Graded Hedgehog (Hh) signaling governs the balance of Gli transcriptional activators and repressors to specify diverse ventral cell fates in the spinal cord. It remains unclear how distinct intracellular Gli activity is generated. Here, we demonstrate that Sufu acts universally as a negative regulator of Hh signaling, whereas Kif7 inhibits Gli activity in cooperation with, and independent of, Sufu. Together, they deter naïve precursors from acquiring increasingly ventral identity. We show that Kif7 is also required to establish high intracellular Gli activity by antagonizing the Sufu-inhibition of Gli2. Strikingly, by abolishing the negative regulatory action of Sufu, diverse ventral cell fates can be specified in the absence of extracellular Hh signaling. These data suggest that Sufu is the primary regulator of graded Hh signaling and establish that the antagonistic and cooperative actions of Kif7 and Sufu are responsible for setting up distinct Gli activity in ventral cell fate specification. Furthermore, a temporal requirement of Shh signaling is involved in the induction of the non-neuronal FP cells. While FP induction depends initially on attaining high levels of Shh signaling, subsequent down-regulation of signaling in presumptive Foxa2+ FP cells is required for their differentiation into mature Shh+/Foxa2+ FP cells; however if this down-regulation is blocked, these cells adopt a p3 fate Sonic hedgehog (Shh) acts as a classical morphogen forming a ventral-to-dorsal signaling gradient to specify diverse cell fates in the spinal cord Shh−/− or Smo−/− neural tubes, FP, p3 and pMN fates are not specified due to the absence of Shh signaling Gli2−/− and Gli1−/−;Gli2+/− embryos Ptch1−/− embryos with elevated levels of Gli activators Mutant studies in mice illustrate that Shh signaling activity is governed by the balance of Gli activators and repressors Sufu−/− embryos exhibit a severely ventralized neural tube, whereas Kif7−/− embryos display a subtle phenotype with a slight expansion of the pMN domain. It is unknown whether Sufu and Kif7 function together during ventral patterning. Here, we show that they possess cooperative as well as antagonistic functions in the specification of graded Gli activity in the ventral neural tube. Strikingly, when Sufu function is eliminated, diverse ventral cell fates can be specified despite the absence of Shh signaling.Sufu and Kif7 are two key conserved regulators of Gli proteins Kif7−/−;Sufu−/− mice and examined the expression of Olig2 and Nkx2.2, which are markers of pMN and p3 cells respectively, at embryonic day (E) 9.5. While Sufu−/− embryos exhibit an increase of both Olig2+ cells (45% increase) and Nkx2.2+ cells (3-fold increase), Kif7−/−embryos only show a slight increase (25%) of Olig2+ cells  at the expense of Olig2+ cells  in the number of FP cells  can be specified despite the absence of graded Shh signaling. Thus, a Shh signaling gradient is no longer necessary to drive graded intracellular Gli activity responsible for the specification of distinct neuronal and non-neuronal cell fates when the inhibitory action of Sufu is eliminated.Our results so far indicate that when Smo is activated, Kif7 regulation of Sufu's inhibitory action on Gli2 is a critical step in Shh signaling. We next investigated the roles of Kif7 and Sufu in cells lacking Smo, in which the pathway is repressed and insensitive to Hh inputs. We have previously shown that ated Smo '. Striki embryos '. Though embryos , the relGli383kD . ImportaSmo−/− embryos), inactivation of Kif7 or Sufu can lead to specification of distinct ventral cell fates. We propose that, through acting on both Gli activators and repressors, Sufu and Kif7 function cooperatively to generate graded intracellular Gli activity  to Sufu-floxed mice Kif7SmoGli2Ptch1Embryos were harvested at E 9.5 and fixed in 4% paraformaldehyde (PFA) in PBS at 4°C overnight, then subjected to ethanol series dehydration, paraffin embedding and sectioning at 7 µm. Immunohistochemistry on anterior spinal cord sections was performed as described Ptch1 single mutant embryos which arrest at 16–20 somite pairs. Cell fate quantification data were expressed and plotted as means ± standard error of the mean (SEM). Statistical analysis for multiple comparisons was completed using one-way ANOVA followed by the Bonferroni post-test , except ost-test  and 3K.  (t-Test . The samShhWhole-mount or section in situ hybridization was performed as described Embryos were snap-frozen, and sonicated in RIPA buffer as described


Herpesviruses constitute a very large and diverse family of DNA viruses, which can generally be subdivided in alpha-, beta- and gammaherpesvirus subfamilies. Increasing evidence indicates that many herpesviruses interact with cytoskeleton-regulating Rho GTPase signaling pathways during different phases of their replication cycle. Because of the large differences between herpesvirus subfamilies, the molecular mechanisms and specific consequences of individual herpesvirus interactions with Rho GTPase signaling may differ. However, some evolutionary distinct but similar general effects on Rho GTPase signaling and the cytoskeleton have also been reported. Examples of these include Rho GTPase-mediated nuclear translocation of virus during entry in a host cell and Rho GTPase-mediated viral cell-to-cell spread during later stages of infection. The current review gives an overview of both general and individual interactions of herpesviruses with Rho GTPase signaling. At the same time, the cytoskeleton constitutes both a formidable barrier and a powerful tool for viruses trying to invade and take over a cell. It is therefore not surprising that herpesviruses have evolved several interactions with the cytoskeleton to facilitate their replication and spread. Increasing evidence indicates that, at multiple stages of their replication cycle, herpesviruses interfere with cellular signaling pathways that affect the structure and organization of the cytoskeleton. The current review will discuss recent progress in our understanding of how herpesviruses interfere with signaling pathways that affect the actin cytoskeleton.Cytoskeletal signaling is mainly initiated by extracellular stimuli that activate cell surface receptors such as G protein coupled receptors (GPCR), integrins and receptor tyrosine kinases (RTK). Signaling downstream of these receptors culminates in activation of specific Rho GTPases –3. Rho GThe current review aims at giving a comprehensive overview of the known interactions between herpesviruses and Rho GTPase signaling, rather than giving an overview on the variety of interactions between these viruses and the actin cytoskeleton. As a consequence, different effects of herpesviruses with the cytoskeleton that have not been related to effects on Rho GTPase signaling will not be discussed. These have been reviewed extensively elsewhere ,16.2.The past years have revealed that herpesvirus entry is often, if not generally, associated with Rho GTPase signaling. This is particularly evident for alpha- and gammaherpesviruses. Less information is available for betaherpesviruses. Herpesviruses gain access to the cytosol of a host cell by fusion of the envelope with a host membrane. Depending on the virus and the type of host cell, this fusion event may occur directly at the cell surface or upon uptake of the virus via endocytosis, macropinocytosis, or phagocytosis. The need and specificity of Rho GTPase signaling and actin rearrangements during herpesvirus entry may very well depend on which of these uptake routes are engaged during infection of a particular cell. Indeed, direct fusion at the cell surface on itself may not require dramatic actin rearrangements. However, this entry route confronts the viral particle with the cortical actin barrier just beneath the plasma membrane, which likely needs to be resolved to some extent by a viral triggered mechanism. Engaging one of the endocytic uptake routes may circumvent this problem, but may still rely on virus-induced Rho GTPase signaling during the initial interaction of the virus with the cell since several of these uptake routes, particularly macropinocytosis and phagocytosis, require Rho GTPase signaling and significant actin rearrangements to proceed ,18. The 2.1.Several lines of evidence suggest that alphaherpesviruses interfere at different points with Rho GTPase signaling pathways during viral entry . AlphaheRegardless of the viral entry pathway, alphaherpesviruses first contact the cell by interaction of viral envelope proteins gB and gC with heparan sulfate moieties on the cell surface. Live cell imaging of HSV-1 infected retinal pigment epithelial cells demonstrated that many virions first attach to filopodia-like structures and move unidirectionally along these filopodia to reach the cell body . HSV notViral induction of filopodia and viral surfing along filopodia likely aid in recruiting high numbers of virus particles towards entry receptors on the cell surface, facilitating subsequent virus entry. Although not formally shown as of yet, it is possible that HSV surfing along filopodia occurs through myosin/actin-dependent retrograde flow, as described for retroviruses . PerhapsAnother alphaherpesvirus envelope protein, gD, has been associated with Rho GTPase signaling during viral entry ,27. The Rho GTPase signaling through gD/nectin-1 has also been reported for a swine alphaherpesvirus, pseudorabies virus (PRV) during viral entry in primary trigeminal ganglion neurons, a central target cell type for different alphaherpesviruses . PRV entAlthough it is obvious that alphaherpesvirus entry in host cells is associated with Rho GTPase signaling, considerable variation has been reported regarding the Rho GTPase members involved. This variation appears to mainly depend on the cell type investigated. Brief Cdc42 activation and sustained RhoA activation occurs during HSV-1 infection of CHO-nectin-1 cells . In MDCKIn order for cell surface receptors to activate Rho GTPase signaling, different intermediate molecules may be involved. Focal adhesion kinases (FAK) and phophatidyl inositol-3 kinase (PI3K) are two prominent intermediate signaling molecules. In CHO-nectin-1 cells, PI3K activity is involved in trafficking of HSV-1 virus particles through the cytosol to the nucleus, but not in viral attachment or penetration . In line2.2.In betaherpesvirus HCMV infection, EGFR serves as a receptor and αvβ3 integrin as a coreceptor. Upon HCMV infection in fibroblasts, viral glycoproteins gB and gH independently bind to EGFR and αvβ3 integrin, respectively, to initiate viral entry and signaling. EGFR activates PI3K, while αvβ3 integrin signals to Src, and their coordinated action results in further downstream signaling. Both signaling pathways are involved in the decreased total and activated RhoA levels observed upon HCMV infection in fibroblasts . HCMV en2.3.per se did not seem to affect viral binding, entry, nuclear transport or infection in fibroblasts [versus endothelial cells [One of the best-documented interactions of herpesviruses with cytoskeletal signaling is triggered by the interaction of the RGD motif of KSHV gB with its cellular entry receptor α3β1integrin. In