Strongyloides stercoralis, which is an intestinal parasitic nematode and the leading cause of strongyloidiasis in humans. Interestingly, HTLV-1 endemic areas coincide with regions citing high prevalence of S. stercoralis infection, making these communities optimal for elucidating the pathogenesis of co-infection and its clinical significance. HTLV-1 co-infection with S. stercoralis has been observed for decades in a number of published patient cases and case series; however, the implications of this co-infection remain elusive. Thus far, data suggest that S. stercoralis increases proviral load in patients co-infected with HTLV-1 compared to HTLV-1 infection alone. Furthermore, co-infection with HTLV-1 has been associated with shifting the immune response from Th2 to Th1, affecting the ability of the immune system to address the helminth infection. Thus, despite this well-known association, further research is required to fully elucidate the impact of each pathogen on disease manifestations in co-infected patients. This review provides an analytical view of studies that have evaluated the variation within HTLV-1 patients in susceptibility to S. stercoralis infection, as well as the effects of strongyloidiasis on HTLV-1 pathogenesis. Further, it provides a compilation of available clinical reports on the epidemiology and pathology of HTLV-1 with parasitic co-infection as well as data from mechanistic studies suggesting possible immunopathogenic mechanisms. Furthermore, specific areas of potential future research have been highlighted to facilitate advancing understanding of the complex interactions between these two pathogens.Infection with human T-cell leukemia/lymphoma virus type 1 (HTLV-1) has been associated with various clinical syndromes including co-infection with  Strongyloides stercoralis. Although the mechanism of the co-infection is not fully understood, it is possible that the microorganisms interact in a complicated manner to influence the course of the combined disease. The epidemiology of HTLV-1 infection is intimately intertwined with its biology. Although worldwide prevalence of HTLV-1 is not at the levels seen for the epidemic of human immunodeficiency virus type 1 (HIV-1), HTLV-1 infection is nonetheless a significant problem affecting millions of individuals across the globe. While the exact numbers of people infected with HTLV-1 remain uncertain, estimates vary from a minimum of 5–10 million, based on data from epidemiologic studies covering areas with approximately 1.5 billion people [A well-characterized disease manifestation associated with human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is the co-infection with n people ,3.The prevalence of infection is heterogeneous across different populations, often achieving high levels (as high as 5–40%) in small population niches, even within an individual country . CompS. stercoralis, have been appreciated for over three decades [S. stercoralis infection, or whether conversely, infection with this nematode might increase the incidence of HTLV-1 infection. Alternatively, it is possible that neither infection specifically alters the incidence of the other, but that co-infection may change the clinical picture of either infection. Overall, data support the later conclusion, including the possibilities that S. stercoralis infection might accelerate the onset of HTLV-1 disease, and that HTLV-1 infection is associated with a higher likelihood of higher parasite burden, more clinically apparent infection, and more severe and life-threatening disease [Since infected individuals are concentrated in discrete geographical locations, and the testing for HTLV-1 across much of the world is often unreliable, there is significant uncertainty in the global extent of HTLV-1 infection. Furthermore, many of the serological prevalence studies have used specific subpopulations, including blood donors or pregnant women seen at specific health care facilities. Data from such groups may not be representative of the general population as a whole. HTLV-1 co-infections with other pathogens, such as nematode  decades ,13,14,15 decades ,32,33,34 disease ,13,35.S. stercoralis. Studies are reviewed to assess the potential role S. stercoralis has in modulating HTLV-1, and subsequently, how HTLV-1 affects the immunological response to strongyloidiasis. A prospective analysis of the clinical significance and pathogenic mechanisms of this co-infection may be studied through populations with a high infectious burden of HTLV-1 and S. stercoralis. Analysis of these studies will help elucidate a potential mechanism for the onset of the co-infection. Final perspectives assess the implications of the combined disease and identify areas of research and model