at the transcriptional and post-transcriptional level. A known regulator of ABI4 includes the PTM transcription factor, which moves from the chloroplast to the nucleus. This transcription factor is a candidate for the transmission of retrograde signals between the plastid and ABI4. The endosymbiotic acquisition of the chloroplast and mitochondria by eukaryotic cells are two of the most important events in the history of life on Earth . ChloropThe plastid genome encodes around 100 genes. Therefore, more than 95% of the proteins required for plastid function are encoded in the nucleus, and the corresponding proteins are imported into the organelle post-translationally. This latter step is achieved through a transit peptide present at the N-terminus that suffices for import into the organelles, found in the majority of nuclear-encoded plastid proteins NEPPs; . HoweverIn silico predictions based on the presence of putative transit peptides yield estimates of 2500–4500 NEPPs (http://ppdb.tc.cornell.edu/) corroborate the complexity of the plastid proteome  mutant more than 30 years ago , which no longer represses NEPPs in the presence of norflurazon, an inhibitor of carotenoid biosynthesis (Cis-acting elements involved in abscisic acid (ABA) responses that are over-represented in the upstream regions of the gun-affected genes and known putative regulatory factors that may interact with these sequences have been identified . The isolation of ABI4 in a screen for ABA-insensitive (abi) mutants was the first evidence linking this factor with ABA signaling . Additional abi4 alleles were isolated in several screenings for mutants with altered responses to sugars . ABI4 and hormones such as ABA and ethylene play an essential role in sugar perception during early seedling development .Over the past decade, ABI4 has emerged as a central player in many signaling processes during plant development  in response to defects in electron transport chain inhibition by rotenone has been commonly used as a marker for mitochondrial retrograde signaling responses  and in mitochondrial retrograde communication. The induction of the nuclear-encoded alternative oxidase gene . PGE appears to be important in coordinating the NEPPs required for different plastid types in early development . Collectively, all these findings support the hypothesis that plastid gene expression mediated by PEP acts as a central integrator to initiate PGE retrograde signaling in response to developmental and metabolic cues.Independent analyses have reported the participation of ABI4 in the PGE and tetrapyrrole retrograde signaling pathways . Accordingly, overexpression of ABI4 suppresses the gun phenotype in the presence of lincomycin .One factor required in PGE signaling is GUN1, a plastid protein and member of the pentatricopeptide repeat (PPR) family. The henotype . Interesncomycin . Howeveranalysis , suggestgun mutants that no longer repressed NEPPs in the presence of norflurazon (gun mutants affected different steps in porphyrin biosynthesis (gun mutant (gun6) showed that excess accumulation of heme acts as an activator of the expression of photosynthetic genes. The role of heme as a signal that positively regulates NEPPs is particularly attractive not only because this molecule is known to be actively transported out of the plastids . However as previously mentioned, the fact that the expression level of genes such as LHCB in the gun1 and abi4 mutants never reaches that observed in control plants without treatment also suggests that additional factors or a more complex mechanism participate in NEPP repression caused by the disruption of chloroplast development.GUN1 is an essential factor in this signaling pathway and has been proposed to function as a node of convergence in the tetrapyrrole and PGE retrograde signaling pathways . The funynthesis . Accordipression . These r tRNAGlu . Several1 mutant . Finallyreatment , supportChanges in chloroplast function in response to high light or environmental fluctuations trigger different retrograde signaling responses classified as operational control pathways . These p1O2), leading to a rapid inhibition of plant growth, the induction of plant cell death and the differential regulation of specific sets of nuclear genes  and non-radical molecules .A second class of ROS that has been shown to elicit specific gene expression responses is hydrogen peroxide H2O2; . Various induced . Activat induced . Mutatiopathways . A key qprin2 mutant disrupt positive signals or induce negative signals that decrease the expression of NEPPs  pool and thioredoxin proteins permit long-term adjustments that result in changes in the accumulation of photosynthetic components such as the NEPPs . Thioredof NEPPs . Collectabi4 mutant . In several repressed genes from different species such as LHCB and RBCS . It has been proposed that ABI4 competition for binding with activators such as G-box binding factors (GBFs) results in ABI4-mediated reduction of gene expression , the ABI4 transcript accumulates again in most of the germinating seedling, and its expression continues through the first days of the seedling emergence. In these stages, ABI4 is expressed in the cotyledons, the hypocotyls, and the root tips, but it is barely detectable in the true leaves . In the ptm mutant, treatments such as lincomycin and norflurazon do not repress LHCB expression. Interestingly, the function of PTM during ABI4 expression appears to be related to histone modification in the ABI4 promoter, a regulatory mechanism that still has not been analyzed. This finding directly links plastid retrograde signals with the expression of ABI4, which in turn impacts on NEPPs. To be more confident in this conclusion, the endopeptidase responsible for PTM activation must be identified and the conditions that regulate its activity defined. It is also important to demonstrate whether ABI4 induction mediated by PTM or other regulators alters active ABI4 protein levels because nuclear gene expression depends on the presence of an active protein.A central question is whether it is possible that any of these regulators can transmit retrograde signals. One of the most interesting findings that potentially links ABI4 and retrograde chloroplast signaling is the recent identification of the transcriptional regulator PTM, which directly activates pression . PTM is pression . Sun et abi and gin phenotypes. These phenotypes apparently are the result of displacement of ABI4 by high ASR1 levels because the binding sites of both factors overlap in some target genes  DNA-binding domain  are less sensitive to norflurazon or lincomycin treatment than wild-type plants, resulting in a weak GUN phenotype similar to that observed in ABI4 mutant .Although ABI4 has emerged as an important integrator of various retrograde signals, there are additional factors regulating these pathways. For example, in the reatment . Some fareatment . These preatment  and direreatment . The GLKreatment . These p pathway , because4 mutant . This re4 mutant . In respTranscription factors that exhibit dual subcellular localization are also good candidates for components of retrograde signaling mechanisms. In addition to PTM, additional factors that exhibit dual localization have been described . One exaABI4 has emerged as a central integrator of essential environmental signals such as light, carbon status, ABA, redox and organelle status, facilitating the coordination of development and central metabolic processes such as photosynthesis. Although our present understanding of the mode of action of this central regulator has advanced, there are still major questions that need to be addressed in the near future, including the impact that the transcriptional regulation of ABI4 has on the levels of its protein. An aspect which is essential to understanding the function of ABI4 as a regulatory node of different signaling pathways, including plastid retrograde signaling, is how it is regulated post-transcriptionally in addition to identifying possible interaction partners. It is still an open question whether different signals can modulate the stability or activity of ABI4 either by protein modification or through direct interaction.After a period of research that was primarily dominated by the description of independent retrograde signaling pathways, recent findings provide compelling evidence that many of those pathways converge into common integrators. Our understanding of retrograde signaling regulation is evolving into a more dynamic process in which multiple signals are produced simultaneously. These signals can affect the expression of nuclear genes in opposing ways through the participation of convergent transcriptional factors such as ABI4. This could be a very powerful strategy to respond to subtle changes in plastid functionality. The current challenges include understanding the molecular mechanism that generates signals during plastid transcription and the molecular nature of these signals. Identification of the direct targets of GUN1 could provide important insights. Although GUN1 is the only PPR protein that has been found to be required in retrograde signaling to date, the function of other related PPRs should be evaluated.gun1 and abi4 mutants, NEPPs repression is not completely lost. This result supports the evidence of the existence of an independent gun1 and abi4 retrograde signaling pathway that modulates NEPPs expression that requires future investigation.Finally, an aspect that has to be taken into consideration is that even in the 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.We would like to thank Dr. Virginia Walbot and Dr. Mari Salmi for their helpful comments and suggestions. This work was supported by Consejo Nacional de Ciencia y Tecnología (127546) and Dirección General de Asuntos para el Personal Académico-UNAM (IN208211-3) grants.