fibroblasts, this binding induces integrin signaling which contributes to viral entry. KSHV-induced integrin signaling in fibroblasts results in phosphorylation of FAK, which leads to the activation and phosphorylation of Src tyrosine kinases and PI3K. This in turn results in the activation of Rho GTPase signaling pathways. Cdc42 was activated from five minutes post infection, followed by sustained RhoA and ezrin activation, resulting in cytoskeletal rearrangements, including stress fiber and filopodia formation –46. Althroblasts . Howeverroblasts . One expal cells . In endoal cells ,48. In lal cells . Howeveral cells . NeverthIn fibroblasts, the role of KSHV-induced Rho GTPase activation on virus entry appears to rely on its subsequent effects on microtubules, another major component of the host cytoskeleton. KSHV-induced Rho GTPase signaling results in acetylation and thereby stabilization of microtubules. Microtubule stabilization facilitated delivery of viral DNA to the nucleus, likely by promoting dynein-mediated transport of virus from the periphery to the nucleus. The stabilization of microtubules was found to be mediated by one of the downstream effectors of RhoA, mDia2, a member of the formin family .Src tyrosine kinases appear to be crucial in KSHV-induced RhoA activation. On the one hand, KSHV induces Src activation, which in turn leads to RhoA activation. On the other hand, RhoA activation of mDia2 results in activation of Src. This creates a positive feedback loop that results in sustained Src activation, which is important during viral entry . Src tyrTaken together, entry by members of the three herpesvirus subfamilies have been reported to be associated with altered Rho GTPase signaling. Activation of FAK and its downstream activation of RhoA seems to be a conserved characteristic of alpha- and gammaherpesviruses, whereas betaherpesvirus entry may be associated with RhoA suppression. In either case, modulation of RhoA signaling enhances trafficking of viral particles to the nucleus.3.Increasing evidence indicates that herpesviruses also trigger Rho GTPase signaling and consequent actin rearrangements later in infection. Again, like for entry, less is known concerning betaherpesvirus interaction with Rho GTPases compared to alpha- and gammaherpesviruses. It will therefore be interesting to determine whether this points to an actual difference in ability of these viruses to interact with Rho GTPase signaling, or whether this particular field of research has been less explored thus far in betaherpesviruses. Both for alpha- and gammaherpesviruses, interactions with Rho GTPase signaling during later stages of infection are associated with the formation of cell projections and are thereby believed to promote viral spread. In addition, for gammaherpesviruses, interactions with Rho GTPase signaling during latency affect cell motility and invasion and may therefore be involved in malignancies associated with these viruses.3.1.et al. (2000). Based on experiments using different dominant active and negative constructs, it was hypothesized that US3 may regulate its effects on the actin cytoskeleton through Cdc42/Rac1 signaling pathways [It is becoming increasingly clear that lytic alphaherpesvirus infection is associated with alterations in Rho GTPase signaling and concomittant drastic rearrangements of the actin cytoskeleton. These rearrangements have been largely attributed to the viral US3 kinase, which is a conserved alphaherpesvirus serine/threonine kinase. For HSV-2, PRV, BHV-1 and MDV, the US3 kinase induces actin rearrangements consisting of the formation of long cellular projections and/or the disassembly of actin stress fibers –60. US3-pathways . More repathways . Activatpathways . PRV US3pathways .Although the conserved phenotype of US3-mediated actin rearrangements suggests a conserved mechanism of action for different alphaherpesviruses, there are indications for differences in the mode of action of US3 of different alphaherpesviruses. Indeed, for PRV, HSV-2, and BHV-1, point mutations in conserved kinase regions of US3 showed that the catalytic kinase activity of US3 is crucial for reorganization of the actin cytoskeleton, while this was not the case for MDV ,60,63,643.2.The gammaherpesvirus Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with a number of malignancies. These cancers are characterized by the proliferation of EBV-infected cells and viral expression in these cells is limited to a subset of latent genes ,67. The A recent report demonstrates that LMP1-mediated actin signaling not only occurs via small Rho GTPases but also through activation of the protein kinase C (PKC) pathway. This leads to phosphorylation of ezrin, a member of the ezrin/moesin/radixin family that acts as a linker between the plasma membrane and the actin cytoskeleton. Phosphorylated ezrin translocates to the plasma membrane and is linked to CD44 and the actin cytoskeleton, which leads to increased cell motility and invasion .During acute infection, gammaherpesviruses have been reported to induce long, branched cell projections, resembling the US3-mediated phenotype in alphaherpesvirus infections described above. Like the alphaherpesvirus US3-induced cell projections, gammaherpesvirus-induced membrane protrusions contain actin and appear to promote intercellular virus spread ,74. Desp4.vice versa. A conserved feature of many, if not all, herpesvirus subfamilies seems to be the role of cytoskeletal signaling in the efficient delivery of virions to the nucleus. PI3K signaling appears to play a central role during this stage of viral infection. The underlying mechanism is not clear, although it may involve increased dynein-mediated transport through acetylation of microtubules [Increasing evidence points at the importance of cytoskeletal signaling in several key phases of herpesvirus infection, including entry, lytic infection and latent infection. However, many questions remain for future research in this relatively young field of research. Herpesviruses may embark on different uptake routes, depending on the virus and the host cell type. Recent data suggest that herpesvirus entry may be generally associated with Rho GTPase signaling, although the specifics and consequences are different for different herpesviruses and different host cells. It will therefore be interesting to further define the impact of the nature of Rho GTPase signaling on the specific entry pathway employed by herpesviruses in different cell types, and otubules ,50.Most of the studies on Rho GTPase signaling during viral entry have focused on signaling initiated by binding of envelope proteins with cell surface receptors. However, herpesvirus access to the cytoplasm involves release of tegument proteins in the cytosol. The potential impact of these tegument proteins on signaling pathways, including Rho GTPase pathways, is largely unknown but may be biologically relevant. The fact that at least one alphaherpesvirus tegument protein, US3, has been reported to interfere with Rho GTPase signaling  may be iDuring acute infection, severe rearrangements of the actin cytoskeleton including actin stress fiber disassembly and the formation of cell projections have been reported for alpha- and gammaherpesviruses. It is not surprising that Rho GTPase signaling plays a critical role in these actin rearrangements, but the underlying mechanisms are not fully understood. Although the role of US3 in the actin reorganization appears to be well conserved in alphaherpesviruses, it remains to be determined whether US3 acts through one conserved mechanism or whether different alphaherpesvirus US3 orthologs employ different mechanisms to manipulate actin. Although gammaherpesviruses induce similar actin rearrangments as observed for alphaherpesviruses, the viral triggers are different. It is unclear at present whether the lack of reports on similar phenotypes in betaherpesviruses indicates that these types of actin rearrangments and viral spread do not occur in this herpesvirus subfamily or whether this has not been investigated specifically as of yet. In this context, it is interesting that viral proteins from evolutionary much more distinct virus families induce similar actin rearrangements, including F11L of vaccinia virus and Nef of HIV –79. Furt


How the Human T lymphotropic virus type 1 (HTLV-1) Tax protein stimulates proliferation while triggering cell cycle arrest and senescence remains puzzling. There is also a debate about the ability of Tax to activate or inhibit the DNA damage response. Here, we comment on these different activities and propose a conceptual rationale for the apparently conflicting observations. First[in vivo ,2.In vivo cell dynamic studies based on deuterated glucose incorporation methods revealed that HTLV-1 persists because Tax, likely in concert with other viral proteins, actively increases infected cell division [Like other oncogenes, the HTLV-1 Tax protein transiently brakes cell cycle progression or even initiates apoptosis and senescence programs -13. On tdivision . Similardivision -20 suppoCell cycle arrest triggered by oncogenes results from an initial highly proliferative state associated with altered DNA replication  and mitochondrial dysfunctions (increased reactive oxygen species (ROS) production) leading to the formation of double strand breaks (DSB). Subsequent engagement of the DNA damage response pathway (DDR) then halts cell cycle progression -23. If DOverall, onset of DNA damage that alerts the DDR machinery is the critical step in oncogene-induced cell cycle arrest. Recently, we and others described two novel mechanisms by which Tax compromises genome stability ,16,24. TARF and Rb/p16INK4a [WAF1/CIP1/p27KIP1 expression and loss of Skp2 E3-ubiquitin ligase [In human cells, DDR signaling cascades converge to trigger two powerful tumor suppressor pathways, p53/p19p16INK4a . It shoup16INK4a . In thisn ligase . If senen ligase ,13. The in vitro HTLV-1 infection halts cells in the G1 phase of the cell cycle [tax/rex, env and gag/pol mRNAs were expressed first and at the highest levels immediately after infection [Ex vivo culture of primary cells from infected patients also demonstrates that the tax/rex mRNA precedes expression of other viral transcripts [in vivo, this process would in fact preclude viral dissemination in the early steps of infection. It is well understood that this phenomenon does not happen. As indicated above, senescence, and more broadly proliferative arrest, occurs in response to a mitotic burst engaged by oncogenic stimuli, a highly proliferative state reproducibly preceding DNA damage formation and DDR stimulation by oncogenes [Although ll cycle , experimnfection . Ex vivonscripts . Assuminncogenes ,31. Imponcogenes ,12,13, wncogenes . At the ncogenes ,16. Thisncogenes ,32. In fAccording to this model, the DDR machinery is a major barrier against Tax-induced proliferation apparently conflicting with a series of reports describing the ability of Tax to inhibit the DDR pathway ,26,33-35As depicted in Figure The authors declare that they have no competing interests.MB and LW wrote the manuscript. Both authors read and approved the final manuscript.