development needed to better understand the co-infection. This may further determine whether HTLV-1 and S. stercoralis are indeed partners in disease pathogenesis.This paper explores the pathophysiology of the co-infection between HTLV-1 and Strongyloides is an intestinal nematode that may cause parasitic infection of humans. Estimates of global prevalence range between 100–370 million people currently infected, mostly in tropical and subtropical regions of the world [S. stercoralis typically presents as urticaria, diarrhea and abdominal pain, although these associations are not universal [S. stercoralis results in multiple potential clinical manifestations with life-threatening consequences resulting in mortality rates of up to 85% to 100% [S. stercoralis hyperinfection. Characterizing the co-infection will require exploring the epidemiology of HTLV and S. stercoralis individually and considering each pathogen’s biology.he world ,37,38. Tniversal ,40,41. H to 100% ,41,42,43The clinical presentations of HTLV-1 infection are broad and varied and have been extensively and elegantly reviewed, as have the key principles developed in the extensive literature on HTLV-1 pathogenesis, so only a limited number of salient features will be addressed here ,45,46,47The most dramatic presentation of HTLV-1 infection is ATLL, an aggressive malignancy of CD4+ T-cells. ATLL occurs in approximately 3–5% of HTLV-1 infected patients, often through breast milk transmission . ATLL caAnother promising approach for ATLL therapy has been the use of antibodies directed against receptors present in high levels on the surface of HTLV-1 malignant T-cells. Early studies targeted the interleukin-2 (IL-2) receptor alpha subunit, anti-CD25, with some success, particularly using antibodies coupled to cytotoxic agents such as cytotoxic toxin subunits ,57. MoreIn addition to malignant disease, HTLTV-1 infection can result in several different inflammatory diseases that may resemble autoimmune disorders. The most prevalent of these inflammatory disorders is HAM/TSP, a demyelinating disorder involving primarily the spinal cord, found in up to 2–4% of HTLV-1-infected patients ,61. HAM/The major treatment of HAM/TSP is corticosteroids, which despite years of use, has only recently demonstrated responses in enough patients to be deemed clinically effective . A littlStaphylococcus aureus and β-hemolytic streptococci . While iewed in . The besStrongyloides co-infections. As expected, patients were identified primarily in areas where HTLV-1 is endemic, and reports of co-infections span a wide patient demographic. Risk factors for strongyloidiasis, in addition to HTLV-1 include corticosteroid use and alcoholism, which often contribute to disease prognosis. Although general helminth infection is associated with HTLV-1 infection [S. stercoralis in particular was identified in the majority of these cases. Thus, case reports and small series have provided a window into the range of manifestations of co-infections as well as of the populations at risk. These cases highlight the broadening geographic distribution of the co-infection, the risk factors present in these patients, the unique disease manifestations, and the changing approach to treatment.Concurrent with initial serologic studies demonstrating co-infection ,15, clinnfection ,72, S. sS. stercoralis co-infection was also observed in immigrants to the UK and to the Netherlands, from Central and South America, respectively. North American immigrants from Trinidad and Jamaica have exhibited co-infection decades following exposure [S. stercoralis throughout the central nervous system, which was presumably enhanced by the HTLV-1 infection and corticosteroid use [As the occurrence of HTLV-1 is increasing in areas that were once not considered endemic areas, as a result of immigration and population migrations, co-infection with helminths is occurring in these areas as well. For example, an older patient, who had immigrated to Canada from the Virgin Islands thirty years prior, exhibited co-infection . HTLV-1 exposure . In the roid use .S. stercoralis infection in these patients. In a number of cases, both corticosteroid use and HTLV-1 status have been documented, during which severe S. stercoralis hyperinfection syndrome resulted from corticosteroid use in the presence of underlying HTLV-1, although rapid ivermectin therapy facilitated recovery [S. stercoralis was detected in the ejaculate and HTLV-1 was positive, but no corticosteroid use was documented for this patient [S. stercoralis-related gastrointestinal (GI) symptoms, such as nausea and vomiting, and corticosteroid use was not documented.Strongyloidiasis often manifests in individuals treated with corticosteroids, although often underlying