The human retroviruses HIV-1 and HTLV-1/HTLV-2 share similar routes of transmission but cause significantly different diseases. In this review we have outlined the immune mediated mechanisms by which HTLVs affect HIV-1 disease in co-infected hosts. During co-infection with HIV-1, HTLV-2 modulates the cellular microenvironment favoring its own viability and inhibiting HIV-1 progression. This is achieved when the HTLV-2 proviral load is higher than that of HIV-1, and thanks to the ability of HTLV-2 to: (i) up-regulate viral suppressive CCL3L1 chemokine expression; (ii) overcome HIV-1 capacity to activate the JAK/STAT pathway; (iii) reduce the activation of T and NK cells; (iv) modulate the host miRNA profiles. These alterations of immune functions have been mainly attributed to the effects of the HTLV-2 regulatory protein Tax and suggest that HTLV-2 exerts a protective role against HIV-1 infection. Contrary to HIV-1/HTLV-2, the effect of HIV-1/HTLV-1 co-infection on immunological and pathological conditions is still controversial. There is evidence that indicates a worsening of HIV-1 infection, while other evidence does not show clinically relevant effects in HIV-positive people. Possible differences on innate immune mechanisms and a particularly impact on NK cells are becoming evident. The differences between the two HIV-1/HTLV-1 and HIV-1/HTLV-2 co-infections are highlighted and further discussed. Microbes that infect the same host may positively influence each other’s replication, or fight for supremacy . In receSince HTLV-1, HTLV-2, and HIV-1 have common modes of transmission, it is not surprising that co-infection is a frequent condition especially among people with high risk behaviors, as needle sharing and unprotected sexual contact. Though human retroviruses have worldwide distribution , dually + and CD8+ T cells. However, the finality of their viral cycle is different. In infected patients, HIV-1 is generally present as a virion (either cell-associated or cell-free) and the provirus is detected clearly in a minority of cells  clones derived from patients with HAM/TSP are actively producing CCL3 and CCL4 chemokines /signal transducer/activator of transcription (STAT) signaling pathway (JAK/STAT) is constitutively activated in HTLV-1-transformed cells. This may occur by autocrine stimulation of IL-2, IL-9, and IL-15 cytokines, and IL-2 and IL-15 receptor expression, as a result of Tax-induced NF-κB expression, which in turn stimulate lymphocyte proliferation  cells, by chronically replicating viruses . Success+ NK cell subsets as is the case for CMV infection  lines (+ CD16+) is decreased in patients with TSP/HAM (Early reports on the definition of NK cell receptors point towards a decreased NK cell activity against HTLV-1-infected MT-2 cell lines . Lysis oL) lines . HTLV-1 L) lines  and in hL) lines . The pro TSP/HAM  and othe TSP/HAM .+ and CD8+ T cell function. In particular, it is possible that different NK cell regulation induces clinical divergence spanning from the lack of symptoms to TSP/HAM and transformation to ATL, similar to what was observed for HCV infected patients clearing infection  that are exploited by HTLV-1 to divert innate immune responses and downstream CD4nfection  or for Hnfection . In addinfection  and in snfection  though bnfection , understMicroRNAs (miRNAs) are small single-strand non-coding RNAs that repress gene expression by inhibiting translation and inducing mRNA degradation . MicroRNMicroRNAs expression was studied during of HTLV-1 or HIV-1 infection but no data were so far reported for the HIV-1/HTLV-1 co-infection. Similarly, studies of miRNAs pattern during HTLV-2/HIV-1 co-infection have not been published up to date.+ purified cells from naive and LTNP HIV-1-mono-infected patients. The hypothesis that miRNAs either could directly influence viral RNA sequences, or could affect cellular factors involved in HIV replication were confirmed by several findings , and the tumor suppressor p53  protein, a key factor to viral transcription, in the presence of the HATs CREB binding protein (CBP), p300, and P/CAF to activate HTLV-1 gene expression while HTLV-2 Tax specifically cooperates with CBP and p300 but not with p300 associated factor to enhance transcription from the viral promoter . Anotherssor p53 . In partssor p53 . Becausessor p53 . One spessor p53 . Similarssor p53  and hemossor p53 . Of partssor p53 . In HIV-ssor p53 . Also, issor p53 . The chrssor p53 .+ T-cells purified from HTLV-2-mono-infected patients and found evidence for a miRNA signature  that discriminates infected from uninfected subjects, similarly to HIV-1 . Interestingly, we found that the most significant GO terms, i.e., positive regulation of macromolecule and cellular biosynthetic process, are related to the formation of substances carried out by individual cells.Recently, we studied miRNA profiles in CD4Table 1; . FurtherTable 2), only miR-155, a key regulatory component of the innate immune response, is differentially expressed in HIV-1 and HTLV-2 infection. Thus, we speculated that a functional impact of cross-talk between miRNA pattern, and the subsequent multifunctional pathways, may occur during HTLVs/HIV-1 co-infection.With specific reference to the deregulated host miRNAs linked to the development of the HTLV-1 oncogenic or neurodegenerative diseases (The assessment of how the altered profiles of miRNA expression can influence viral replication and latency, as well as the efficiency of host defenses, may be useful for understanding the basis of the retroviral related modifications of cellular pathobiology and immunologic control.Is the retroviral interference relevant to HIV-1 infection? In this review we discussed how HIV-1/HTLVs co-infection can either positively or negatively affect the course of HIV-1 disease. In particular, co-infection with HTLV-2 seems to confer immunological benefits in patients with HIV-1. By contrast, HTLV-1 is mainly associated to HIV-1 disease progression and to an increased risk of TSP/HAM and ATL.+ or CD8+ T lymphocytes (An overall picture of different effects of HTLVs on HIV infection is emerging. The widely diverging effect of HTLV-1 and HTLV-2 on the clinical course of HIV-1 progression is remarkable, and the findings initially reported for HTLV-2 are particularly surprising. The possibility that specific co-infections may improve the clinical course of HIV-1 infection by directly or indirectly interfering with HIV-1 cell entry, replication and spread, originally proposed by the study of HIV-1/HTLV-2-infected cells  has beenphocytes .+ T cells can give a boost to HIV-1 replication.Modulation of the cytokine/chemokine network represents a major element of shift dynamics that regulates the co-existence of several infections. Thus, cytokines and chemokines might be considered strategic weapons in the bid to gain benefits to the infecting agents. Since a poor Th1 response and a dominant Th2 response have been implicated in the pathogenesis and progression of HIV infection , 1994, HAn enhanced secretion of CC-chemokines, in particular of CCL3L1, was ascribed to the transactivating function of Tax-2 and the original studies of HTLV-2/HIV-1 co-infection proposed this as a key mechanism of retroviral interference. The CCL3L1 isoform down-regulates CCR5 co-receptor for HIV-1 entry leading to a LTNP status in co-infected individuals with high HTLV-2 proviral load .In the same manner, GBV-C acquisition via blood transfusion increases the secretion of CCL5, CCL3, CCL4, and CXCL12 and by means of their NS5A and E2 proteins support the deregulation of co-receptors, thus inhibiting HIV entry and resulting in a reduced mortality in patients with advanced HIV-1 disease . SimilarAnalysis of JAK/STAT regulation during HTLV-2 infection provides some clues of intervention to interfere with HIV-1 replication by taking advantage of pathway interference instead of enzymatic inhibition of viral enzymes. The lack of knowledge of HTLV-1 mediated activation pathway interference has probably limited efforts in this direction.Concerning innate immune responses in HIV-1 co-infection with either HTLV-1 or HTLV-2, no data are so far available for NK cell function, and little is known on other innate immune cellular mechanisms. The observed effect of proviral load, but not of Tax-1, on NK cell proliferation during HTLV-1 infection , is likeSince miRNAs have been correlated with viral life cycle, they represent good candidates among the top cellular factors to be used by HTLVs to favor their own replication. In the case of co-infection with HIV-1, HTLV proteins were found to interact with cellular chromatin modifying enzymes and with cellular transcription and other immune factors. The interaction of HTLV Tax proteins with cellular factors results in the alteration of miRNAs profile that in turns can activate transcription and consequently viral replication. Furthermore a likely interplay between two competing mechanisms is taking place: the ability of Tax to manipulate chromatin structure and the innate host cellular defense mechanism of RNAi to regulate pathogen gene expression.mac251 was not accompanied by an exacerbation of SIVmac251 infection (HTLV/HIV-1 co-infection can be considered as a useful model for the study of new strategic approaches for HIV-1 vaccine development, as suggested by the finding that the exposure of HTLV-2 infected macaques to SIVnfection .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.


We further showed the specificity of β2AR in this crosstalk as treatment with formoterol, a β2AR-selective agonist, but not dobutamine, a predominantly β1AR agonist, activated cardiac Akt and ERK1/2. Acute βAR stimulation also significantly increased the phosphorylation of mTOR , P70S6K, ribosomal protein S6, GSK-3α/β (glycogen synthase kinase-3α/β), and FOXO1/3a (the forkhead box family of transcription factors 1 and 3a). Moreover, acute βAR stimulation time-dependently decreased the mRNA levels of the muscle-specific E3 ligases atrogin-1 and muscle ring finger protein-1 (MuRF1) in mouse heart. Our results indicate that acute βAR stimulation in vivo affects multiple cardiac signaling cascades, including the PI3K signaling pathway, ERK1/2, atrogin-1 and MuRF1. These data 1) provide convincing evidence for the crosstalk between βAR and PI3K signaling pathways; 2) confirm the β2AR specificity in this crosstalk in vivo; and 3) identify novel signaling factors involved in cardiac hypertrophy and LV unloading. Understanding of the intricate interplay between β2AR activation and these signaling cascades should provide critical clues to the pathogenesis of cardiac hypertrophy and enable identification of targets for early clinical interaction of cardiac lesions.Sustained β-adrenergic receptors (βAR) activation leads to cardiac hypertrophy and prevents left ventricular (LV) atrophy during LV unloading. The immediate signaling pathways downstream from βAR stimulation, however, have not been well investigated. The current study was to examine the early cardiac signaling mechanism(s) following βAR stimulation. In adult C57BL/6 mice, acute βAR stimulation induced significant increases in PI3K activity and activation of Akt and ERK1/2 in the heart, but not in lungs or livers. In contrast, the same treatment did not elicit these changes in β Moreover, the early signaling events following βAR stimulation preceding the onset of cardiac hypertrophy has not been demonstrated. A more detailed picture of these early signaling events would enable identification of targets for early clinical interaction for the βAR-related cardiac lesions.