A combination therapy of interferon-α (IFN-α) and zidovudin (AZT) shows therapeutic effects in ATL patients, although its mechanism is also obscure. We previously found that viral gene expression in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients was markedly suppressed by stromal cells through a type I IFN response. Here, we investigated the effects of IFN-α with or without AZT on viral gene expression and cell growth in ILTs.Human T-cell leukemia virus type-1 (HTLV-1) is the causative retrovirus of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 gene expression is maintained at low levels ILTs expressed variable but lower amounts of HTLV-1 Tax protein than HTLV-1-transformed HUT102 cells. Following the addition of IFN-α, the amounts of HTLV-1 p19 in the supernatants of these cells decreased in three days, while HTLV-1 gene expression decreased only in ILTs but not HUT102 cells. IFN-α also suppressed the spontaneous HTLV-1 induction in primary ATL cells cultured for 24 h. A time course study using ILTs revealed that the levels of intracellular Tax proteins decreased in the first 24 h after addition of IFN-α, before the reduction in HTLV-1 mRNA levels. The initial decreases of Tax protein following IFN-α treatment were observed in 6 of 7 ILT lines tested, although the reduction rates varied among ILT lines. An RNA-dependent protein kinase (PKR)-inhibitor reversed IFN-mediated suppression of Tax in ILTs. IFN-α also induced cell cycle arrest at the G0/G1 phase and suppressed NF-κB activities in these cells. AZT alone did not affect HTLV-1 gene expression, cell viability or NF-κB activities. AZT combined with IFN-α markedly induced cell apoptosis associated with phosphorylation of p53 and induction of p53-responsive genes in ILTs.IFN-α suppressed HTLV-1 gene expression at least through a PKR-mediated mechanism, and also induced cell cycle arrest in ILTs. In combination with AZT, IFN-α further induced p53 signaling and cell apoptosis in these cells. These findings suggest that HTLV-1-infected cells at an IL-2-dependent stage retain susceptibility to type I IFN-mediated regulation of viral expression, and partly explain how AZT/IFN-α produces therapeutic effects in ATL. HTLV-1 mRNA, but not proteins, are detectable in peripheral blood mononuclear cells (PBMCs) of HTLV-1-infected individuals [in vivo, as HTLV-1-infected individuals maintain antibodies against HTLV-1 structural proteins and Tax protein-specific cytotoxic T lymphocytes.Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL) , a maligividuals . AlthougEx vivo studies have indicated that graft-versus-tumor responses including anti-Tax cytotoxic T-cells were potentially involved in the therapeutic mechanisms of allo-HSCT [in vitro[in vitro[in vivo.Recent therapeutic approaches, such as allogeneic hematopoietic stem cell transplantation  ,8, a humllo-HSCT , and thallo-HSCT . However[in vitro, and the[in vitro. The com[in vitro,19. As Iin vitro. For example, IFN-α reduced the virus release but not viral protein synthesis in HTLV-1-transformed HUT102 or MT-2 cells [in vitro, while conflicting with the clinical effects of AZT/IFN-α therapy in ATL patients. This discrepancy between in vivo and in vitro systems can be partially attributed to differences in status of HTLV-1-infected cells between the two systems.It is well established that HTLV-1-infected cells are resistant to type I IFNs -2 cells . The mec-2 cells , Tax-med-2 cells , Tax-med-2 cells ,24, and -2 cells , all of in vitro. Our experimental system differed from previous studies in two ways. First, we used IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients, while previous studies used IL-2-independent HTLV-1-transformed cell lines such as HUT102. Second, we used stromal cells as effectors; these mediated the type I IFN response, but could have also produced multiple factors other than IFNs.We previously found that HTLV-1-infected cells could induce type I IFN responses in co-cultured stromal cells . We alsoIn the present study, we investigated whether purified type I-IFNs can affect viral expression and cell growth of HTLV-1-infected cells by using various ILTs. Here we report a novel finding that IFN-α suppresses intracellular Tax expression at a translational level at least through PKR. We further demonstrate that IFN-α activates p53 pathways in cooperation with AZT, partly explaining the mechanisms of the therapeutic effects of AZT/IFN-α in ATL.We evaluated the baseline levels of HTLV-1 gene expression in HUT102, ILT-Hod and ILT-#29 cell lines  on HUT102, ILT-Hod, and ILT-#29 cells  or HAM/TSP patients  were maintained in RPMI 1640 medium  containing 10% fetal calf serum , Antibiotic Antimycotic Solution (Sigma Aldrich) and 30–100 IU/ml of recombinant human IL-2 . The IL-2-independent HTLV-1-infected T-cell line, HUT102, derived from a patient with mycosis fungoides  and MOLTAlexa Fluor 488-conjugated Lt-4 , a mouse13H8N4OS; Calbiochem) and its negative control inhibitor  were dissolved in DMSO and added at 500 nM in culture 2 h before IFN-α treatment and carried over through the culture. Pifithrin-α p-nitro cyclic, a chemical p53-inhibitor , was used at 1 μM.Natural type human IFN-α  was added to cell cultures at various concentrations. Zidovudin (AZT)  was used at 10 μM, a concentration inhibiting reverse transcription without cell toxicity . When cuAliquots (0.5 μg) of total RNA extracted from cells using Isogen  were treated with DNase  and subjected to reverse transcription (RT) with oligo(dT)20 primers followed by PCR using THUNDERBIRD qPCR Mix . To quantify HTLV-1 mRNAs, three primer sets were used; Gag primers  located at Gag region, pX primers  located at pX region, and RPX primers  located at upstream and downstream of the second splice junction site of tax/rex mRNA . The priFor intracellular staining of HTLV-1 antigens, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 100% methanol for 10 min on ice. To detect the Gag protein, cells were serially incubated with GIN-7 or control ascites and FITC-conjugated goat anti-mouse IgG + IgM. To detect the Tax protein, cells were incubated with Alexa Fluor 488-labelled Lt-4 or isotype control antibody. To stain intracellular Ki-67, cells were fixed with 4% paraformaldehyde and permeabilized with a BD Perm/Wash™ Buffer Kit (BD Pharmingen), then incubated with an R-PE-conjugated anti-human Ki-67 mouse mAb or isotype control mouse IgG1, κ antibody. For staining intracellular phosphorylated p53, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 100% methanol for 10 min on ice prior to incubation with antibody. Stained cells were analyzed with a flow cytometer  using FlowJo software (Tree Star).HTLV-1-infected or uninfected cells were dissolved in Cell Culture Lysis Reagent  containing 25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100, with protease inhibitor cocktail , and were incubated on ice for 1 hour. The cell lysates were cleared by centrifugation, denatured with SDS sample buffer  and 2.5% 2-mercaptethanol  at 70°C for 15 min, and 17 μg of proteins were electrophoresed on polyacrylamide gel , and then transferred to PVDF membrane . The membranes was blocked with Block Ace  overnight, and reacted with mouse anti-Tax and anti-α-Tubulin  antibodies as primary antibodies overnight, followed by exposure to horseradish peroxidase-conjugated sheep anti-mouse IgG whole antibody  as a second antibody. The reacted bands were visualized by enhanced chemiluminescence using Novel® ECL  and analyzed on Image Quant mini LAS 4000 .Cells were cultured in the presence of 10 μM bromodeoxyuridine (BrdU) for 24 h, fixed and then permeabilized, followed by incubation with a mouse anti-BrdU mAb and 7AAD from BrdU flow Kits (BD Pharmingen), according to the manufacturers’ instructions. Stained cells were analyzed with a flow cytometer using CellQuest software (Becton Dickinson). To evaluate cell growth, Trypan blue exclusion test and a colorimetric assay using Cell Counting Kit-8  based on formazan color development were used.The concentration of HTLV-1 p19 in the supernatants from ILT-Hod, ILT-#29 or HUT102 cultures were measured using a RETRO-tek HTLV-1/II p19 antigen ELISA  according to the manufacturer’s instructions.Reporter cell lines (ILT-Hod/NF-κB-Luc and ILT-#29/NF-κB-Luc) were established by using a Cignal Lenti-NF-κB reporter Luc Kit  and Cignal Lenti thymidine kinase (TK)-Renilla control (Qiagen). Luciferase assays were conducted with Luciferase or Renilla luciferase assay systems  on cell lysates in Renilla luciferase lysis buffer (Promega). Relative NF-κB activity was calculated as the ratio of firefly luciferase to renilla luciferase activities in the same sample.t-test was performed for statistical significance, and P values less than 0.05 were considered significant.The unpaired ATL: Adult T-cell leukemia/lymphoma; AZT: Zidovudin (3'-Azido-3'-deoxythymidine); BrdU: Bromodeoxyuridine; ELISA: Enzyme-linked immunosorbent assay; FCS: Fetal calf serum; FITC: Fluorescein-isothiocyanate; HAM/TSP: HTLV-1-associated myelopathy/tropical spastic paraparesis; HSCT: Hematopoietic stem cell transplantation; HTLV-1: Human T-cell leukemia virus type-1; IFN-α: Interferon-α; IgG: Immunoglobulin G; ILT: IL-2-dependent HTLV-1-infected T-cell; mAb: Monoclonal antibody; MFI: Mean fluorescence intensity; PBMC: Peripheral blood mononuclear cell; PKR: RNA-dependent protein kinase; PMA: Phorbol 12-myristate 13-acetate; RT: Reverse transcription; TK: Thymidine kinase; VEGF: Vascular endothelial growth factorThe authors declare that they have no competing interests.SK carried out most of the experiments, analyzed data, and wrote the manuscript; MKi and AT carried out certain aspects of the experiments; AH advised on flow cytometry analysis; AS advised on signaling analysis; TM advised on RT-PCR analysis; YT provided HTLV-1-specific monoclonal antibodies; AU provided clinical samples; MKa designed the study, analyzed data, and wrote the manuscript; all authors reviewed and approved the final manuscript.