HTLV-1 infection is also present as a risk factor. Corticosteroids has been historically associated more with helminthic infection, and the risk factors are often conflated. For example, corticosteroids are a main stay of therapy for HAM/TSP, thus likely providing an additional risk factor for aggressive recovery ,22. It irecovery . In this patient . SimilarS. stercoralis was observed in a pregnant woman following corticosteroid treatment, and it was subsequently determined that she was an HTLV-1 carrier [S. stercoralis worms, larvae, and eggs [A common theme of many of these case reports is that more severe symptoms of the helminth infection are often noted in the presence of HTLV-1 infection. Obstructive GI disease has been characterized in a number of other co-infected patients ,17. In o carrier . Anotherand eggs .S. stercoralis co-infection are very often documented to have fully developed HTLV-1-associated diseases such as ATLL [S. stercoralis disease [S. stercoralis has not been as well-documented. Therefore HIV-1 patients with S. stercoralis should be screened for HTLV-1 as well. These scattered case reports, as well as some limited cohorts (see below) suggest the possibility of more severe disease presentations in co-infected patients, although definitive studies to prove this are yet to be done.Although most individuals with HTLV-1 remain asymptomatic carriers, patients with the  as ATLL ,24. For  disease . Other p disease . Another disease  while an disease . HTLV-1 S. stercoralis, with poor response to ivermectin therapy, and ultimately was diagnosed with HTLV-1 infection [S. stercoralis ELISA, presumably due to the profound immunosuppression [S. stercoralis infection after receiving a hematopoietic stem cell transplant [Klebsiella, along with chronic S. stercoralis infection. This combination of infections leads to a complicated and ultimately fatal clinical course [Some co-infected patients have presented with symptoms initially not suspected to be related to strongyloidiasis and without known HTLV-1 infection. For example, in one case, a patient with GI issues and suspected vasculitis was treated with steroids, and subsequently developed nfection . Anotherpression . He subsansplant . In anotl course . These aS. stercoralis infection, while albendazole is sometimes included in combination as a second agent or used as monotherapy. Daily oral ivermectin is normally indicated, but subcutaneous ivermectin has shown success in patients with severe strongyloidiasis and HTLV-1 co-infection in whom oral medications were not tolerated or absorbed [S. stercoralis co-infected patient has been reported who failed to respond to ivermectin, requiring addition of albendazole to the therapeutic regimen; it is possible (see below) that the immunomodulatory effects of HTLV-1 may have contributed to the aggressive S. stercoralis infection [S. stercoralis may include Klebsiella meningitis, acute respiratory distress syndrome, and Clostridium difficile and Escherichia coli infections [A number of case reports have raised the possibility that co-infection with HTLV-1 may alter the response to helminth treatment. Ivermectin is the standard treatment for absorbed . Interesnfection . Complicfections ,32,33.S. stercoralis co-infection. HTLV-1-associated leukemia patients show upregulation of CD25+/FOXP3+ T-cells which may increase risk for S. stercoralis co-infection [S. stercoralis infection was determined to have elevated IL-17 levels following ivermectin treatment [S. stercoralis.Case studies are useful for assessing clinical data but do not allow for robust mechanistic studies of disease pathogenesis. Regardless, several case studies, accompanied by careful laboratory analyses have provided insights into a potential mechanism for the effects of HTLV-1 and nfection . IL-2 renfection . Recentlreatment . More stS. stercoralis infection with HTLV-1 infection [S. stercoralis co-infection, Yamaguchi et al hypothesized that this interaction may be responsible for the earlier onset of ATLL seen in Caribbean and African patients  as compared with non-co-infected patients  as comp43 years ). Furthe0 years, ). In thiS. stercoralis has on HTLV-1 has often been assessed through comparison of HTLV-1 carriers with and without helminths. Gabet et al. used quantitative PCR methods to study HTLV-1 DNA proviral load in 18 patients with S. stercoralis+/HTLV-1+ along with 15 patients with HTLV-1 alone from French Guyana and Martinique [S. stercoralis infection stimulates the oligoclonal proliferation of HTLV-1-infected cells. This was hypothesized to account for the possibly shorter latency period in those that develop ATLL; expanding oligoclonal populations of HTLV-1 infected cells undergoing increased rounds of