βAR stimulation has been shown to affect insulin signaling via PI3K/Akt in cardiomyocytes 1/β2AR double knockout (β1/β2 DKO) mice were employed. We demonstrated that multiple signaling molecules downstream of PI3K are affected following acute βAR stimulation, including Akt, P70S6/mTOR/S6 axis, GSK3α/β and FOXOs. More important, two novel players, atrigin-1 and MuRF1, were shown to be part of the early signaling cascade.To elucidate the cardiac signaling mechanisms responsible for the crosstalk between βAR and PI3K signaling pathway after acute βAR agonist administration, wild type adult male C57BL/6 and βThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Lifespan . All efforts were made to minimize suffering.1AR agonist 2AR-selective agonist , was obtained from Tocris Bioscience, Ellisville, MO. LY294002, a potent inhibitor of PI3K was purchased from Santa Cruz Biotechnology, inc. (sc-201426). All other chemicals and reagents were obtained from Sigma unless stated otherwise.Isoproterenol (ISO), a nonspecific βAR agonist, and dobutamine, a predominantly β1/β2 DKO mice  were used. Animals were fed standard laboratory chow ad libitum. Mice were injected intraperitoneally (i.p.) with ISO (1.25 mg/kg), dobutamine , formoterol  or saline for 30 min (unless otherwise specified) before euthanized as previously described Three months old male C57BL/6 wild-type mice  and βin vitro immunoprecipitation lipid kinase assay as described previously 32P ATP , the final extracted reaction mixtures were spotted onto silica gel-coated TLC plates  and run in TLC buffer . The results were analyzed by phosphorimaging (Bio-Rad Laboratories).Left ventricular lysates were prepared as described, and protein concentrations were determined with the bicinchoninic acid assay Protein expression was evaluated by Western blotting as previously described -ΔΔCt). Each sample was analyzed in triplicate.Total RNA was extracted from ventricular tissue using TRIzol reagent , and further purified using RNeasy MinElute Cleanup Kit . RNA concentration and purity were determined by spectrophotometry . The reverse transcription was performed using the Superscript III (Invitrogen) and Oligo (dT) primers from total RNA (1 µg). Quantitative determination of muscle-specific E3 ligases atrogin-1 (atrogin-1) and muscle ring finger protein-1 (MuRF1) mRNA levels was performed by real-time quantitative-PCR using specific TaqMan probes and GAPDH as the endogenous control. The following primer/probe pairs were used: atrogin-1, forward, 5′-ACC AAA ACT CAG TAC TTC CAT CAA GA-3′, reverse, 5′-TGT TGA AAG CTT CCC CCA AA-3′, probe,5′-FAM-CAA AGG AAG TAC GAA GGA GCG CCA TG-TAMRA-3′; MuRF1, forward, 5′-ACA CAA CCT CTG CCG GAA GT-3′, reverse, 5′-ACG GAA ACG ACC TCC AGA CA-3′, probe, 5′-FAM-AGG CTG CGA ATC CCT ACT GGA CCA A-TAMRA-3′; GAPDH, forward, 5′-ATG TTC CAG TAT GAC TCC ACT CAC G-3′, reverse, 5′-GAA GAC ACC AGT AGA CTC CAC GAC A-3′, probe, 5′-FAM-AAG CCC ATC ACC ATC TTC CAG GAG CGA GA-TAMRA-3′ t-test. A probability of p<0.05 was considered statistically significant.All data were expressed as mean ± S.E. based on data derived from multiple independent experiments. The intensity of bands from Western blots was scanned with densitometry and digitally analyzed using the Image J software (NIH). The statistical significance was tested by Student's 1/β2 DKO mice , and ERK1/2 phosphorylation in adult mice . PretreaDKO mice /B. AcuteDKO mice . To deteDKO mice  or liverDKO mice . It is wDKO mice .This sug1AR and β2AR are expressed in the heart. To assess which subtype of βAR is responsible for ISO-induced increase in phosphorylation of Akt and ERK1/2, mice were treated with saline, dobutamine (a predominantly β1AR agonist) or formoterol (a β2AR-selective agonist) for 30 min. Acute β2AR stimulation with formoterol induced significant increases in PI3K activity  is an important downstream effector of PI3K/Akt pathway Glycogen synthase kinase-3 (GSK-3) is another critical downstream element of the PI3K/Akt signaling pathway. These kinases are key signaling mediators in myocardial hypertrophy and progressive heart failure in vivo (The forkhead box family of transcription factors (FOXOs) is implicated in the regulation of a variety of cellular processes, including the cell cycle, apoptosis, DNA repair, stress resistance, and metabolism. FOXOs proteins are negatively regulated by the PI3K/Akt signaling pathway in vivo . Taken tIt has been shown that atrogin-1 and MuRF1 are associated with hypertrophy and atrophy processes in skeletal muscle through the PI3K/Akt signaling pathway in vivo evidence for the crosstalk between βAR and PI3K signaling pathway. We demonstrate that the βAR transactivation of PI3K/Akt pathway is β2AR-specific and confines to the heart. Many known signaling factors downstream of PI3K, including the mTOR/P70S6K/S6 axis, GSK-3α/β, and FOXO1/FOXO3a, were shown to be involved. Although these cascades have been well investigated, the link between βAR and these signaling pathways has not been systematically characterized in vivo, especially in the cardiac context. More important, we demonstrate, for the first time, that atrogin-1 and MuRF1 are affected by acute βAR stimulation in mouse heart.In the present study, we set out to elucidate the early signaling events that mediate the effect of acute βAR stimulation in mouse heart. Our results provide convincing in vitro and in vivo evidence showing that βAR stimulation induces activation of cardiac PI3Kα but not the other isoforms of PI3K The levels and activity of the cardiac PI3K signaling pathway are tightly controlled during development. The highest activity is found in late gestation and early postnatal life and then decreases dramatically in older animals Akt is a serine/threonine protein kinase downstream of PI3K. Full activation of Akt requires phosphorylation at both Thr308 [by 3–phosphoinositide–dependent protein kinase 1 (PDPK1)] and Ser473 (by a putative PDPK2) 1/β2 DKO mice are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities 1/β2AR loss on basal physiological functions such as heart rate, blood pressure, or metabolic rate is remarkably modest. Hence, β1/β2 DKO mice represent a useful model for the study of βAR-modulated cardiac functions in vivo1/β2 DKO mice, providing further evidence for the cardiac βAR-PI3K crosstalk in vivo.β1AR and β2AR in terms of regulation of cardiac function and alterations in cardiac structure 1AR has been shown to mediate pro-apoptotic responses while β2AR mediates anti-apoptotic responses in cardiomyocytes 2AR signaling has a lower overall effect on contractility than β1AR signaling does i, β2AR signaling has been demonstrated to induce the recruitment and activation of other signaling molecules, including PI3K/Akt and ERK 2AR-specific in H9c2 cardiomyoblasts, a β2AR predominant cell line 1AR comprises approximately 70–80% of the total βAR population in healthy heart 2AR-specific. It is unknown, however, whether or not chronic βAR stimulation-induced cardiac hypertrophy is solely mediated by β2AR. It has been reported in cultured rat neonatal cardiomyocytes that βAR-induced hypertrophy is mediated primarily by the β1AR subtype in vitro and in vivo systems. The in vivo data shown here are novel and add new insights to our understanding regarding the characteristics among βAR subtypes.There is increasing evidence to support a differential role of βThis crosstalk may be tissue-specific since the same βAR stimulation did not activate Akt in the lungs, kidneys  or liverActivation of Akt and mTOR plays a crucial part in the regulation of cell growth and proliferation by monitoring nutrient availability, cellular energy levels, oxygen levels and mitogenic signals in vivo.Akt-mediated phosphorylation of GSK-3 is inhibitory Phosphorylation of FOXOs by Akt is inhibitory and leads to nuclear exclusion and the inhibition of the forkhead transcriptional program 2AR–agonists has also been shown to increase skeletal muscle strength in normal volunteers It has been extensively investigated that atrogin-1 and MuRF1 are common mediators in several models of skeletal muscle hypertrophy and atrophy. The levels of atrogin-1 and MuRF1 are regulated by the growth factor/Akt signaling axis through direct transcriptional regulation by FOXOs in vivo. Our data support a model depicted in 2AR activates PI3K, which affects the activities of ERK1/2, Akt and its downstream signaling cascades, including mTOR/P70S6K/S6 axis, GSK-3α/β, FOXOs, atrogin-1 and MuRF1. These data provide mechanistic and signaling insights for the pathophysiological consequences induced by chronic βAR stimulation.In summary, our data suggest that crosstalk between βAR and PI3K signaling pathway activates multiple downstream signaling effectors and cascades


The human T-cell leukemia virus type 1 (HTLV-1), identified as the first human oncogenic retrovirus 30 years ago, is not an ubiquitous virus. HTLV-1 is present throughout the world, with clusters of high endemicity located often nearby areas where the virus is nearly absent. The main HTLV-1 highly endemic regions are the Southwestern part of Japan, sub-Saharan Africa and South America, the Caribbean area, and foci in Middle East and Australo-Melanesia. The origin of this puzzling geographical or rather ethnic repartition is probably linked to a founder effect in some groups with the persistence of a high viral transmission rate. Despite different socio-economic and cultural environments, the HTLV-1 prevalence increases gradually with age, especially among women in all highly endemic areas. The three modes of HTLV-1 transmission are mother to child, sexual transmission, and transmission with contaminated blood products. Twenty years ago, de Thé and Bomford estimated the total number of HTLV-1 carriers to be 10–20 millions people. At that time, large regions had not been investigated, few population-based studies were available and the assays used for HTLV-1 serology were not enough specific. Despite the fact that there is still a lot of data lacking in large areas of the world and that most of the HTLV-1 studies concern only blood donors, pregnant women, or different selected patients or high-risk groups, we shall try based on the most recent data, to revisit the world distribution and the estimates of the number of HTLV-1 infected persons. Our best estimates range from 5–10 millions HTLV-1 infected individuals. However, these results were based on only approximately 1.5 billion of individuals originating from known HTLV-1 endemic areas with reliable available epidemiological data. Correct estimates in other highly populated regions, such as China, India, the Maghreb, and East Africa, is currently not possible, thus, the current number of HTLV-1 carriers is very probably much higher. Very rapidly after HTLV-1 discovery and its association with adult T-cell leukemia (ATL), several studies were initiated both by American and Japanese researchers, to get insights into the distribution, the transmission modes, as well as the origin of HTLV-1.Thus, as soon as 1982–1984, important works demonstrated clearly that Japan was a high endemic area for HTLV-1. Interestingly, since the first studies, the geographic distribution of HTLV-1 carriers is quite uneven in Japan and the greatest prevalence is observed in Southwestern Japan (Kyushu island and the Okinawa archipelago). The origin of such a peculiar distribution is still the matter of several hypotheses  Several large regions/areas have not been investigated for HTLV-1 infection. Thus, the prevalence remains barely unknown in many areas of the world. This is evident in some highly populated regions of Asia or in East and North Africa. (2) The assays used for HTLV-1 serology exhibited some lack of specificity leading in the 1980s–1990s to an overestimated HTLV-1 prevalence. (3) Most of the works performed to appreciate the HTLV-1 prevalence are based on study of series of blood donors, pregnant women, or hospitalized patients. Population-based studies trying to estimate HTLV-1 prevalence in large areas, or even at a country level, remain very rare. (4) A very important last point is that, in most of the studied areas, HTLV-1 distribution is not homogeneous. HTLV-1 is indeed present mainly as relatively small foci or clusters with a high or very high prevalence of infection, with nearby quite low endemic areas. This has been very well exemplified in Southern Japan and in some areas of South America. Thus, to give a precise estimation of HTLV-1 prevalence in a specific country or area is relatively difficult and, in some cases, nearly impossible.Very few studies