Objective(s): HTLV-I and HIV virus quantification is an important marker for assessment of virus activities. Since there is a direct relationship between the number of virus and disease progression, HTLV-I and HIV co-infection might have an influence on the development of viral associated diseases, thus, viral replication of these viruses and co-infection were evaluated.Materials and Methods: In this study, 40 subjects were selected; 14 HIV infected, 20 HTLV-I infected and 6 HTLV-I/HIV co-infected subjects. The amount of viruses was measured using qPCR TaqMan method and CD4 and CD8 lymphocytes were assessed by flow cytometry.Results: The mean viral load of HIV infected subjects and HTLV-I infected individuals were 134626.07±60031.07 copies/ml and 373.6±143.3 copies/104 cells, respectively. The mean HIV viral load in co-infected group was 158947±78203.59 copies/ml which is higher than HIV infected group. The mean proviral load of HTLV-I in co-infected group was 222.33±82.56 copies/ml which is lower than HTLV-I infected group (P<0.05). Also, the mean white blood cell count was higher in co-infected group (5666.67±1146.49 cells/μl). However, the differences between these subjects did not reach to a statistical significance within 95% confidence interval level (P =0.1). No significant differences were observed regarding CD4 and CD8 positive lymphocytes between these groups.Conclusion: HTLV-I/HIV co-infection might promote HIV replication and could reduce the HTLV-I proviral load, in infected cells. Considering the presence of both viruses in Khorasan provinces, it encourages researchers and health administrators to have a better understanding of co-infection outcome. Retroviridae family. This virus mostly infects CD4+ T lymphocytes which have a crucial role in immune regulation and causes immunodeficiency syndrome (AIDS) by severe lymphocyte depletion. A global total of 40 million people were diagnosed with HIV infection in 2012 and almost 5 million people were reported as newly infected individuals (-). Based on the AIDS progress report in 2012, HIV prevalence in Iran has been significantly increased. Therefore, effective control and prevention of HIV and also appropriate monitoring of HIV infected individuals must be implemented as major health programs in Iran (Human immunodeficiency virus (HIV) is a lentivirus belonging to  in Iran .Retroviridae family and associated with two main types of diseases; adult T-cell leukemia (ATL) and the inflammatory condition named HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Human T lymphotropic virus type I (HTLV-I) is a retrovirus belonging to HAM/TSP) -8. This HAM/TSP) -10. HTLVHAM/TSP) .HTLV-I proviral load is a major determinant of HTLV-I infection outcome and higher HTLV-I proviral load as well as the interaction between the virus and the host have crucial role in the development of HTLV-I associated diseases -16.Furthermore, an increased frequency of other clinical complications has been reported in HIV–HTLV-I/II co-infection . It seems that introduction of highly active antiretroviral therapy (HAART), has been led to longer survival for HIV-infected individuals and stops HIV replication, but does not have any protective impact on HTLV proviral load, CD4 cell count and HAM/TSP development  and those newly diagnosed asymptomatic HTLV-I cases were included in this study. Including criteria were as follow; newly diagnosed untreated subjects, non IV drug users, Khorasani residents and healthy carriers without any diseases symptoms at the time of sampling. Forty subjects met our study criteria including; 14 HIV, 20 HTLV-I and 6 HTLV-I/HIV co-infected subjects. For each subject, clinical staging was assigned based on the revised criteria of WHO staging system  by an inThe study was approved by the Research Ethics Committee of Mashhad University of Medical Sciences (No: 88797). Participants had full authority to participate in study. An informed consent was taken from participants. All information were recorded and tagged with proper identification codes to identify participants for confidentiality. , lymphadenopathy, ext.) and general complications. All participants were completed a standard questionnaire including demographic information and the history of any current illness. Specialized physicians examined the patients and recorded the results that were including complications related to immune system  and anti-HIV  by enzyme-linked immunosorbent assay (ELISA) according to manufacturer instruct-tions in Center of Infectious Diseases . Positive specimens were confirmed by a conventional polymerase chain reaction (PCR). To confirm HTLV-I infection a conventional PCR was carried out for Tax and LTR region on PBMCs extracted DNA. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) using an available commercial kit  and PCR amplification was performed using specific primers for tax (5-AGGGTTTGGACA GAGTCTT-3 and 5-AAGGACCTTGAGGGTCTTA- 3) and LTR regions (5-CATAAGCTCAGACCTCCGGG-3 and 5- GGATGGCGGCCTCAGGTAGG-3).HTLV-I proviral load 4 and expressed as the number of HTLV-I provirus per 104 PBMCs . Genomic DNA was extracted from PBMCs using an enzymatic method (QIAamp® DNA Mini Kit) according to the manufacturer instructions. A real time PCR (Taqman probe) using an absolute quantification kit was carried out to measure the HTLV-I proviral load in PBMCs using specific primers and a fluorogenic probe which have been tested on Rotorgen Q 6000 machine  The HTLV-I copy number was reported as an actual amount of cellular DNA by means of the quantification of the albumin gene as the reference gene. HTLV-I and albumin DNA concentrations were calculated from two 5-point standard curves. The normalized value of the HTLV-I proviral load was calculated as the ratio of  ×1004 PBMCs .HIV viral loadTo examine the HIV viral load, RNA was extracted from plasma using Viral RNA extraction Mini Kit . A Real Time PCR (Taqman probe) using a commercial absolute quantification kit  was carried out to measure HIV viral load by a Rotorgen Q machine . Evaluation of CD4 and CD8 countsCD4, CD8 and CD3 cells were stained by conjugated antibodies FITC, PE and PreCP, respectively . Cells were analyzed using a FACS Calibour  flow cytometer.Statistical analysisP- values of < 0.05 were considered statistically significant.Data were analyzed by Mann-Whitney and Independent Sample T Tests using SPSS/ver.16 software. Descriptive data were summarized as mean with standard deviation (SD) and standard error of mean (SEM). Thirty seven subjects with mean age of 35.88 ± 1.89 years (ranging 22-50 years) were studied. The mean age of HIV, HTLV-I and HTLV-I/HIV co-infected subjects were 36.43 ± 2.66, 35.71 ± 3.87 and 33.67 ± 2.33 years, respectively.p < 0.05). The mean HIV viral load in HIV group was 134626.07 ± 60031.07 and in HTLV-I/HIV co-infected group was 158947 ± 78203.59. There was no significant difference between two groups.The mean HTLV-I proviral load in HTLV-I infected individuals was 373.6 ± 143.3 and in HTLV-I/HIV co-infected group was 222.333 ± 82.56, the difference was statistically significant (p < 0.05). The results of this study show that HIV viral load in HTLV-I/HIV co-infected patients (158947 ± 78203.59) was higher than in HIV infected patients (134626.07 ± 60031.07). Furthermore, HTLV-I proviral load in HTLV-I/HIV co-infected patients (222.3333 ± 82.56) is lower than in HTLV-I infected patients (373.6 ± 143.3) (p = 0.1) level.The mean white blood cell count was higher in co-infected group (5666.67±1146.492 cells/μL) than both groups (P = 0.1). However, the differences between these subjects did not reach a statistical significance within 95% confidence interval (The CD4+ cells count in HTLV-I infected (659.9 ± 110.7) and co-infected patients (431.43 ± 120) was higher than HIV infected group (414 ± 97.5), however, the differences were not statistically significant.HIV and HTLV-I are two human retroviruses that mostly contaminate CD4+ helper lymphocytes. HTLV-I/HIV co-infection has been reported in different parts of the world. Since there is a direct relationship between HIV- RNA plasma level and the clinical progression of AIDS. High HTLV-I proviral has been detected in HTLV-I related diseases which are implicated in clinical progression and severity of these diseases , 32-35. In the present study, HTLV-I proviral load in HTLV-I infected patients was higher than co-infected patients , which wHelper T lymphocytes (CD4+) are the main targets for HTLV-I and HIV and in case of HTLV-I and HIV; it leads to T cell proliferation , 8 and dIn a recent study, evaluation of absolute lymphocyte count among HTLV-I/HIV co-infected and HIV infected groups showed that, lymphocyte reduction in co-infected patients occurs more slowly than in HIV infected group. This could explain the higher HTLV-I proviral load in co-infected patients due to increasing number of host cells for HIV virus. In contrast, progressive depletion of CD4+ T cells (target cells for HTLV-I virus infection) might explain the reduction of HTLV-I provirus load in the co-infected patients . Furthermore, in the present study like some previous results, HTLV-I proviral load in women was higher than men and in contrast, HIV viral load in men was higher than women  which maHTLV-I/HIV co-infection could possibly increase the HIV viral load and reduce the HTLV-I proviral load. Since HTLV-I/HIV co-infection is increasing in different parts of the world, larger studies should be conducted to discover the interactions between HIV and HTLV-I and host to have a better understanding of clinical co-infection outcome, transmission and monitoring HTLV-I/HIV co-infection subjects.


Recent findings suggest that both subcellular compartmentation and route of sucrolysis are important for plant development, growth, and yield. Signaling effects are dependent on the tissue, cell type, and stage of development. Downstream effects also depend on the amount and localization of hexoses and disaccharides. All enzymes of sucrose metabolism  are not produced from single genes, but from paralog families in plant genomes. Each paralog has unique expression across plant organs and developmental stages. Multiple isoforms can be targeted to different cellular compartments . Many of the key enzymes are regulated by post-transcriptional modifications and associate in multimeric protein complexes. Some isoforms have regulatory functions, either in addition to or in replacement of their catalytic activity. This explains why some isozymes are not redundant, but also complicates elucidation of their specific involvement in sugar signaling. The subcellular compartmentation of sucrose metabolism forces refinement of some of the paradigms of sugar signaling during physiological processes. For example, the catalytic and signaling functions of diverse paralogs needs to be more carefully analyzed in the context of post-genomic biology. It is important to note that it is the differential localization of both the sugars themselves as well as the sugar-metabolizing enzymes that ultimately led to sugar signaling. We conclude that a combination of subcellular complexity and gene duplication/subfunctionalization gave rise to sugar signaling as a regulatory mechanism in plant cells. Through this review, we show how one should consider both extensive gene duplication and extensive cellular compartmentation of plant metabolism to fully understand sugar signaling. Establishment of sugar gradients across different subcellular compartments, cells, and organs is a central issue of plant physiology; therefore, we address the multiplicity of sucrolytic pathways in sink and source tissues.a priori that sugar perception and signal transduction is also compartmentalized  is typically harvested and homogenized to determine metabolite levels as a bulk megacompartment, thereby both ignoring diversity of cell types and further mixing subcellular organelles  is a powerful technique for separating subcellular compartments under conditions in which biological activities are completely arrested . This metabolism . Metaboltabolism , Arabidos leaves , potato s leaves , and pots leaves , 2001, ss leaves .Improved methods for single-cell transcriptomics, proteomics, and metabolomics are needed for a holistic understanding of sugar signaling . FluoresFigure 1). AtHXK1 is predominantly associated with the mitochondria but can also occur in the nucleus  are crucial for sucrose loading into the phloem, but their role as sensors remains elusive. In yeast, the glucose transporters RGT2 and SNF3 act as low- and high-affinity glucose sensors, respectively  is needed for reproductive development in maize  and Arabbidopsis , and is bidopsis  and sugabidopsis . T6P is bidopsis . Supplyibidopsis , althougbidopsis . T6P maybidopsis .Overexpression of yeast invertase (INV) and bacterial glucokinase in the cytosol was intended as a strategy to increase sucrose import and sink strength in potato tubers . ContrarHeterologous expression of sucrose phosphorylase (SuPho) was used to bypass SUS (sucrose synthase)/fructokinase (FK) and INV/HXK routes . It decrFigure 1). A low sucrose/hexose ratio promotes respiration over starch synthesis . Neutral/alkaline INVs have ascribed functions for energy metabolism and oxidative stress in the mitochondria .Sucrose 6-phosphate synthase (SPSs) are encoded by a multigene family whose tissue- and developmental stage-specific expression patterns appear to have functional significance . The actGenome-scale metabolic modeling is well-established for microbes , but is not yet established in plants, which posses many different isozymes and subcellular compartments . VariousSugar signaling research has advanced rapidly in recent years. Learning more will require acknowledging the importance of subcellular compartmentation and routinely implementing all available methods . A key fSince carbon metabolism occurs simultaneously in different organelles, different sensors may be required under specific conditions or circumstances. Specific isoforms of sucrolytic enzymes are therefore not redundant but complementary for signaling or catalysis. We propose that different sucrolytic enzymes are important for channeling carbon into different metabolic routes, and we further postulate that sugar signals are specific for each paralog/pathway.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.