replication were hypothesized to have acquired additional cellular mutations enhancing the likelihood of malignant transformation. A mechanism of how S. stercoralis led to clonal expansion was not elucidated. As noted above, enhanced viral load has been associated with more severe infection and potential clinical manifestations. Gabet et al. looked only at HTLV-1 carriers without clinical manifestations of ATLL or HAM/TSP leaving open the possibility that the interactions between viral dynamics and nematode infection may differ in those already experiencing an HTLV-1-associated disease manifestation.The effect rtinique . Their mS. stercoralis and HTLV-1 were included. This study had the advantage of samples representing of several clinical presentations of HTLV-1 patients, including uveitis and neurological diseases. An increased number of HTLV-1-infected clones was observed in S. stercoralis patients. In addition, there was higher proviral load in co-infected patients due to increased mean clone abundance . This indicates that in the presence of S. stercoralis infection, either endogenous restrictions on clonal proliferation are over-ridden or there is some spur to increased proliferation in already proliferating clones. A surprising observation from this study was that the previously identified association between HTLV-1 infection and the genomic context of proviral integration, was not observed in proliferating clones in the setting of S. stercoralis co-infection. Both of these observations suggest that in the presence of Strongyloides co-infection there is an additional drive for proliferation of subsets of HTLV-1 infected cells, which in turn may provide a relevant contribution to the early observations of earlier onset of ATLL in co-infected patients.Subsequent studies have also correlated viral load and clonal proliferation of HTLV-1 in co-infected patients using a larger sample size, including individuals with HTLV-1-specific disease presentations. Applying the pioneering deep sequencing of HTLV-1 integrations sites, 61 individuals with HTLV-1 alone , were studied . This inS. stercoralis on HTLV-1 viral load, the immune response in these patients is of great interest. The shift to a Th1 response is well-documented in HTLV-1 carriers, and this is likely involved in the mechanism of co-infection with helminths because the body cannot induce an effective Th2 response against S. stercoralis. It is worth mentioning, however, that some studies have shown increased Th2 responses in HTLV-1 patients with the parasites compared to those without them. Porto et al. studied 310 HTLV-1 carriers and 32 HAM/TSP patients in Brazil [S. stercoralis showed a lower HTLV-1 proviral load. The lower viral burden may have been the reason for the predominant Th2 response rather than Th1. These observations raise questions as to whether Strongyloides always induces proliferation of HTLV-1-infected cells, and which T-cell response predominates.In addition to the impact of n Brazil . SpecifiS. stercoralis co-infected patients had higher proviral load and increased CD4+25+ T-cells, compared to patients with HTLV-1 alone [S. stercoralis antigen activated the IL-2 promoter, supporting growth of cell lines with HTLV-1 or transduced with Tax. Overall, this was a new finding and showed that S. stercoralis can induce polyclonal expansion of HTLV-1 cells, through activation of the IL-2/IL-2R receptor system. Later work in Brazil came to similar findings through the study of 30 patients with Strongyloides and HTLV-1 and 60 patients with HTLV-1 only [S. stercoralis. Treatment of helminth infection decreased these levels, again suggesting a role of the parasites in modulating HTLV-1 infection. This further suggests a role of helminthic infection in enhancing T-cell proliferation through IL-2 induction and induction of IL-2 receptor levels when co-infected with HTLV-1. Further studies that might integrate this work on the effects of S. stercoralis on IL-2/IL-2R function with the apparent increased drive for HTLV-1 clonal abundance and proliferation in the setting of co-infection [S. stercoralis infection and HTLV-1-associated disease. It also would have implications for both T-cell functions and T-cell oncogenesis. In addition to potential effects on oncogenesis, other studies are exploring the impact of HTLV-1 and S. stercoralis infections on the incidence of other malignancies. Co-infection has been independently associated with a number of different cancers other than ATLL; for example, HTLV-1 infection has been associated with increased non-ATLL lymphomas and hepatocellular carcinoma in a long-term follow-up of a cohort in Okinawa [Other work has sought to better understand the role of IL-2 in the mechanism of the co-infection. Satoh