have thus given an estimation of the HTLV-1 world prevalence. Japan and the African continent have been rapidly considered, as the two regions in which HTLV-1 infected persons were the most numerous. Then, South America has also been considered as a very important focus of HTLV-1 carriers and associated diseases , a chronic severe neuromyelopathy). One of the most frequently quoted estimation number of infected persons is based on a paper, published in 1993 by G. de Thé and R. Bomford from our group in Pasteur  most of the 1,100 papers referenced in PubMed on such a topic, (2) most of the available book chapters concerning HTLV-1 epidemiology but also (3) on all the abstracts (of the epidemiology sections) of the International Conference on HTLV and related viruses held since 1985.This study was, of course, possible thanks to our expertise and experience in the field since 30 years for one of us and nearly 10 years for the other one.After presenting the different methods used for the epidemiological studies of HTLV-1 infection, we shall describe briefly the major epidemiological determinants and the different transmission modes of this human retroviral infection. Then, a small chapter will report the major findings concerning the molecular epidemiology of HTLV-1, which possesses an unusual great genetic stability for a retrovirus. Lastly, we shall try to present, with a table and a map, the HTLV-1 worldwide distribution and its prevalence estimates by continent and by country when possible.Most of the studies were performed on series of blood donors, pregnant women, or hospitalized patients. In few cases, population-based studies were done in some villages, towns, or even regions of a given country. The epidemiological and demographic characteristics of blood donors could be very different according to the countries: indeed, they can be quite representative of the middle-class population in some countries but in other areas, either they are mainly family members of hospitalized patients, or they originate from low socio-economic populations and are sometimes paid to give blood and, lastly, in several areas  they are mainly young men. HTLV-1 prevalence varies according to age, sex, and economic level in most of HTLV-1 endemic areas. Thus, the prevalence found in blood donors can be, of course, useful, but provides not always the best data to estimate precisely the HTLV-1 prevalence in a given country. The real prevalence is very probably higher than that found in blood donors in most of the cases. Thus, the data based on pregnant women are generally more useful to compare the situation between different areas or countries as first, it is quite representative of a given region and second, the mean age of pregnant women is generally comparable (about 22–26 years) in most of the countries. The studies in general populations, in or out-adult patients, can also be very useful to try to appreciate the HTLV-1 prevalence in a given area as, in the great majority, the tested patients do not suffer of the very rare diseases linked specifically to HTLV-1 infection.Plasmodium falciparum in Central Africa and Indonesia  or Particle Agglutination (PA). Confirmatory tests are immunofluorescence (IFA), but mostly Western Blot (WB), or Innogenetics line immunoassay (INNO-LIA). Moreover, research of integrated provirus, in the DNA from peripheral blood cells, could be done by qualitative and/or quantitative polymerase chain reaction (PCR). Despite some improvements in the WB assays specificity during the last two decades, indeterminate serological patterns remain frequent following WB analysis, and represent an important concern for routine screening in blood banks in Europe, the Americas, and some parts of Africa. It is also of course a major issue for comparative analyses between epidemiological studies performed in both low and high endemic areas, especially in intertropical areas  and of the middle East (such as the Mashad region in Iran), and rare isolated clusters in Australo-Melanesia. In Europe, only Romania seems to represent an HTLV-1 endemic region. The origin of this puzzling geographical or rather ethnic repartition is not well understood but is probably linked to a founder effect in some groups, followed by the persistence of a high viral transmission rate.Interestingly and despite different socio-economic and cultural environments, HTLV-1 seroprevalence increases gradually with age, especially in women, in all the highly endemic areas. The general increase with age may be related to a cohort effect, as well as demonstrated in Japan, while the increase in women might also be due to an accumulation of sexual exposures with age  Mother to child transmission, which is mainly linked to a prolonged breast-feeding after 6 months of age Hino, . Ten to On a molecular point of view, HTLV-1 possesses a remarkable genetic stability, an unusual feature for a retrovirus. Viral amplification via clonal expansion of infected cells, rather than by reverse transcription is, very probably, the reason for this striking genetic stability. The low sequence variation of HTLV-1 can be used as a molecular tool to follow the migrations of infected populations in the recent or distant past and thus to gain new insights into the origin, evolution, and modes of dissemination of such retroviruses and of their hosts , according to five large geo-climatological areas: North Africa, West Africa, Central Africa, East Africa, and lastly South Africa. Then, we shall try to estimate the number of HTLV-1 infected persons in some specific countries for which reliable data are available.The very few studies concern mainly blood donors or multi-transfused patients from Egypt, Morocco, and Tunisia where the HTLV-1 seroprevalence appears to be very low or negative , Chad, Democratic Republic of the Congo (DRC) formerly Republic of Zaire, Equatorial Guinea, Gabon, Republic of the Congo, Sao-Tome-et-Principe. This represents around 130 millions of persons. Several important epidemiological studies have been performed in Cameroon and especially in Gabon. Cases of ATL and/or TSP/HAM have been reported mainly in Cameroon, Gabon and the DRC  have been performed in such a large and highly populated region . Moreover, only sporadic cases of ATL and/or TSP/HAM have been reported from East African countries. Lastly, this region appears globally much less endemic for HTLV-1 than West and Central Africa. The reasons of such a situation are unknown. Indeed, all the studies performed in the countries, North of Malawi, indicate a very low or an absence of HTLV-1 infection. As examples, a nationwide community-based survey performed in Rwanda on more than 2,500 samples indicated an HTLV-1 seroprevalence of 0.2% Group, , and in This area comprises only three countries: Botswana, Namibia, and South Africa. Several studies have been performed but concern only South Africa, a country of 48 millions of inhabitants.Indeed, several sporadic case or series of ATL, TSP/HAM, or infective dermatitis have been reported in South African patients  and the Seychelles archipelago. Indeed, in the Reunion Island, despite that few cases of TSP/HAM and ATL have been reported  showed evidence of HTLV-1 infection  and show the absence of HTLV-1 infection among healthy women as well as in blood donors . In 2012, its estimated population is 42.6 millions. Despite the fact that some of these countries have strong commercial and cultural ties with the highly HTLV-1 endemic Caribbean Islands, relatively few studies concerning the HTLV-1 infection and the associated diseases have been performed except in the Honduras, Panama and, to a lesser extent, in Costa Rica. In these countries, the HTLV-1 seroprevalence is rather low but with significant differences between the populations tested. Thus, the HTLV-1 prevalence is significantly higher in non-Mestizo population, especially individuals living in the coastal cities of Honduras, than in the Mestizo ethnic groups , among the Nicaraguan and Costa Rican adult populations , Guyana, Paraguay, Peru, Surinam, Uruguay, and Venezuela. This represents around 400 millions of persons. Numerous studies on HTLV-1 prevalence have been performed in many of these countries, especially in Brazil, Peru, Columbia, Chile, Argentina, and French Guyana. This vast continent represents, as a whole, a major endemic area for HTLV-1 infection and associated diseases. Even more than in other world regions, it is very difficult to appreciate the general HTLV-1 prevalence in a given country, as specific high endemic foci have been frequently reported in several South American countries. This is exemplified in Peru, in some of the Quechua population groups, and also in Columbia (Tumaco area) or in French Guyana (Noir-Marron population), where some specific groups of African origin are highly endemic for HTLV-I infection and associated diseases.In Peru, a real multiethnic country mainly inhabited by Mestizos and Amerindians, HTLV-1 prevalence in blood donors ranges from 1.2 to 1.7% depending on the region , overseas departments  and dependencies , totalizing around 40 millions of inhabitants. The majority of these islands have a relatively small population . Only Cuba, Haiti/Dominican Republic, and Jamaica have a population of more than two millions. All the islands, which are mainly peopled with persons of African ancestry, represent a high endemic region for HTLV-1 infection and associated diseases.Jamaica has been extensively studied concerning HTLV-1 infection . Here we arbitrarily defined four subregions: (1) East Asia , (2) North (Siberia), Central  and Southwest Asia , (3) South Asia  and (4) Southeast Asia . Except for some very important endemic foci including mainly Japan and Iran, the prevalence of HTLV-1 and associated diseases in Asia seems very low. However, in most of the areas, due to the lack of large and representative studies, the situation remains still poorly known.East Asia comprises more than 1.4 billion people (about 20% of the world population). In Mainland China, no general population-based-study has been performed to our knowledge regarding HTLV-1 prevalence. However, a large-scale study concerning 145,293 blood donors representative of 13 provinces revealed a very low global prevalence  reported among blood donors . Thus, the HTLV-1 prevalence must be appreciated according to the donors’ countries of origin. However, the HTLV-1 prevalence in blood donors born in Israel reached 0.001% . In this region, the studies concerning HTLV-1 prevalence are scarce and the most recent one among Indian blood donors revealed a 0.14% prevalence rate  was found among patients with motor neuron disease called neurolathyrism , Australo-Melanesia , and Polynesia . The population is around 35 millions individuals with two third of Australian origin.Only few population-based-study using solid serological confirmation criteria have been performed regarding HTLV-1 prevalence in the Australo-Melanesian region. Interestingly, few studies, conducted among Papuan aboriginal populations, demonstrated that some remote tribes as the Hagahai population living in the highlands (Madang province), exhibited higher HTLV-1 prevalence rate than population groups originating from different Papuan provinces such as the Sepik province  revealed that HTLV-1 prevalence rate is very low, comprised between 0.001 and 0.032% Whyte, . In contThe few reliable studies performed in Polynesia/Micronesia regarding the HTLV-1 prevalence among native-born Polynesian individuals, indicated that the infection is absent or very rare (Nicholson et al., Our best estimates range approximately from 5 to 10 millions HTLV-1 infected individuals. However, these results were only based on nearly 1.5 billion of individuals originating from known HTLV-1 endemic areas with reliable available epidemiological data. Correct estimates in other highly populated regions, such as China, India, the Maghreb, and East Africa, is currently not possible. The real number of HTLV-1 carriers is thus very probably much higher.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.


Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown.in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival.Utilizing This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors. Colorectal cancer (CRC) is one of the most common malignancies with high incidence rates globally Tumor suppressor genes (TSG) contribute to the induction of apoptosis in response to stress. The failure to induce apoptosis in response to various types of cellular damage has been long recognized as contributing to oncogenesis. One example of TSG loss contributing to cancer formation and progression is TGFβ signaling We have shown that TGFβ signaling in an early stage non-metastatic colon carcinoma model leads to cell death in colon cancer cells in response to stress in association with inactivation of pAKT and inhibition of the expression of the IAP protein survivin 20TGFβ signaling has been shown to activate cAMP- dependent Protein Kinase A (PKA) in vivo. Rescue of specific tumor suppressor aspects of the TGFβ signaling pathway may provide therapeutic benefits without promoting the cell survival and metastatic effects of TGFβ. If these TGFβ tumor suppressor effects can be mimicked in late stage tumors, therapeutic value against metastatic CRC might ultimately be obtained.The molecular mechanisms involved in the TGFβ mediated downregulation of IAP were investigated in order to determine the effects of TGFβ receptor signaling on metastasis in a metastatic orthotopic model of colon carcinoma. We now report the identification of a novel TGFβ/PKA/AKAP mediated transduceome that converges on XIAP function in controlling aberrant cell survival. Additionally, we have shown that TGFβ receptor rescue in highly metastatic cells with epigenetic silencing of TGFβ receptors leads to decreased metastases in a TGFβ/PKA signaling pathway dependent mechanism in a highly metastatic orthotopic colon cancer models A critical report from the Simeone laboratory noted that TGFβ signaling activates PKA in Mv1Lu cells and mediates control of TGFβ growth inhibitory responses 20 in the cytosol, but not in mitochondria 20). Earlier reports indicate that PKA phosphorylates the Rpt6 ATPase, which unfolds and transports substrates into the proteasome and this phosphorylation enhances the chymotrypsin proteasomal activities of the proteasome XIAP and survivin are well characterized IAP family members recently documented as metastatic genes Collectively, these data lead to the hypothesis that the TGFβ/PKA signaling regulates XIAP expression at multiple levels with activation of PKA leading to phosphorylation of key proteasomal components that promote proteasomal degradation of XIAP.There is an abundance of A-kinase anchoring proteins (AKAPs) which compartmentalize the PKA and other enzymes to the vicinity of specific cellular organelles for carrying out enzyme function. We hypothesized that AKAPs are a critical component in TGFβ/PKA mediated XIAP downregulation. It is well documented that the subcellular localization of PKA regulatory subunits (PKARI and/or PKARII) is mediated through interactions with AKAPs Since the AKAP family includes more than 50 members We next questioned whether the effect of AKAP149 was selective in this TGFβ/PKA effect. While AKAP-PKA interaction is a global phenomenon, validation of AKAP149 as selectively regulating TGFβ/PKA signaling would be a novel extension of our understanding of the TGFβ/PKA mediated XIAP downregulation. AKAP220 has been shown to regulate protein phosphatase 1 (PP1) activity by coordinating the location of PKA and PP1 catalytic subunit It has been observed that activation of PKA leads to the inactivation of AKT through dephosphorylation by protein phosphatase 2A (PP2A) To further validate the role of PP2A in TGFβ/PKA signaling, we generated stable shRNA PP2A catalytic subunit knockdown in FET cells designated FET PP2A Cat KD . TGFβ tr87 which leads to increased stabilization of XIAP and decreased apoptosis of ovarian cancer cells in response to cisplatin While inhibition of AKT activation would have potential for many effects on cell survival, one effect that is of particular interest as related to TGFβ/PKA function is in the context that it targets XIAP stability. A previous study reported that XIAP is phosphorylated by AKT on Serin vivo  subunits bound to two (R) subunits (RIα/β and RIIα/β) that renders it inactive An intriguing observation made in our studies which originally showed that TGFβ signaling inhibits survivin expression was that TGFβ signaling also mediated inactivation of AKT 87, thereby stabilizing XIAP and preventing it from auto-ubiquitination We have shown that XIAP is associated with pAKT and this association is disrupted following TGFβ treatment . AKT aloIn association with direct regulation of XIAP by TGFβ/PKA signaling, we also demonstrated that chymotrypsin-like activity of the proteasome is induced by TGFβ in a PKA dependent manner. XIAP can be auto-ubiquitinated and degraded when treated with DNA damaging agents like chemotherapeutic drugs We have made the novel observation that reconstitution of TGFβ receptor TGFβRI in highly metastatic GEO colon cancer cells rescues TGFβ signaling and inhibited metastatic colonization from orthotopic xenografts. This observation raises the therapeutic possibility that TGFβ can suppress metastatic competence after this advanced state of progression is attained in cells with attenuated type I and/or II TGFβ receptor expression (designated TGFβRI or TGFβRII).in vivo even though invasion at the primary tumor site was maintained (We have made the novel observation that rescue of attenuated receptor expression does inhibit metastatic capability in GEORI (reconstituted with RI) transplanted orthotopic models intained . This fiStudies on different cancer types provide extensive support for loss of TGFβ receptor expression as an important contributor to tumor progression in subgroups of several types of cancer In an effort to determine the relationship between the TGFβ/PKA transduceome and the capability of a tumor for metastatic spread, we investigated the molecular aspects of the TGFβ/PKA transduceome signaling in the context of metastatic progression in highly metastatic GEO and CBS cells and their poorly metastatic counterpart GEORI and CBSRII where receptor reconstitution has led to a decrease in metastatic spread. Since metastatic deposits, like primary cancers, are faced with hypoxic stress and must face the additional challenge of growth in a foreign microenvironment, it seems likely that even established metastases must retain their aberrant survival characteristics.Striking difference in TGFβ/PKA signaling activity and downstream signaling leading to XIAP downregulation was observed when highly metastatic  and their poorly metastatic colon cancer counterparts (GEORI and CBSRII) were compared. Therefore, reconstitution of TGFβ receptors in metastatic models with attenuated TGFβ signaling leading to metastasis might also reconstitute TGFβ/PKA signaling in established metastases which could affect their survival. These lines of evidence lead to the consideration that the TGFβ/PKA transduceome is a critical component in steady-state suppression of abnormal survival signaling that prevents formation of metastases by disrupting the XIAP/survivin cell survival pathway.In conclusion, understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases (most prominent cause of death from solid cancers). The role of loss of tumor suppressor activities in the metastatic process is essentially unknown. Presently, loss of TGFβ signaling is largely regarded as a driver for transition from a benign to a malignant state subsequently leading to metastatic spread by other mechanisms or even by usurping aspects of TGFβ signaling for oncogenic mechanisms such as the acquisition of EMT. However, we have now shown that loss of TGFβ tumor suppressor signaling is a driver for metastatic colonization of distant organs from the primary tumor. Importantly, loss of TGFβ signaling does not affect invasion as reflected by retention of a 100% rate of invasion at the primary site of metastatic cells in which TGFβ signaling has been rescued and metastatic potential inhibited. This indicates that while loss of TGFβ signaling enables the transition from a benign to a malignant state its loss does not drive the earliest step of the metastatic process.However, we have shown here that loss of TGFβ signaling is necessary to allow the last step of the metastatic process- colonization of the metastatic site. Moreover, we have identified a novel TGFβ tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. This survival mechanism is apparently not necessary for malignant behavior at the primary site once the cancer cells have made the transition from the benign state since its inhibition by restoration of TGFβ tumor suppressor signaling does not alter malignant behavior at the primary site.Our work indicates the potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by this novel, multifaceted TGFβ induced pathway. Our previous body of work showing that HDACi's act at least in part through the rescue of TGFβ receptor I and II expression and TGFβ tumor suppressor activity in a wide variety of cancer cells indicates the clinical potential of this concept.Since TGFβ signaling responses can be supportive of events that support malignant progression such as EMT and suppression of TGFβ signaling as a therapy for cancer has been supported by experimental evidence, therapeutic strategies for the inhibition of TGFβ signaling in cancer therapy are currently being pursued. However, our work indicates that the relationship between TGFβ signaling and tumor suppressor function may be governed by context even in the later stages of cancer. The concept that the disruption of the balance between TGFβ tumor suppressor activity and the survivin/XIAP cell stress response enables metastatic colonization of distant organs raises the concern that therapies aimed at inhibition of TGFβ signaling may be deleterious to at least a subset of cancer patients. For example, it has been shown in several types of cancer that progressive epigenetic silencing of TGFβ receptor I and/or II correlates with malignant progression. Pharmacological inhibition of TGFβ receptor signaling may promote metastases in these patients.All experiments involving animals were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC #: 07-047-08-FC).The FET Cells were lysed in a Tris-HCl based buffer containing 0.5% NP-40 and appropriate protease and phosphate inhibitors as described previously Smad 3 (sc-38376-SH), PKA Catalytic α (sc-36240-SH) and PP2A Catalytic α (sc-43509-SH) shRNA was obtained from Santa Cruz. These vectors were co-transfected by electroporation as described previously PKA activity was measured using the PepTag non-radioactive protein kinase assay (Promega) using kemptide (LRRASLG) following the manufacturer's protocol. For quantitative determination of cellular cAMP, the non-radioactive Direct Cyclic AMP Enzyme Immunoassay kit (Assay Design) was used.For quantitative determination of cAMP, non-radioactive Direct Cyclic AMP Enzyme Immunoassay kit (Assay Design) was utilized. Manufacturer's protocol was followed for the assay.Protein Phosphatase 2A (PP2A) activity was measured using the non-radioactive PP2A immunoprecipitation assay (Millipore).The fluorometric Proteasome substrate III for chymotrypsin (SUC-LLVY-AMC) and Peptidyl-glutamyl peptide hydrolyzing (PGPH)-caspase-like activity proteasome substrate II (Z-LLG-AMC) and the corresponding inhibitors were obtained from Calbiochem. FET cells were plated at 800,000 cells/100 mm dish in SF media. On day 5, cells were treated with TGFβ (5 ng/ml) with or without H89 (15 µM) pretreatment for 1 h and harvested in proteasomal assay buffer . After quantification of protein concentration, cell lysates were pretreated with 25 uM of the chymotrypsin-like activity inhibitor Benzyloxy carbonyl Leu Leu phenylalaninal Inhibitor (ZLLF-CHO) or the PGPH activity inhibitor Z-Gly-Pro-Phe-Leu-CH (Z-GPFL-CHO) followed by incubation with chymotrypsin or PGPH substrate for 1 h at 37°C as described by Apoptosis was measured by the Cell Death Detection ELISA Plus kit (Roche) as described previously 3 pieces. These pieces were then orthotopically implanted into other BALB/c nude mice. For operative procedures, animals were anesthetized with isofluorane inhalation. A 1 cm laparotomy was performed and both the cecum and ascending colon were exteriorized. Using 7× magnification and microsurgical techniques, the serosa was disrupted by scraping in two locations. The 1 mm3 pieces of xenograft were sub-serosally implanted using an 8-0 nylon suture at the two points of serosal disruption. The bowel was then returned to the peritoneal cavity and the abdomen was closed with 5-0 vicryl suture. Subsequently, animals were anesthetized with a 1∶1 mixture of ketamine (10 mg/ml) and xylazine (1 mg/ml) by intraperitoneal injection (0.01 ml/mg) and weekly GFP fluorescence imaging was performed for up to 5 weeksWeekly GFP fluorescence imaging was performed for up to 5 weeks. Excitation of GFP in the light box facilitated identification of primary and metastatic disease by direct near-real time visualization of fluorescence in live animals. Both GEO and GEORI cells gave rise to an invasive primary tumor in 100% of the animals implanted.All experiments involving animals were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. The orthotopic implantation methodology has been described in details in the following publications from Brattain laboratory Approximately 50 d post-implantation, the animals were euthanized following proper IACUC protocol. The colon (with primary tumor), liver, lungs, and heart were harvested. Following the harvesting, organs were explanted, imaged, and immediately placed in 10% neutral buffered formalin fixative for 24 h. This was followed by tissues processing and embedding in paraffin. Slides were then cut for hematoxylin and eosin (H and E) and immunohistochemical characterizations. Serial sections were cut to complement the H and E sections and were stained with an IgG1 rabbit polyclonal antibody for Ki-67 . Slides from paraffin embedded tissue blocks were stained according to the Apotag  terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) assay kit. Detailed methodology and quantitative analysis for these individual experiments has been described in details in the Figure S1Activation of pSmad2.(TIF)Click here for additional data file.Figure S2PKA catalytic subunit shRNA knockdown in FET cells.(TIF)Click here for additional data file.Figure S3No change in IκB-α with TGFβ treatment.(TIF)Click here for additional data file.Figure S4Smad3 shRNA knockdown in FET cells.(TIF)Click here for additional data file.Figure S5TGFβ treatment downregulates survivin. Blocking the proteasome with MG132 abrogates the TGFβ mediated survivin loss.(TIF)Click here for additional data file.