To estimate the risk for developing a defined neurodegenerative syndrome in a large cohort of idiopathic REM sleep behavior disorder (IRBD) patients with long follow-up.Using the Kaplan-Meier method, we estimated the disease-free survival rate from defined neurodegenerative syndromes in all the consecutive IRBD patients diagnosed and followed-up in our tertiary referal sleep center between November 1991 and July 2013.The cohort comprises 174 patients with a median age at diagnosis of IRBD of 69 years and a median follow-up of four years. The risk of a defined neurodegenerative syndrome from the time of IRBD diagnosis was 33.1% at five years, 75.7% at ten years, and 90.9% at 14 years. The median conversion time was 7.5 years. Emerging diagnoses (37.4%) were dementia with Lewy bodies (DLB) in 29 subjects, Parkinson disease (PD) in 22, multiple system atrophy (MSA) in two, and mild cognitive impairment (MCI) in 12. In six cases, in whom postmortem was performed, neuropathological examination disclosed neuronal loss and widespread Lewy-type pathology in the brain in each case.In a large IRBD cohort diagnosed in a tertiary referal sleep center, prolonged follow-up indicated that the majority of patients are eventually diagnosed with the synucleinopathies PD, DLB and less frequently MSA. IRBD represented the prodromal period of these conditions. Our findings in IRBD have important implications in clinical practice, in the investigation of the early pathological events occurring in the synucleinopathies, and for the design of interventions with potential disease-modifying agents. REM sleep behavior disorder (RBD) is a parasomnia characterized by dream-enacting behaviors associated with REM sleep without muscle atonia In the present study we determined the risk of development a defined neurodegenerative syndrome in all the consecutive 174 subjects that were diagnosed with IRBD between November 1991 and July 2013.In all instances the diagnosis of IRBD required history of dream-enacting behaviors, video-polysomnography detection of REM sleep with increased muscular activity associated with abnormal behaviors, absence of known neurodegenerative diseases, lack of motor and cognitive complaints, normal neurological examination, and RBD not explained by a brain lesion  or by the introduction or withdrawal of any medication or substance  The methods of clinical assessment at first and follow-up visits have been described elsewhere In the current study, to assess the presence and nature of a defined neurodegenerative syndrome, clinical records were reviewed, and all available patients were scheduled at our clinic (or at their homes or nursing homes) between February and August 2013. During these assessments, we conducted a detailed medical history, a neurological examination and neuropsychological testing when cognitive symptoms were first reported or noticed. The clinical outcome of the patients  was determined in all cases by means of direct, face-to-face clinical interviews, neurological examinations and neuropsychological tests. Diagnoses were not done by telephone interviews. We also reviewed the neuropathological findings  and the time of the last follow-up assessment or death. The date of IRBD diagnosis was defined as the date when video-polysomnography demonstrated REM sleep without atonia linked to abnormal behaviors. Follow-up duration was defined as the interval from diagnosis of IRBD to the time of the last visit or death. The onset of a defined neurodegenerative syndrome  was determined as the date when the diagnosis was made according to accepted clinical criteria. Duration of any emerging defined neurodegenerative syndrome was defined as the interval between the time of its diagnosis and the time of the last follow-up assessment or death.14 Disease-free survival rate was assessed from the date of IRBD diagnosis to the date of the diagnosis of defined neurodegenerative syndrome or to the last follow-up visit for censored observations (subjects who died or were lost to follow-up).The risk of developing a defined neurodegenerative syndrome in the 174 subjects from the cohort was estimated by means of the Kaplan–Meier method.t test and χ2 test, as appropriate. The risk of developing a neurodegenerative syndrome was calculated for the two groups, and comparisons were assessed with the log rank test.Demographic and clinical variables were compared between the first 44 subjects reported previously P values less than 0.05 were considered to be significant. All analyses were done with SPSS version 20.0.In our sleep center, 174 individuals were diagnosed with IRBD between November 1991 and July 2013. With the exceptions of the duration of follow-up and the duration of RBD, there were no demographic and clinical differences between the first 44 subjects who were diagnosed between November 1991 and March 2003 and the remaining 130 who were diagnosed between April 2003 and July 2013 .Patients were 136 (78.2%) men and 38 (21.8%) women with a median age at estimated RBD onset of 62.0  years, median age at diagnosis of IRBD of 69.0  years, and median interval between estimated RBD onset and IRBD diagnosis of 4.0  years. The median age at last visit was 74.0  years and the median follow-up from IRBD diagnosis to the last visit or to death was 4.0  years.By the 2013 assessment, 65 (37.4%) patients were diagnosed with a defined neurodegenerative syndrome, 103 (59.2%) remained disease-free, and 6 (3.4%) were lost to follow-up with the diagnosis of IRBD at their last visit. Emerging diagnoses were DLB in 29 subjects, PD in 22 (6 developed dementia after the diagnosis of PD), MCI in 12 and MSA in two. These two subjects were diagnosed with MSA of the cerebellar subtype based on the presence of cerebellar signs plus dysautonomic features . Follow-up visits disclosed the development of rigid-akinetic parkinsonism unresponsive to levodopa in both, and stridor due to bilateral vocal cord paralysis in one. None of our IRBD patients were diagnosed with pure autonomic failure. In 25 of the 29 subjects who were diagnosed with DLB, a previous period of MCI was recognized, and the median interval between the diagnosis of MCI and DLB was 2.0  years. Two patients with the diagnosis of MCI developed PD after two years, and they have not developed dementia or visual hallucinations after 2.4 and 3.1 years of follow-up.In the 65 subjects diagnosed with a defined neurodegenerative syndrome, the median interval between estimated RBD onset and diagnosis of a defined neurodegenerative syndrome was 11.0  years, and the median interval between IRBD diagnosis and diagnosis of a defined neurodegenerative syndrome was 4.0  years. The risk of a defined neurodegenerative syndrome from IRBD diagnosis was 33.1% at five years, 75.7% at ten years, and 90.9% at 14 years . The medDuring the follow-up period, 32 (18.4%) subjects were deceased. Twenty-four of them were clinically diagnosed during life with a defined neurodegenerative syndrome , and the remaining eight had the diagnosis of IRBD at their last follow-up visit. Six patients with the antemortem diagnoses of DLB (n = 3), PD  and MCI (n = 1) were brain donors. In two of them  the peripheral autonomic nervous system was also available for examination. In the brain, all six cases presented variable neuronal loss, gliosis and alpha-synuclein immunoreactive Lewy pathology (Lewy bodies and Lewy neurites) in vulnerable regions such as the olfactory bulb, dorsal motor nucleus vagal nerve, central raphe nucleus, the nuclei that regulate REM sleep muscle atonia , substantia nigra pars compacta, limbic structures , and nucleus basalis of Meynert. Lewy pathology was also detected in the neocortex of the three patients with the antemortem diagnoses of DLB and of the PD patient who developed dementia, corresponding in all of them to Lewy body Braak stage 5 This is the largest study with long term follow-up of IRBD. In a cohort of 174 patients that were closely followed during a median period of 4.0 years, survival curves showed that the estimated risk of a defined neurodegenerative syndrome from the time of IRBD diagnosis was 33.1% at five years, 75.7% at ten years, and 90.9% at 14 years. Patients were clinically diagnosed with PD, DLB, MSA and MCI. In patients who underwent postmortem brain examination, the antemortem diagnoses of PD and DLB were confirmed showing neuronal loss and widespread Lewy-type pathology in selective brain regions. The patient with MCI and lack of parkinsonism and dementia also had widespread Lewy type pathology. The current study confirms our first observation Similar results have been found by two other studies where IRBD patients diagnosed in sleep tertiary centers were clinically followed. The nature of disorders (synucleinopathies) evolving from these two cohorts is very similar to what we have found in our study. In the first study, Schenck et al. found that parkinsonism developed in 11 of 29 (38%) IRBD subjects nearly four years after the diagnosis of IRBD and almost 13 years after the onset of RBD Our findings indicate that there is a strong and specific association of IRBD with PD and DLB, and that this parasomnia can be considered in most cases a prodromal manifestation. In PD, other clinical features known to precede the motor symptoms such as hyposmia, constipation, depression and hypersomnia are much less specific since only a small number of individuals with these symptoms are eventually diagnosed with PD The findings of our study have clinical relevance and research implications. The investigation of patients with IRBD offers a unique opportunity to obtain information of the aetiology, pathogenesis and progression of the prodromal stage of the synucleinopathies. In IRBD, clinical, neuroimaging, biochemical, proteomic and genetic studies will help to better understand the mechanisms of neurodegeneration and to design interventions with potential disease modifying properties. To date, several studies have added weight to the concept that IRBD represents in most cases an evolving synucleinopathy in its earliest phases. Several studies have found that IRBD patients display clinical (e.g., hyposmia The finding that IRBD patients are later frequently diagnosed with DLB and PD with associated dementia suggests that both disorders may represent different phenotypes from the same condition. MCI is an intermediate stage between normal cognitive function and dementia where individuals have cognitive complaints, deficits in neuropsychological tests and their daily living activities are preserved Limitations of our study should be acknowledged. Our study did not include a control group of healthy subjects without RBD who were followed-up on during a similar time period. In the present study, the proportion of IRBD subjects that developed a neurological condition was 37% over a median follow-up period of 4 years. This proportion is greatly superior to the estimated prevalence of these neurological disorders in a population of individuals of similar age and sex distribution In summary, our study shows that a majority of IRBD patients with prolonged follow-up are diagnosed with the synucleinopathies PD, DLB and MSA. IRBD represents the prodromal period of these conditions. Our findings in IRBD have important implications in clinical practice, in the investigation of the early pathological events occurring in the synucleinopathies, and for the design of future trials with potential disease-modifying agents.


Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type I (HTLV-1) infection and often occurs in HTLV-1-endemic areas, such as southwestern Japan, the Caribbean islands, Central and South America, Intertropical Africa, and Middle East. To date, many epidemiological studies have been conducted to investigate the incidence of ATL among general population or HTLV-1 carriers and to identify a variety of laboratory, molecular, and host-specific markers to be possible predictive factors for developing ATL because HTLV-1 infection alone is not sufficient to develop ATL. This literature review focuses on the epidemiology of ATL and the risk factors for the development of ATL from HTLV-1 carriers, while keeping information on the epidemiology of HTLV-1 to a minimum. The main lines of epidemiological evidence are: (1) ATL occurs mostly in adults, at least 20–30 years after the HTLV-1 infection, (2) age at onset differs across geographic areas: the average age in the Central and South America (around 40 years old) is younger than that in Japan (around 60 years old), (3) ATL occurs in those infected in childhood, but seldom occurs in those infected in adulthood, (4) male carriers have about a three- to fivefold higher risk of developing ATL than female, (5) the estimated lifetime risk of developing ATL in HTLV-1 carriers is 6–7% for men and 2–3% for women in Japan, (6) a low anti-Tax reactivity, a high soluble interleukin-2 receptor level, a high anti-HTLV-1 titer, and high levels of circulating abnormal lymphocytes and white blood cell count are accepted risk factors for the development of ATL, and (7) a higher proviral load  is an independent risk factor for progression of ATL. Nevertheless, the current epidemiological evidence is insufficient to fully understand the oncogenesis of ATL. Further well-designed epidemiological studies are needed. Adult T-cell leukemia (ATL) was first reported as a distinct clinical entity in Japan in 1977  all patients with ATL have antibodies against HTLV-1  in HTLV-1 endemic areas in the Kyushu district, southwest Japan  reported that the annual incidence of ATL was estimated to be approximately 6.1 in adults aged over 30 years per 100,000 populations . Nevertheless, several case series of ATL were available. The first case of ATL was reported in Taiwan in 1985  and Urmia are known to be an endemic area for HTLV-1, where the prevalence of HTLV-1 infection was reported to be 0.34–0.77% in blood donors , especially in Rio de Janeiro and Salvador (1.8%) on the northeast coast of the country where the population is largely of African descent. ATL accounts for approximately 30% of patients with T-cell malignancies in Brazil , an overseas French administrative district located on the northeast coast of the South American continent between Brazil and Surinam, is also known to be an area of high endemicity for HTLV-I  has been reported in sub-Saharan African countries, especially in Gabon  may be at higher risk for developing ATL  system in dually infected carriers, which may be a precipitating factor for ATL. The immunosuppressive state has been reported to potentially contribute to ATL development in HTLV-1 carriers. There were several case reports of ATL developed in HTLV-1 carriers undergoing immunosuppressive treatment after living-donor liver transplantation  reported that an HTLV-1 carrier with a high anti-HTLV-1 titer , a high number of circulating abnormal lymphocytes, and a low anti-Tax reactivity were associated with a greater risk of developing ATL  of patients presenting a monoclonal integration of HTLV-1 proviral DNA in their blood lymphocytes. Carvalho and Da Fonseca Porto  are also evaluated in some epidemiological and clinical studies to support the hypothesis that increased HTLV-1 proviral load level is an important predictor of developing ATL.A cross-sectional study , was initiated . Further viral markers are needed to determine the function of a higher HTLV-1 proviral load to direct the way to developing ATL or developing HAM/TSP from HTLV-1 carriers.Although many prior studies found important epidemiological evidence on ATL and risk factors for the development of ATL in HTLV-1 carriers, limited data are available on the valid annual incidence of ATL from longitudinal prospective studies. Existing predisposing factors are still insufficient to explain the characteristics of ATL oncogenesis. Unknown risk factors may be involved in the acquisition of malignant characteristics of HTLV-1 infected cells. Further well-designed epidemiological studies are needed to fully understand the oncogenesis of ATL.http://www.kantei.go.jp/foreign/kan/actions/201009/13htlv_e.html). The challenge in the next few years will be to reduce the number of HTLV-1 carriers, to develop an easy method that allows identification of high-risk carriers, and to implement earlier therapeutic interventions for carriers with high-risk markers.Even though the incidence of ATL is relatively low among HTLV-1 carriers and a novel promising agent, mogamulizumab , is released (Ishida et al., 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.


This review summarizes recent in vitro and in vivo data demonstrating that TGFβ-induced myofibroblast differentiation is driven by a prooxidant shift in redox homeostasis. Elevated NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) supported by concomitant decreases in nitric oxide (NO) signaling and reactive oxygen species scavengers are central factors in the molecular pathogenesis of fibrosis in numerous tissues and organs. Moreover, complex interplay between NOX4-derived H2O2 and NO signaling regulates myofibroblast differentiation. Restoring redox homeostasis via antioxidants or NOX4 inactivation as well as by enhancing NO signaling via activation of soluble guanylyl cyclases or inhibition of phosphodiesterases can inhibit and reverse myofibroblast differentiation. Thus, dysregulated redox signaling represents a potential therapeutic target for the treatment of wide variety of different degenerative fibrotic disorders.Degenerative fibrotic diseases encompass numerous systemic and organ-specific disorders. Despite their associated significant morbidity and mortality, there is currently no effective antifibrotic treatment. Fibrosis is characterized by the development and persistence of myofibroblasts, whose unregulated deposition of extracellular matrix components disrupts signaling cascades and normal tissue architecture leading to organ failure and death. The profibrotic cytokine transforming growth factor beta (TGF The wound healing response in which damaged/dead cells are replaced following acute injury  is essential to maintain tissue architecture and function –4. HowevThere are numerous degenerative fibrotic diseases, including multisystemic disorders such as systemic sclerosis, chronic graft versus host disease, and nephrogenic systemic fibrosis as well as organ-specific diseases, for example, cardiac fibrosis, idiopathic pulmonary fibrosis (IPF), intestinal fibrosis, liver cirrhosis, progressive kidney disease, macular degeneration, and benign prostatic hyperplasia (BPH) –3, 5–12.Despite the considerable morbidity and mortality caused by fibrosis, there are currently no effective treatments for many of these diseases and no approved antifibrotic therapies. In part, this is due to our current lack of knowledge regarding (i) the precise etiology of the initiating injury/infection and (ii) the mechanisms that drive fibrosis progression. Thus, a better understanding of the molecular pathways underlying fibrosis and the initiating signals/causes is urgently required for the development of effective therapeutic strategies. This review focuses on accumulating evidence that redox signaling plays a fundamental and integral role in the molecular pathogenesis of fibrosis in many different tissues and organs and as such represents a potential therapeutic target for the treatment of wide variety of different fibrotic disorders.Fibrotic diseases are clearly distinct in their etiology and clinical manifestation. Nonetheless, fibrogenesis in most organs and tissues progresses in a remarkably similar manner, characterized in particular by the development and persistence of large numbers of myofibroblasts , 9, 12. de novo expression of alpha-smooth muscle cell actin  in stress fibers and (ii) contractile force. The cellular origin of myofibroblasts remains somewhat controversial but may differ depending on the organ and/or the initiating stimulus  as reviewed recently . Myofibrreviewed ). Myofibreviewed , 23. Howβ, which are produced initially by platelets and infiltrating leukocytes at the wound site, are major inducers of fibroblast-to-myofibroblast differentiation [β thus generating an autocrine feed-forward loop driving continued myofibroblast differentiation [Fibroblast-to-myofibroblast differentiation occurs via a two-step process. Following injury or during chronic inflammation, changes in mechanical tension of the ECM are transmitted to the fibroblast cytoskeleton via RhoA/ROCK signaling . Consequntiation . Howeverntiation , 27  and PI3 kinase/Akt pathways [Although several TGFreviewed , 35), TGnd liver –42. Consisorders , 43–50. pathways , 51. Colpathways , 52. Notpathways . On the pathways . Moreovepathways . Thus, rβ undoubtedly plays a pivotal role in pathogenic fibrogenesis. Therapeutic approaches designed to interfere with downstream TGFβ signaling processes that culminate in myofibroblast activation may represent an alternative viable strategy for the treatment of fibrotic disease. In this respect, a convincing body of data implicates dysregulated redox signaling by NADPH oxidase 4 (NOX4) and nitric oxide (NO) in the pathophysiology of fibrosis.TGF2•−) [2O2), although this most likely is a result of rapid superoxide dismutation [2O2 compared to superoxide is consistent with a signaling function of NOX4-derived ROS [High levels of free radicals can result in nonspecific oxidative damage to cell structures and biomolecules. However, when produced in a regulated manner, reactive oxygen species (ROS), NO, and reactive nitrogen species play a critical role as biological second messengers in a variety of cellular processes, including myofibroblast differentiation . NADPH o2•−) . Howevermutation –61. It iived ROS , 62, 63.ived ROS .β, angiotensin II, and platelet-derived growth factor [NOX4 mRNA levels specifically correlated with the myofibroblast phenotype in benign prostatic tissue [α-SMA, were observed in liver biopsy samples from patients with autoimmune hepatitis [β1 [β1-induced cytoskeletal remodeling and maintains the differentiated phenotype of vascular SMCs [Unlike other NOX isoforms, NOX4 is constitutively active with primary regulation occurring at the transcriptional level , 65. NOXc tissue . Similarc tissue . In addic tissue . Recentlepatitis . These oepatitis , 67–71. itis [β1 . Similaritis [β1 , 67, 72.lar SMCs , 74.in vivo studies have provided more definitive evidence that NOX4-derived ROS play a direct role in the pathogenesis of fibrosis. For example, inhibition of NOX4 via genetic deletion, antisense oligonucleotides, siRNA, or NOX inhibitors attenuated disease progression in rodent models of pulmonary, renal, and liver fibrosis [Several fibrosis , 82–84.β remain(s) largely unknown. However, TGFβ1-induced NOX4-derived ROS have been shown to directly oxidatively inactivate MKP1, a dual specificity MAPK phosphatase that targets JNK and p38 [β1-induced myofibroblast differentiation of prostatic fibroblasts and cardiomyocyte differentiation of pluripotent embryonal carcinoma cells [β from its latency association peptide (LAP) are also induced by oxidative modification of LAP with free radicals capable of stimulating TGFβ expression and secretion in many cell types [NOX4 induction appears to contribute to fibrogenesis not via oxidative stress-induced damage , 62, 63, and p38 . Consistma cells , 80. Othma cells , 81. Thull types , 85 -dependent enzymes. We observed downregulation of the Se transporter SEPP1 and Se-containing ROS scavengers glutathione peroxidase 3 (GPX3) and thioredoxin reductase 1 (TXNRD1) during TGFntiation . Moreoventiation . Se is antiation . Consistntiation , 90, exoroblasts . Similarte cells . Furtherstimulus , 93. Constimulus .in vitro and in vivo data indicates that myofibroblast differentiation in fibrotic disorders and tumor-reactive stroma is driven by a prooxidant shift in intracellular redox signaling caused by elevated ROS and/or reduced antioxidative potential. NOX4 appears to be the major source of elevated ROS and central mediator of TGFβ-induced myofibroblast differentiation in diverse tissues. Thus, restoring cellular redox homeostasis by (i) targeting NOX4, (ii) Se supplementation, and/or (iii) application of antioxidants may represent a promising therapeutic strategy for fibrotic disease , involving the oxidation of NADPH and the reduction of molecular oxygen. NO signaling is mediated via activation of soluble guanylyl cyclase (sGC). The second messenger cyclic guanosine monophosphate (cGMP) that is subsequently generated by sGC regulates the activity of cGMP-dependent protein kinases such as protein kinase G (PKG), cyclic nucleotide phosphodiesterases (PDEs), and cation channels and may have other unknown effects [The free radical NO is an important signaling molecule in a variety of biological processes that is biosynthesized  effects .