The aim of the study was to evaluate the cause of the lost to follow-up in the newly diagnosed HIV positive patients, after the first visit in the HIV clinic.We retrospectively reviewed the clinical charts of the adult patients (P) who consulted for the first time in the Infectious Disease Clinic of the university associated hospital at Buenos Aires, Argentina, between June 1st 2005 and May 31st 2008. We identified between the P who were newly diagnosed with HIV infection, those who attended the clinic only once; we considered lose to follow up those P who never come back in a year time. We exclude those P who attended other clinic before coming to ours. We contacted via telephone those P who had only one visit and never came back, to know the reason why they never came back to the clinic; we compared the age, gender, and socioeconomic characteristics between the group of patient who continued medical care (those who are currently attending our clinic and were diagnosed in the same period) and those who lost to follow up. We considered the first visit, the one in which the P is informed about his HIV seropositive condition.We included 227 adult P who consulted the clinic for the first time. We identified 123 P (54%) who never came back to consultation after the first visit. The main cause of lost to follow up was because the patient felt good enough and thought they do not need medical attention (31/123); 25/123 P gave us a wrong number so we could not contact them. Most of the P were under no medical attention at the time we called them (61/123). We compare those P with 104 P who continued attending our clinic; the group of P who continued under medical care were older (median age 40.4 vs 34.2), patient with 40 years or more were 50/104 in the first group and 24/123 in those lost to follow up (p=0.0000048 OR 3.82 CI 2.04-7.20), and were in most cases women . P who continue follow up were working in most cases , live with family/partner (94/104 vs. 77/123 p=0.01 OR 0.67) and got better scholarship degree (university studies 21/104 vs 7/123 p=0.000 OR 4.19 CI 1.60-11.43) than P that did not continue follow up.We must reinforce the medical care need in recently diagnosed HIV patients during the first visit to avoid the loss to follow up, especially in those patients who are younger and socially excluded.


Vibrio species, CqsS is a membrane-bound histidine kinase that acts as the receptor for the CAI-1 autoinducer which is produced by the CqsA synthase. In Vibrio cholerae, CAI-1 is (S)-3-hydroxytridecan-4-one. The C170 residue of V. cholerae CqsS specifies a preference for a ligand with a 10-carbon tail length. However, a phenylalanine is present at this position in Vibrio harveyi CqsS and other homologues, suggesting that a shorter CAI-1-like molecule functions as the signal. To investigate this, we purified the V. harveyi CqsS ligand, and determined that it is (Z)-3-aminoundec-2-en-4-one (Ea-C8-CAI-1) carrying an 8-carbon tail. The V. harveyi CqsA/CqsS system is exquisitely selective for production and detection of this ligand, while the V. cholerae CqsA/CqsS counterparts show relaxed specificity in both production and detection. We isolated CqsS mutants in each species that display reversed specificity for ligands. Our analysis provides insight into how fidelity is maintained in signal transduction systems.Quorum sensing is a process of bacterial cell–cell communication that enables populations of cells to carry out behaviours in unison. Quorum sensing involves detection of the density-dependent accumulation of extracellular signal molecules called autoinducers that elicit population-wide changes in gene expression. In  Many bacteria produce and release extracellular signalling molecules called autoinducers. By monitoring the accumulation of autoinducers, bacteria track changes in population density and species complexity in the vicinity. This process, known as quorum sensing, enables groups of bacteria to synchronize gene expression and carry out collective behaviours such as bioluminescence, biofilm formation, competence and virulence factor production, which presumably are not productive when performed by a single bacterium .Vibrio species possess two or more quorum-sensing systems that channel multiple autoinducer inputs into the output response -2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate in Vibrios, is produced by the LuxS enzyme and detected by the LuxPQ receptor (V. cholerae as (S)-3-hydroxytridecan-4-one, is produced by the CqsA synthase and detected by the CqsS receptor. The CqsA/CqsS system is conserved in many Vibrio species  has not been examined.Many response . For exaand AI-2 ; whereasVh-CAI-1   is shuttled into a common phosphorelay signal transduction pathway (V. cholerae). LuxR (HapR) acts as both a transcriptional activator and a transcriptional repressor. In V. harveyi, LuxR activates genes for bioluminescence and regulates at least 50 additional targets -3-aminoundec-2-en-4-one -3-hydroxytridecan-4-one and (S)-3-aminotridecan-4-one . Sterile medium and spent culture fluid from a ΔcqsA strain do not induce light production in JMH626. However, addition of 10% (v/v) spent culture fluid prepared from wild-type V. harveyi induces light production 3000-fold in JMH626 . These results suggest that V. harveyi CqsS does not detect the V. cholerae CAI-1 molecule (10-carbon tail) as an agonist, and that C8-CAI-1 is only a weak agonist for V. harveyi CqsS since it cannot induce maximal light production even at very high concentrations. We therefore reason that V. harveyi must produce a CAI-1 like molecule that likely has an 8-carbon tail with additional modification(s) that do not exist in CAI-1 or in C8-CAI-1.We chose n JMH626 . Presuman JMH626 . Howevern JMH626 . Increasn JMH626 . CombiniV. harveyi CqsS receptor, we overexpressed the V. harveyi cqsA gene in Escherichia coli (strain WN1327). Spent culture fluid (10% v/v) from WN1327, but not from E. coli carrying the empty vector, induced light production in JMH626 to the maximal level (data not shown). Moreover, activity could be quantitatively extracted with dichloromethane (DCM), which allowed us to separate the activity from other components. Concentration of the DCM extract to complete dryness led to a significant loss in activity; therefore in all our analyses, we avoided steps that resulted in activity decreases by monitoring the activity in each step.In order to identify the molecule detected by the Supporting information) were used to isolate fractions capable of inducing maximal light production in V. harveyi JMH626. The purity of the active fractions was determined by ESI-TOF HRMS. One fraction that could induce maximal light production in JMH626 possessed a single ion peak with m/z [M+H+] of 184.1699, corresponding to a molecular formula of C11H21NO. A second fraction was weakly active and possessed a molecular ion peak with m/z [M+H+] of 187.1694, corresponding to a molecular formula of C11H22O2. Concentration of this second fraction allowed us to use NMR to determine that it contains 3-hydroxyundecan-4-one (C8-CAI-1) and its regioisomer 4-hydroxyundecan-3-one in approximately a 1:1 ratio. Through chemical synthesis and consistent with the above results, we found that C8-CAI-1 is only a weak agonist for the V. harveyi CqsS receptor. The regioisomer, 4-hydroxyundecan-3-one, is inactive . The resulting maximally active fraction contains a molecule with a single olefinic proton by 1H-NMR  (Supporting information). Taken together with the molecular formula, the splitting pattern and chemical shift of this signal allows us to propose that the structure of the highly active V. harveyi CqsS agonist is (Z)-3-aminoundec-2-en-4-one  and determined that, by 1H-NMR, the synthetic material contains the diagnostic olefinic quartet observed in the natural sample. Additionally, the synthetic compound has identical activity to the ligand-containing spent culture fluid harvested from wild-type V. harveyi; that is, they both induce maximal light production in JMH626  and quite sensitive to Ea-C8-CAI-1, we hypothesized that V. cholerae must also produce and detect Ea-C8-CAI-1. Based on the above results, we also wondered if V. cholerae produces an enamino molecule . CAI-1 type molecules were extracted with DCM for HRMS analysis. The concentration of each CAI-1 type molecule was calculated by comparison to the intensity of each ion peak to that of d2-CAI-1 (double deuterium labelled CAI-1), which was added as an internal standard at 500 nM prior to the extraction. Correction for the different ionization potentials of each of the CAI-1 type molecules was achieved using a mixed sample containing a known amount of each of the molecules .To examine these possibilities, we determined how much of each CAI-1 type molecule is produced by wild-type V. cholerae spent culture fluids, we detected the molecular ions from Ea-C8-CAI-1, Ea-CAI-1 and CAI-1 at an average concentration of 19 nM, 139 nM and 222 nM respectively. We did not detect any C8-CAI-1 ion in the V. cholerae sample .In the e sample . In the detected . The detV. harveyi and V. cholerae  that carries the V. harveyi luxCDABE operon (luciferase) and responds exclusively to exogenously supplied CqsS ligands. The V. harveyi wild-type CqsS receptor is highly sensitive to synthetic Ea-C8-CAI-1  but only responds weakly to C8-CAI-1 (EC50 = 5 µM). The V. harveyi CqsS receptor is insensitive to Ea-CAI-1 and CAI-1 (V. cholerae CqsS receptor responds to a wider array of molecules including Ea-C8-CAI-1 (EC50 = 36 nM), Ea-CAI-1 (EC50 = 4 nM) and CAI-1 (EC50 = 62 nM), but is only weakly sensitive to C8-CAI-1 (EC50 = 1.2 µM)  on cholerae , Table 1nd CAI-1 , Table 1V. harveyi only detects Ea-C8-CAI-1 through CqsS to activate quorum sensing. Although C8-CAI-1 is a weak agonist, the EC50 value is significantly larger than the concentration of C8-CAI-1 released by V. harveyi. Using the same reasoning, V. cholerae likely detects three molecules, Ea-CAI-1, CAI-1 and Ea-C8-CAI-1, but not C8-CAI-1. Therefore, V. harveyi CqsS has a stringent specificity for its ligand, while V. cholerae CqsS is somewhat promiscuous. In addition, our results show that there is a common molecule, Ea-C8-CAI-1, produced and detected efficiently by both V. cholerae and V. harveyi CqsS  and decanoyl coenzyme A (C10-CoA) as substrates  (Supporting information). However, these findings do not account for all the CAI-1 type molecules we detect in cell-free culture fluids. Specifically, V. cholerae must also produce Ea-C8-CAI-1. We wondered therefore if similar to their cognate CqsS receptors, V. harveyi and V. cholerae CqsA enzymes possess high and low substrate specificity respectively. To examine this possibility, the two CqsA enzymes were purified and used in a coupled-enzyme assay  to determine the kinetic parameters with respect to processing C8-CoA or C10-CoA as substrates.In another study, we showed that Vibrio cholerae CqsA uses C10-CoA and C8-CoA to make Ea-CAI-1 and Ea-C8-CAI-1 respectively . In contrast, the V. harveyi