β1 significantly reduced NOS activity and NO levels, whereas the NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) synergistically potentiated TGFβ1-induced collagen production [β1-induced myofibroblast differentiation of human prostatic fibroblasts in vitro [β1-induced collagen production by SNP in dermal fibroblasts in vitro and attenuation of fibrosis in rodent model systems using the NOS substate L-arginine or the NO donor S-Nitroso-N-acetylcysteine (SNAC), respectively [In terms of fibroblast-to-myofibroblast differentiation, NO signaling appears to be a central pathway associated with the fibroblast phenotype . Treatmeoduction . Consistoduction –99. We pin vitro . These fectively , 98, 101ectively .in vitro myofibroblast differentiation of cardiac fibroblasts and dermal fibroblasts from healthy subjects and patients with systemic sclerosis [In vivo BAY 41-2272 limited disease progression in models of renal, cardiac, and dermal fibrosis [Since NO activates sGC, increasing sGC activity via NO-independent heme-dependent sGC stimulators represents an alternative approach to enhance NO signaling . Similarclerosis , 104. Infibrosis –106 and fibrosis , 108. Infibrosis . Under cfibrosis . Of notefibrosis , whilst fibrosis . in vivo, indicating an active role of PDE5 in fibrogenesis [β1-induced myofibroblast differentiation of prostatic fibroblasts in vitro [in vitro and counteracted fibrosis in TGFβ1-induced Peyronie's disease-like plaques in rats [The fact that treatment with the cell-permeable cGMP analog 8-bromo-cGMP is able to mimic the inhibitory effects of enhanced NO/sGC signaling on myofibroblast differentiation clearly indicates that inhibition is mediated downstream via cGMP , 112. Thogenesis . We previn vitro . Likewis in rats , 118. Mo in rats .in vitro differentiated prostatic myofibroblasts restored morphological and molecular characteristics typical of the fibroblast phenotype, indicating that enhancement of NO signaling not only prevents but also might reverse fibrosis [in vitro [in vivo sGC stimulation by BAY 41-8543 decreased tubulointerstitial fibrosis after relief of unilateral ureteral obstruction in rats [β1-induced Peyronie's disease-like plaques in rats [β1-induced Peyronie's disease-like plaques in rats [Similarly to exogenous Se, we recently reported that PDE5 inhibition in fibrosis . Consistin vitro  and in v in rats . Similar in rats  and PDE5 in rats . Of note in rats , 118, inin vitro and in vivo model systems indicate that the fibroblast phenotype is maintained by NO signaling and that myofibroblast differentiation is associated with an attenuation/inhibition of the NO/sGC/cGMP signaling cascade, while stimulation of NO signaling is capable of even reverting myofibroblast differentiation. Thus, enhancement of NO signaling by NO donors, stimulators, and activators of sGC or inhibition of cGMP degradation via PDE inhibitors might be of therapeutic benefit for patients suffering from degenerative fibrotic disease , thereby depleting NO levels [4) leading to NOS uncoupling and superoxide generation rather than NO production [2O2 production [β-induced myofibroblast differentiation by NO and NOX4-derived ROS signaling presumably predominantly occurs via distinct mechanisms  [2O2 production. Since treatment with 8-bromo-cGMP is sufficient to inhibit myofibroblast differentiation [2O2-counteracting effects of elevated NO signaling appear to be mediated via downstream cGMP-dependent mechanisms and not via the NO radical per se.Taken together these findings clearly indicates that Hroblasts . Thus, svations) , indicatntiation , the H2O2O2 and NO signaling, has significant clinical implications. Targeting redox signaling, for example, via inhibitors of NOX4 or PDE5, antioxidants such as Se or enhancers of NO signaling, represents a promising therapeutic strategy to modulate the fibroblast/myofibroblast ratio in pathological conditions such as degenerative fibrotic diseases . However, in some cases it may be necessary/advantageous to identify tissue-specific signaling mechanisms/inducers whose specific targeting is less likely to be associated with adverse side effects on healthy tissues. The observation that fibroblasts and myofibroblasts are interconvertible phenotypes, with phenotypic switching apparently regulated via crosstalk between NOX4/Hdiseases .β unequivocally plays a central role in fibrogenesis in diverse tissues and organs. However, given its essential role in a wide range of fundamental cellular functions, there are concerns that systemic approaches directly targeting TGFβ for the treatment of fibrotic conditions will potentially exert undesirable toxic effects [β agents have been completed and several others are underway. Unfortunately, to date, these trials have largely yielded disappointing results despite promising in vitro observations .2O2 and reduced NO signaling are apparently central downstream components of TGFβ-mediated myofibroblast differentiation in diverse tissues and organs. Thus, therapeutic targeting of redox homeostasis in degenerative fibrotic diseases might also be expected to elicit broad and undesirable effects. However, it should be noted that, unlike TGFβ that drives myofibroblast differentiation and fibrosis via Smad-dependent and -independent pathways [β1 in prostatic fibroblasts [Induction of NOX4-derived Hpathways , 141, NOroblasts , 40, 65.roblasts , 57. Furroblasts –115.Despite intense research efforts, there currently remains no NOX4-specific inhibitor and several attempts to generate peptides that disrupt NOX4 function have been unsuccessful with the authors concluding that, unlike other NOX isoforms, NOX4 exists in a tightly assembled and active conformation, which cannot be disrupted by conventional means , 143. Noβ-dependent manner in rats [Given that the signaling potential of NOX4-derived ROS is regulated by antioxidant systems, enhancing the activity of ROS scavenging enzymes may represent an alternative potential therapeutic strategy . Animal  in rats . However in rats ; thus fuInhibitors of PDE5 are clinically approved for the treatment of erectile dysfunction, pulmonary arterial hypertension, and BPH –115 and 2O2 and NO signaling. In addition, the fact that these enzymes belong to multimembered families may be clinically exploited to selectively target tissue or disease-specific isoforms. For example, selective targeting of PDE1A, that appears to play a critical role in cardiac fibrosis, led to regression of cardiac remodeling in rodents [Due to the presence of multiple NOX, PDE, and GC isoforms, modulation of NOX4, PDE5, and/or sGC activities may permit continued physiological H rodents .β production leads to the development and persistence of large numbers of myofibroblasts, a hallmark of fibrotic disorders  targeting NOX4 directly using specific inhibitors, (ii) indirectly inhibiting NOX4 using antioxidants or Se to scavenge ROS/H2O2, (iii) enhancing NO signaling via NO-donors, stimulators/activators of sGC, and/or (iv) preventing cGMP degradation using PDE inhibitors. It is hoped that the recent findings summarized herein can be applied and translated into effective therapeutic strategies for the treatment of debilitating fibrotic disorders.Fibrogenesis is widely considered the result of a dysregulated wound healing response. In particular, failure of the wave of myofibroblast apoptosis during wound healing combined with an autocrine feed-forward loop of TGFisorders . TGFβ plived ROS  and suppived ROS . Interesntiation . Moreove


This study compares two methods for testing fertility trends and fertility stalls using Demographic and Health Surveys data. The first method is based on linear regression and uses the equivalence of period and cohort estimates with the same cumulative fertility at age 40, the same number of births, and the same distribution of women by parity. The second method is based on logistic regression. It assumes that the age pattern of fertility is constant over short periods of time. Both methods were applied to fertility trends in several African countries . The two methods were found to predict similar values of cumulative fertility, to produce consistent slopes, to document fertility trends the same way, and to characterize fertility stalls with similar statistical evidence. They can also be used to refute apparent fertility stalls obtained when comparing two point estimates from two successive surveys. The United Nations Population Division anticipates that fertility will continue to decline in the next 40 years in the world as a whole. However, the fertility transition will not be completed by 2050 in countries that are currently classified as "high fertility, " most of them in sub-Saharan Africa. In these countries, one expects a total fertility rate (TFR) of 2.8 children per woman, whereas elsewhere fertility will be below replacement, with TFR equaling 1.8 by 2050 [Fertility decline from high values typical of natural fertility to near- or below- replacement fertility is a quasi-universal phenomenon. Fertility decline started in almost all countries in the world at some point in the 19 by 2050 .However, in some cases the transition is not continuous, and long periods of fertility stalls have been documented. A typical case of fertility stall is that of Argentina, where fertility dropped from 7.0 children per woman in 1895 to 3.2 children per woman in 1947 and stayed at the same level for about 30 years before resuming its path in recent years towards replacement fertility [Sub-Saharan Africa is the continent that has started its fertility transition most recently. Fertility levels are still high but tended to decline since the 1970s, and declines occurred even earlier in urban areas of the most advanced countries. Most documented cases of fertility decline in Africa seem to be smooth and continuous, although it has been argued that fertility has stalled in a number of countries -16. A coThe aim of this paper is to present and compare two simple methods of analyzing slopes of fertility decline and testing fertility stalls. The first method is based on a demographic reconstruction of synthetic cohorts, and the second is based on a direct analysis of fertility rates. This work is a follow-up of earlier work on the same topic -24.The Demographic and Health Surveys (DHS) are standard surveys based on representative samples of national populations. Among other information, they provide maternity histories for women aged 15 to 49 with details on the date of each birth and on the age of the mother at time of each birth. This is enough information to compute person-years lived and births by age and time period and therefore age-specific fertility rates by period for the years preceding the survey. Note that since these maternity histories are closed cohorts, these calculations can be done with basic tabulation and simple tools such as a spreadsheet or basic computer programming. The only limitation of this type of information is the truncation effect: one knows the fertility "x" years ago only up to women aged "50 - x" years, because women who were interviewed were required to be under 50 years of age. Therefore, we limited our analysis to fertility up to age 40 and for the 10 years preceding each survey for which information is complete. This truncation of retrospective surveys is inevitable; for example, a woman who gave birth nine years ago at age 41 was not interviewed, as she was 50 at the time of the survey. This effect is better explained in other documents ,22. DHS If several surveys were available in a country, events and person-years were cumulated in order to provide annual fertility rates for longer periods. The cumulating of several surveys reduces fluctuations due to sample size and on average tends to compensate for minor biases associated with sampling. When displayed in a figure, estimates of cumulative fertility by age 40 grouped by two-year period tend to be quite regular and reveal the major trends in fertility, whether increasing, decreasing, or remaining steady. Of course, only formal statistical testing of slopes allows one to demonstrate an increase in fertility, a fertility decline, or a fertility stall. This method has been explained in more details in other documents ,22,23.For this study, we used the following DHS: Ghana: 1988, 1993, 1999, 2003, 2008; Kenya: 1989, 1993, 1998, 2003, 2008; Madagascar: 1992, 1997, 2003; Nigeria: 1990, 1999, 2003, 2008; Rwanda: 1992, 2000, 2005; Senegal: 1986, 1993, 1997, 2005; Tanzania: 1991, 1996, 1999, 2004, 2007; Zambia: 1992, 1996, 2001, 2007.All computations were done separately for urban and rural areas, since the trends were often divergent at the beginning of the transition, with an earlier decline in urban areas while fertility continued to rise or stagnated in rural areas.The criteria used for defining a fertility stall were similar to those proposed by