CqsA enzyme only uses C8-CoA to produce Ea-C8-CAI-1 and is unable to use C10-CoA as a substrate -3-hydroxytridecan-4-one -3-aminoundec-2-en-4-one . In contrast, CqsA/CqsS systems similar to the V. cholerae system are relaxed . Both C8-CoA and C10-CoA can be used as substrates for CqsA, and multiple signals including CAI-1, Ea-CAI-1 and Ea-C8-CAI-1 can be detected by CqsS. Ea-C8-CAI-1 links the two types of systems as it is produced and detected by both.Although we focused in this study on  by CqsS  and 2. Ve ligand  and 2. V harveyi  and 2. BV. cholerae CqsS, and thus we wondered why we did not identify these enamino compounds previously  has been studied  or at least in close proximity with an occasional insertion of an accessory gene (e.g. in L. pneumophila and B. xenovorans). Finally, signal biosynthesis and signal detection profiles in V. cholerae and V. harveyi match closely . JMH626 is a V. harveyiΔcqsAΔluxQΔluxN mutant that does not produce Vh-CAI-1 due to the cqsA mutation. In addition, JMH626 does not detect HAI-1 and AI-2 because it lacks both the LuxN and LuxQ receptors  or LM with tetracycline (for V. cholerae carrying pBB1 and V. harveyi carrying cqsS on pLAFR2) and diluted 20-fold with sterile medium. Approximately 10–30% (v/v) of spent culture fluids were added to the diluted reporter strains. Bioluminescence and OD600 were measured in an Envison Multilabel Reader following 4 h incubation at 30°C with shaking. Sterile medium was used as the negative control. Synthetically prepared CAI-1 analogues were dissolved in DMSO and supplied at varying concentrations to the reporter strains. DMSO was used as the negative control.Culture fluids were prepared from overnight cultures unless otherwise specified. Cells were removed from the fluids by centrifugation and subsequent filtration through 0.22 µm filters. To assess CAI-1 activity in these cell-free fluids, overnight cultures of reporter strains were grown in LM medium (for Escherichia coli overexpressing V. harveyi cqsA (WN1327) was grown overnight in LB with kanamycin at 30°C with shaking. The culture was diluted 1000-fold in M9 minimal salts medium (Sigma) supplemented with 0.05% (w/v) leucine, 2 mM MgCl2, 0.1 mM CaCl2, 0.01% (w/v) thiamine and 0.4% (w/v) glucose with 10 mg l−1 kanamycin. The culture was incubated overnight at 30°C with shaking. Cells were removed by centrifugation and the cleared fluid was extracted by DCM. The extract was carefully concentrated without heating to a small volume (5 ml) in a Rotovap. The concentrated extract was fractionated using HPLC (see Supporting information). Each HPLC fraction was assayed in triplicate for V. harveyi CqsS agonist activity using the reporter strain JMH626 as described above. Fractions containing activity were further characterized (Supporting information).V. cholerae CqsA enzyme was purified as previously described  resulting in strain WN1666. Strain WN1666 was grown in LB with kanamycin at 30°C with shaking until the OD600 of the culture reached ∼1.0. IPTG was added at a final concentration of 100 µM, and the culture was incubated for an additional 4 h at 30°C with shaking. Cells were harvested by centrifugation, suspended in lysis buffer , and lysed using a Cell Cracker. Soluble materials were loaded onto a HiTrap chelating column charged with nickel, the column was washed extensively with lysis buffer, and His6-tagged V. harveyi CqsA was eluted using a linear gradient of increasing concentration of imidazole dissolved in lysis buffer. Fractions containing CqsA were pooled, the buffer was exchanged, and protein was stored at −20°C in storage buffer . Protein concentrations were determined by Bio-Rad Protein Assay Reagent.The + to form succinyl CoA, NADH and CO2 when excess α-ketoglutarate dehydrogenase is supplied. The rate of NADH production is followed at 340 nm using a spectrophotometer. Briefly, the rates of CoA release from V. cholerae and V. harveyi CqsA enzymes were measured in reactions containing 20 mM potassium phosphate buffer pH 7.0, 3 mM MgCl2, 1 mM NAD+, 1 mM α-ketoglutarate, 200 µM SAM, 1 unit of α-ketoglutarate dehydrogenase (Sigma) and 60–300 nM CqsA. Decanoyl CoA or octanoyl CoA was added to the reactions at concentrations of 1–200 µM. The rate of CoA production was monitored for 30 min. Data were fitted using Graphpad Prism to obtain the kinetic parameters.A coupled-enzyme assay , 0.3 M NaCl, 0.5% (w/v) glucose and incubated at 30°C with shaking. Cell growth was monitored at OD600. When OD600 reached ∼1.0, cells were removed by centrifugation. Pure d2-CAI-1 was added to 9 ml of collected cell-free fluids at 500 nM as an internal control. The mixtures were extracted into 500 µl of DCM. The organic layer was separated and 100 µl of this extract was diluted into 100 µl of HRMS solvent (9:1 acetonitrile : H2O with 0.1% formic acid), and this sample was injected directly for HRMS analysis. Due to variability in the aqueous/organic partitioning during extraction and the different ionization potentials of the CAI-1 type molecules, we determined a correction factor for each molecule by preparing a mixed sample containing known concentrations of each of the CAI-1 type molecules together with an internal standard (d2-CAI-1) in M9 medium. This sample was treated in an identical manner to the cell-free fluids described above.Bacteria were grown in LM medium at 30°C overnight with shaking. The cultures were diluted 200-fold in M9 minimal salts medium (Sigma) supplemented with 2 mM MgClSupporting information.All chemical syntheses and analytical methods are provided in


There are four known strains of HTLV including HTLV type 1 (HTLV-1), HTLV-2, HTLV-3 and HTLV-4. HTLV-1 is primarily associated with adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-2 is rarely pathogenic and is sporadically associated with neurological disorders. There have been no diseases associated with HTLV-3 or HTLV-4 to date. Due to the difference in the disease manifestation between HTLV-1 and HTLV-2, a clear understanding of their individual pathobiologies and the role of various viral proteins in transformation should provide insights into better prognosis and prevention strategies. In this review, we aim to summarize the data accumulated so far in the transformation and pathogenesis of HTLV-1, focusing on the viral Tax and HBZ and citing appropriate comparisons to HTLV-2.Human T lymphotropic viruses (HTLVs) are complex deltaretroviruses that do not contain a proto-oncogene in their genome, yet are capable of transforming primary T lymphocytes both  There are four known strains of HTLV, of which HTLV-1 and HTLV-2 are the most prevalent worldwide. HTLV-1 was originally identified in 1980 in a T cell line derived from a patient with cutaneous T cell lymphoma . SubsequHTLV-1 is an enveloped virus that is approximately 100 nm in diameter. The inner membrane of the virion envelope is lined by the viral matrix protein (MA). This structure encloses the viral capsid (CA), which carries two identical strands of the genomic RNA as well as functional protease (Pro), integrase (IN), and reverse transcriptase (RT) enzymes. A newly synthesized viral particle attaches to the target cell receptor through the viral envelope (Env) and enters via fusion, which is followed by the uncoating of the capsid and the release of its contents into the cell cytoplasm. The viral RNA is reverse transcribed into double stranded DNA by the RT. This double stranded DNA is then transported to the nucleus and becomes integrated into the host chromosome forming the provirus. The provirus contains the promoter and enhancer elements for transcription initiation in the long terminal repeats (LTR); the polyadenylation signal for plus strand transcription is located in the 3′LTR ). Once these complexes stabilize, Tax recruits the co-activators CBP/p300 and P-CAF to activate transcription [The Tax response element (TxRE) in the unique 3′ (U3) region of the 5′LTR is a 21-bp triple repeat, which contains an octamer motif TGACG(T/A)(C/G)(T/A) that is flanked by a G stretch at the 5′end and a C stretch at the 3′end. This octamer motif is homologous to the cAMP response element (CRE) 5′-TGACGTCA-3′ ,176. Taxith TxRE –183. Allith TxRE . Tax intith TxRE –190. Taxith TxRE ,192. Taxith TxRE ,194. Thrnd c-myb ,196. Taxnd c-myb ,197. Mornd c-myb –203. The8.2.Apart from activating viral gene transcription, Tax induces various cellular functions in the HTLV-1 infected cells and thus, renders them susceptible to viral persistence and thereby initiates neoplastic transformation. NF-κB is a key player in Tax-induced T cell transformation. The NF-κB family of transcription factors includes five structurally related members – RelA, RelB, c-Rel, NF-κB1 (p50/p105) and NF-κB2 (p52/p100) –206. Thein vitro and in vivo has been demonstrated by several groups [In naïve T cells, the activity of NF-κB pathways is tightly controlled. NF-κB is transiently activated upon immune stimulation, but constitutively activated in HTLV-1 infected T cells –213. Conl groups ,30,227. l groups –245. App8.3.INK4A and p15INK4B [Tax has two main functions in the pathogenesis of HTLV-1. First is constitutive cell cycle progression and the second is resistance to apoptosis. Tax induces cell cycle progression by protein-protein interaction and transcriptional regulation of cell cycle-associated proteins. Specifically, Tax stimulates the transition of cells from G1 to S phase through the hyper-phosphorylation of Rb and activation of E2F transcription factors and by the induction of cyclin D2 and cyclin-dependent kinase 6 (CDK6) via the NF-κB pathway . In addition, Tax activates CDK4/CDK6 through the direct interaction with CDK4, CDK6 and CDK inhibitors like CDKN1A, p16p15INK4B –255. Thup15INK4B ; Tax indp15INK4B ; Tax infp15INK4B ,258–261.p15INK4B –267. Botp53. The biological activity of p53 is central to the integrity of a cell in that any loss of its function either due to mutation or inactivation increases the chance of genetic instability leading to oncogenesis. The subcellular localization and phosphorylation status of p53 are critical for its function. In a majority of HTLV-1 infected cell lines, p53 function is impaired although the protein itself is sufficiently expressed and stable. Two mechanisms have been identified for the abrogation of p53 function by Tax. First, it was demonstrated that the expression of Tax-1 or Tax-2 impaired the functionally relevant phosphorylation of p53 at serines 15 and 392; Tax activation of the NF-κB pathway was essential for this activity [Tax exhibits its oncogenic potential by both clastogenic DNA damage and aneuploidic effects ,268,269.activity ,274. Secactivity ,276.Drosophila discs large protein (DLG) and epithelial tight junction protein (Zonula Occludens-1). The PDZ domain is commonly used in eukaryotic cells to recruit and organize proteins to sites of cellular signaling. Using both virus gene knockout and knockin approaches, Xie et al. investigated the role of the Tax PBM in both cell culture and infected animals. These authors