Gendell (1985): fertility decline must have started for some years, then the decline must stop for a few years, and if the stall had come to an end, the fertility decline must have resumed. In terms of slopes, this requires an initial period with a significant negative slope, a second period with a net zero or positive slope, a significant change in slopes between the first and second periods, and, when applicable, a third period with a significant negative slope, with a significant change in slopes between the second and third period (p < 0.05 using 2-tailed tests). We used only linear trends for testing the changes in slopes, since these summarize well the changing slopes and are easy to compute. Note that in any country, changes in total fertility rates can almost always be approximated by linear trends over monotonic periods. The knots defining monotonic periods were chosen visually after plotting the yearly cumulative fertility on a graph and then fine-tuned by computing the intersection points of the two regression lines. The slopes were calculated one by one, on each monotonic segment, with the same linear trend.Our methods focused on slopes computed over periods for which annual fertility rates were available. These methods are far more stable than simply comparing point estimates. For example, in a DHS based on 6, 000 women, a TFR of 3.50 over the three years preceding the survey could be given with a confidence interval of 3.15 to 3.85 (about 0.25 due to sample size and 0.10 due to design effect). If two surveys are available five years apart, it is almost impossible to test a trend from those two points, unless the difference is very large (> 0.50). Even if the second survey indicates a TFR of 3.10, one cannot rigorously conclude whether fertility declined or stayed constant. The testing of slopes is very different, since it includes all points over the period covered, 10 years before each survey, totaling 15 years if two surveys are available. Furthermore, merging datasets for computing slopes allows one to smooth out erratic values of point estimates: these erratic fluctuations include fluctuations due to sample size and design effect, so that a simple test is enough to prove the slope or the changing of slopes.The first method used for testing slopes and changes in slopes of fertility trends follows a demographic approach and uses the property of period fertility rates. The concept of TFR is abstract and refers to what is called a "synthetic cohort." In other words, it computes what would be the cumulative fertility of a real cohort if it had the same age-specific fertility rates as those observed over a given period. Here, of course, one ignores mortality, as if all women survive up to age 40, as one would do in a real cohort of women who already reached age 40. One could test the trends in period cumulative fertility (period TFR) as if they were trends in cohort cumulative fertility  with the same level and the same number of births. For example, a period TFR(40) of 5.0 based on 1, 000 births is considered to be equivalent to a cohort cumulative fertility of 5.0 among 200 women, who would have had 1000 births by age 40. Testing trends in cohort fertility therefore requires the distribution of completed family size by parity. As in the real world, when an average completed family size is 5.0, the sample includes women with 0, 1, 2 ... 16+ children ever born, with an average of 5.0. Here the period TFR(40) was simply distributed accordingly, by assuming that at the same level of cumulative fertility, the distribution of women by parity was the same in a period and in a cohort (from 0 to 16+ children ever born). This procedure allows one to obtain a direct measure of the slope and its variance, based on individual women, as one would do in a cohort.In practice, in Method 1 one proceeds the following way. First, one computes the cumulative fertility, TFR(40), from age-specific fertility rates by single calendar year and five-year age group. Then, one computes the corresponding number of women in the synthetic cohort. These women are distributed by parity using a simple relationship linking the proportion of women with (i) children to the completed family size. These relationships were computed from cohort data using the same DHS, from parity 0 to parity 16+. Then, the sample is analyzed as a cohort sample, and cumulative fertility is related to time in a straightforward linear regression:The model allows us to calculate cumulative fertility by year after linear fitting and the precise fertility trend, to provide confidence intervals for slopes , and to test for changing slopes using standard Student T-tests. This method requires no hypothesis other than the equivalence between period and cohort, which is the rationale for computing period fertility rates. A regression is also calculated for each monotonic period.The second approach focuses on age-specific fertility rates and is based on the fact that women are likely to have only one or no delivery over a period of one year, depending on age and period. Therefore, the method chosen is a linear-logistic model, or logistic regression, where the dependent variable is 1 for a birth and 0 for no birth, and the weights are proportionate to the exact person-years lived over the period. The age pattern of fertility is complex and not easy to parameterize, so age groups  are introduced as dummy variables, with the 25-29 age group taken as the reference category because it has the largest number of births and is therefore the most stable. The model is:i is the dummy variables associated with each age group, from 1 = 12-14, 2 = 15-19, etc. and 6 = 35-39, with the fourth group (ages 25-29) omitted as reference category.where i is the age group and XThis model allows one to compute age-specific fertility rates by period, to recalculate the cumulative fertility by age 40, and to estimate the trends. As in the first model, it provides a confidence interval for the slopes and allows simple testing for fertility stalls. This method requires only two basic hypotheses: homogeneity in risk of bearing a child over a short period of time and a constant age pattern of fertility over short periods of time, both of which appear realistic.-6 in both cases). Results based on the logistic regression  confirm these findings with the same predicted values of cumulative fertility over the same periods, same trends, consistent slopes, and similar levels of significance.Both fitting methods were first applied to the case of Kenya, a country for which most authors agree that there was a fertility stall in the recent past. As expected, formal calculations demonstrate the fertility stall for the 1994 to 2002 period [see Table -3 [see Table The same procedure was repeated for a number of situations where fertility stalls have been documented or suspected. Urban and rural areas were analyzed separately, because they were found to have divergent fertility trends . FertiliA number of fertility stalls were proposed by some authors but refuted by others . These sIn this exercise, we showed that two different methods led to basically the same conclusions with respect to fertility stalls and to equivalent slopes and confidence intervals. This is reassuring, since cumulative period fertility can be considered as a synthetic cohort or as the sum of age-specific fertility rates. This finding also indicates that basic assumptions underlying both methods are likely to be fulfilled, namely the equivalence of period and cohort and the constant age pattern of fertility during the process of fertility changes. What the preferable method of testing is can be discussed endlessly. The logistic regression method tends to provide lower p-values, which suggests that it is more sensitive. However, the linear regression method might be more specific and seems to provide a higher p-value when the case is borderline, therefore ignoring false positives (as seen for Mozambique over the period 1995 to 2003). Therefore, it may be more realistic. In any case, both methods led to the same conclusions in most instances.Data quality is usually not an issue in DHS. Completed family size (or TFR) values tend to be very consistent for the same cohorts (or periods) in the same country, and comparisons with more precise data from demographic surveillance systems did not reveal errors that could not be explained by random fluctuations due to sample size and to cluster sampling . Minor eOther authors have used Poisson regression, or negative-binomial regression, to do similar testing of slopes. There is no obvious comparative advantage to using Poisson or negative-binomial regressions when calculating yearly fertility rates, since most women will have only one delivery at most over a 12-month period (the case would be different when using five-year periods or longer). Therefore, a simple outcome of 0/1 as used in the logistic regression method seems to be more appropriate. We did some basic comparisons of Poisson regression versus logistic regression and found similar results in the case of Kenya displayed in Table Other options are available for further testing fertility trends. For instance, one could use an age pattern of fertility in each situation in order to make the testing more precise. However, this is likely to be difficult. A Coale-Trussell function could be tried, but is likely to miss premarital fertility, which accounts for 20% to 40% of total fertility in some southern African countries . SimplerSophisticated statistical models have been developed in the past 20 years for testing changing slopes of a response variable in a variety of situations. Some of these models, such as "switching regression" or "change point regression" could also be tried to estimate fertility trends from DHS -29.Using retrospective data leads necessarily to some minor mortality biases, compared with full-scale vital registration or prospective data. However, these biases are likely to be small, since mortality between age 12 and 40 is usually very low. In Africa, the high prevalence of HIV/AIDS and high mortality among young women could lead to larger biases. This would lead to overestimating fertility levels in recent years, since the fertility of HIV-infected women tends to be lower than that of others. In theory, this could produce some apparent fertility stalls in retrospective surveys, but in our studies we did not find any obvious correlation with HIV prevalence nor with HIV mortality. This point could be further investigated when more data become available.Fertility stalls appear uncommon in African countries. Of the 31 countries investigated in our earlier studies, only eight exhibited some kind of fertility stall, of which five were restricted to either urban areas  or to rural areas , while the other three cases affected both urban and rural areas . Most of these stalls were of short duration (< 10 years) or had been occurring for less than 10 years before the last survey. These stalls of short duration do not compare with formal stalls such as that of Argentina, which lasted for about 30 years (over an entire generation) at a much lower level of TFR (about three children per women). Fertility stalls in Africa appear so far to be minor accidents in the course of the fertility transition. However, if they last longer, they could have serious consequences for long-term demographic dynamics, especially when they occur at relatively high levels of fertility. Furthermore, African countries are still in the middle of the fertility transition, and anything could happen in the future. A recent study in the Pacific Islands showed that new forms of fertility stalls or of fertility reversals could happen as a result of deliberate reproductive strategies of couples. Because couples might have an economic advantage to produce children who will be sent later in migration and who could remit money to the family, they may choose to have more children [Some of the fertility stalls proposed by other authors appeared undocumented in our analysis. This is due to the differences in case definition and in statistical testing. Using only two successive surveys with wide confidence intervals and point estimates based on a period of three years before a survey could be misleading when compared with a detailed analysis of fertility trends using all data available based on longer periods of 10 years or more. The case is even more delicate when comparisons are made on smaller sample sizes or when stalls are studied at the regional level or according to socioeconomic characteristics.More research could be conducted on the rationale for these well documented stalls. In an earlier study, we showed that country situations were highly diverse, and one could use a variety of factors to explain them without any consistent pattern .More research could also be conducted on the provision of family planning services, both in terms of quantity and in quality. Some authors have suggested that reduced financing for family planning services could explain the fertility stalls . This coThe author declares that they have no competing interests.