demonstrated that the PBM of HTLV-1 Tax significantly increased HTLV-1-induced primary T cell proliferation in vitro. Moreover, HTLV-1 proviruses containing a deletion in this four amino acid motif had severely attenuated infectivity and persistence in vivo [in vitro, thus lending further support that this domain plays a key role in Tax pathogenic activity[A PDZ binding motif (PBM) or domain, which is comprised of a four amino acid sequence at the C-terminus of HTLV-1 Tax, has been implicated in T cell proliferation and transformation. The PDZ domain is named after the first identified PDZ-containing proteins, post-synaptic density protein (PSD-95),  in vivo . HTLV-2  activity. The thr activity–281. HTL activity; PBM com activity; and PBM activity, all of  activity,284,285. activity,286,287.9.Hbz transcription is driven by a TATA-less promoter in the 3′ end of the proviral genome activated primarily by Sp1 [usHbz) and two spliced forms (sHbz) – major and minor, as identified by 5′ rapid amplification of cDNA ends (RACE) [sHbz transcripts have multiple initiation sites in the unique 5′ (U5) and R regions of the 3′LTR, whereas the usHbz initiates from within the tax gene. The TxRE in the 3′LTR functions as the promoter enhancer element for Hbz transcription, although its function is much weaker compared to the 5′LTR TxRE [tax and Hbz are inversely proportional during the course of HTLV-1 infection and disease progression. There are marked differences between the unspliced and spliced HBZ isoforms both at the RNA and protein levels  in the sense strand. In the antisense strand, this region forms a variable stem loop structure that might interact with host factors to induce the proliferation of ATL cells in an IL-2-independent manner [et al. have reported a correlation between the Hbz transcript levels and the severity of HAM/TSP, which suggests a possible role for HBZ in the pathogenesis of HAM/TSP [HBZ is encoded by an mRNA transcribed from the minus strand of the proviral genome. Transcription from the antisense strand of HTLV-1 was first reported in 1989 . Almost y by Sp1 . HBZ is s (RACE) ,290,291.LTR TxRE ,292. Thin levels . Hbz tran levels . Total Hcription . The dift manner ,289. Sai HAM/TSP .MafB is a transcriptional factor that is responsible for lineage-specific hematopoiesis. Additional investigations are required to delineate the effect of this suppression [Western blot analyses have shown the expression of sHBZ protein in almost all ATL cell lines. The HBZ protein contains three domains – activation, central and basic leucine zipper (bZIP) ,295. HBZpression .in vitro. When exposed to DCs, HBZ was processed and presented in the context of HLA-A*0201. HBZ peptides induced specific cytotoxic T lymphocytes (CTL), but they failed to lyse HLA-A*0201-positive HTLV-1-infected T cell lines and ATL cells. This could be because the amount of HBZ protein expressed by HTLV-1 infected cells is not sufficient to be recognized by the HBZ-specific CTLs. HBZ-specific CTLs are detected in very sparse numbers in ATL patients and healthy carriers [Although no antibody responses against HBZ have been detected so far from HTLV-1-infected carriers , recent carriers .Hbz mRNA levels are significantly lower than tax/rex mRNA levels [Hbz was dispensable for immortalization of primary human T cells in vitro. Stable T cell clones generated with Hbz defective proviruses produced significantly higher levels of p19 Gag protein indicating increased Tax-mediated viral gene expression [Hbz RNA, specifically a stem loop structure near the amino terminus of the transcript, and not the protein, increased T cell proliferation by increasing the transcription of the E2F1 gene [Kinetic analyses of gene expression levels in HTLV-1 proviral plasmid over-expression studies in cell culture revealed that A levels . In the pression . Stable pression  and incrpression . Knockdopression . Hbz RNA2F1 gene . HBZ bin2F1 gene ,308. NF-et al. have demonstrated that siRNA-mediated knockdown of CBP/p300 or a truncated form of CBP/p300 containing the KIX domain abrogated the expression of Dickkopf-1 (Dkk-1), a Wnt signaling inhibitor in HBZ transfected cells [DKK-1 mRNA levels were positively correlated with the expression of Hbz in HTLV-1 infected cell lines. Interestingly, Tax represses the expression of Dkk-1 consistent with the opposing forces of Tax and Hbz within the cell. Taken together, in the late stages of ATL, when Tax is repressed, HBZ supports cell proliferation and the maintenance of the leukemic cell.Recently, Polakowski ed cells . Dkk-1 hIn vivo studies in rabbits revealed a significant reduction in proviral load and attenuated antibody response against the viral proteins correlating with the loss of HBZ function [in vitro data that Hbz mRNA levels were low early after infection and then increased to a stable plateau. This trend was similar to that of the proviral loads and was the reverse of the tax/rex mRNA levels [+ T cells in the spleens of transgenic mice expressing HBZ under the control of the mouse CD4 promoter/enhancer [γchain−/− mice showed that HBZ knockdown in a transformed T cell line significantly reduced tumor formation and organ infiltration [function . ProviraA levels . Thus, Henhancer . Anotherltration . Togethe10.tax have been documented; DNA methylation of tax has been shown in another 15% of ATL patients; and deletion of the 5′LTR and promoter region has been shown in an additional 27% of ATL cases [tax/rex mRNA in the nucleus [tax/rex mRNA [A number of mechanisms involving Tax and HBZ have been demonstrated to play a role in HTLV-1-induced oncogenesis . Tax is TL cases –316. In  nucleus ,103. By rex mRNA . TogetheHbz is detected during the later stages of HTLV infection, unlike tax/rex and gag/pol, which are expressed at high levels during the early stages of infection. Expression of HBZ is correlated positively with proviral loads and negatively with tax/rex and gag/pol expression [tax gene and/or the 5′LTR. Neither the Hbz gene nor the 3′LTR are affected by any of these epigenetic silencing mechanisms. Miyazaki et al. showed that in one ATL case, the polyadenylation site of Hbz gene was deleted. However, the Hbz gene utilized a downstream polyadenylation signal for transcription [In HTLV-1, the 3′LTR is identical to the 5′LTR. Both carry the TxRE sequences that have the enhancer elements for transcription. However, the enhancing elements of the antisense transcript are much weaker than the sense transcript. This could be part of the reason why pression . HBZ exppression ,293. HBZcription .Tax initially increases hTERT expression facilitating lifespan extension and immortalization of cells. Once the virus establishes persistence, Tax represses hTERT via an E-box present in the hTERT promoter probably through competition for CBP/p300, and thus favors accumulation of chromosomal rearrangements and then the transformation of the infected cells towards a malignant phenotype. Subsequent epigenetic silencing of Tax in the leukemic cells reactivates hTERT, which in turn, stimulates the proliferative potential of the infected cells leading to ATL. The reactivation of hTERT is a key event in the induction of ATL progression, which is probably tailored by HBZ .et al. [env, tax, p13 and p30 to be responsible for the reduced viral gene expression in these cells. The generation of nonsense mutations does not affect the Hbz gene, which codes from the minus strand. Moreover, there are very few mutation sites in the opposite strand of the HBZ coding region. Therefore, the functional activity of HBZ is not affected. It also was shown that these mutations occur both in ATL cells and HTLV-1 infected cells from asymptomatic carriers, indicating that these mutations do not occur during oncogenesis but are present even during the carrier state. The mutations were present in the leukemic cells and not in the non-leukemic HTLV-1 infected cells, suggesting that there is a selection for these cells during leukemogenesis, which likely favors the virus by promoting immune evasion [tax, rex and p30 genes were frequently deleted, and thus Tax-mediated activation of NF-κB and CREB pathways was abrogated. In all these cases, the coding sequences of the Hbz gene were intact [Another possible mechanism that could cause epigenetic silencing has been elucidated recently by Fan et al. . They sh evasion . Evidence intact .Taken together, the possible mechanism of HTLV-1-induced oncogenesis suggests that Tax is expressed initially to induce transformation and cell proliferation. Tax-specific CTLs mediate the death of virus infected cells. Thus, the virus is forced to down-regulate Tax expression to below detectable levels by the expression of HBZ in order to persist in the host. Since HBZ-specific CTLs do not lyse ATL cells and HBZ can promote and maintain the leukemic state of these cells, this mechanism ought to be favored by the virus in order to evade immune surveillance and continue to persist in the host. However, why it would be in the best interest of the virus to cause cancer, which is a dead end situation for itself, although in a very small percentage of infected individuals, is still an unanswered question.11.HTLVs are complex retroviruses with unique proteins that have oncogenic potential. There are four known strains of HTLV, HTLV-1, HTLV-2, HTLV-3, HTLV-4; only HTLV-1 and HTLV-2 have been consistently associated with disease in humans. HTLV-1 mainly causes ATL and HAM/TSP. HTLV-2 is not etiologically oncogenic and has been associated with some neurological disorders. HTLV-3 and HTLV-4 were identified in African primate hunters without any subsequent etiological disease associations so far.A complete understanding of the functions of the viral genes would give insights into the pathogenic mechanisms by which HTLV-1 induces oncogenesis. In this review we summarize the data published so far in this field with pertinent comparisons to HTLV-2, the non-leukemic counterpart. Like simple retroviruses, HTLV-1 expresses structural and enzymatic proteins for its assembly and maturation, and for entry into new target cells. HTLV-1 also expresses regulatory and accessory proteins that are essential for viral persistence, immune evasion and ultimately, leukemogenesis. Although, the exact mechanisms and pathways have not been fully elucidated, much is known thus far. For instance, Tax is expressed in the early stages of infection to establish viral transcription and induce T cell transformation by regulating cellular transcription factors, inducing G1 to S phase transition and DNA damage resulting in genetic instability, and promoting proliferation of genetically altered (leukemic) cells. Subsequently, HBZ suppresses Tax expression to evade immune elimination by Tax-specific CTLs, and also complements for Tax to support proliferation; it provides a second oncogenic signal required for the maintenance of the leukemic cell. Further investigations are warranted to determine the additional events in the minor population that progresses to ATL.