et al. studied 123 patients in Okinawa, showing that HTLV-1/-1 alone . This coV-1 only . Co-infenfection  will be  Okinawa . HoweverS. stercoralis in patients with the co-infection. Clinically, this appears to be manifest as a significant association between HTLV-1 infection and the occurrence of the aggressive and potentially life-threatening clinical syndrome of S. stercoralis hyperinfection [S. stercoralis co-infected patients to aggressive parasite infection, strongly suggests that HTLV-1 infection may compromise an effective host response limiting nematode infection. A series of studies have measured various cytokine and IgE levels to determine possible associations between cytokine production and parasite expulsion in co-infection. In typical immune responses to the presence of S. stercoralis, a Th2 response includes an increase in the production of IL-4, which promotes isotype switching in B cells and the production of IgE [S. stercoralis infection.HTLV-1 appears to have specific effects on how the body reacts to nfection ,83. In pnfection . The incn of IgE . SubsequS. stercoralis-infected individuals from Japan [S. stercoralis negative individuals, of whom, 9 were HTLV-1 positive, were also included [S. stercoralis, and the effect of treatment of strongyloidiasis in HTLV-1 carriers was observed. This study observed serum IgG4 levels were higher in patients with co-infection, supporting the hypothesis that IgG4 may block production of IgE. Increased IFN-γ and tumor growth factor beta (TGF-β) were also seen in co-infected patients, followed by a decrease in IL-4. As noted above IL-4 is required for B cells to undergo isotype switching to produce IgE. Another study examined the effect of HTLV-1 infection on serologic and skin tests for strongyloidiasis to assess the impact on IgE and eosinophils [S. stercoralis endemic area in Salvador, Brazil who tested positive for helminth infection via fecal examination; and patients hospitalized for severe infection. This study concluded that HTLV-1 reduced the sensitivity of parasite specific IgE testing as well as immediate hypersensitivity skin test. The decrease in IL-4 also apparently prevented an increase in intestinal fluid content associated with parasite rejection. These findings were in accordance with prior knowledge of the role of IL-4. Observed effects were attributed to the impaired isotype switching from IgG to IgE as illustrated in S. stercoralis infection through eosinophil activation and specific immunoglobulin production. Subsequently, the patient may develop disseminated strongyloidiasis and further complications. The same data set was also used to measure various cytokine and IgE levels to determine other possible association between cytokine production and parasite expulsion. The results included a negative correlation between IFN-γ and total IgE, which was in accordance with predicted levels in co-infection. Other results included an increase in IL-10 and a decrease in IL-13, which corresponded with the expected Th2 to Th1 shift.Several studies observed that with increased production of IFN-γ characteristic of a Th1 response, HTLV-1 infection results in decreases in IL-4, IL-5, and IgE ,85. Thesom Japan . Furtherincluded . PatientS. stercoralis treated between 1991–2014 [Strongyloides to HTLV-1 proviral load; therefore, it is unclear if worsening HTLV-1 infection causes an incremental impairment of immune response to the helminths.Despite many studies that showed a change in IgE production, a recent study found unchanged serum IgE levels with co-infection. The study observed Japanese patients with 991–2014 . PatientS. stercoralis infection. However, other changes in the CD4+ T-cell population have also been noted. A current area of interest is the impact HTLV-1 has on Treg cells. Several studies show associations between Treg cells and IgE expression, which may contribute to the pathogenic mechanisms of HTLV-1/S. stercoralis co-infection [S. stercoralis larval antigen-driven production of IL-5. This decrease in IL-5 production is potentially also followed by decreased eosinophil differentiation, decreased helminth killing, and decreased mast cell degranulation. Malpica et al. [S. stercoralis in the infected intestine. The study demonstrated increased overall CD3, CD8 and Treg cell responses in duodenal biopsies of co-infected patients versus non-specific duodenitis or normal patients. However, there was also a relative decrease of the aforementioned cells and eosinophils adjacent to the parasites. Recently, intriguing follow-up studies showed that successful treatment of S. stercoralis infection in co-infected patients resulted in decreased Tregs, however the defect in IL-5 production in response to Strongyloides antigen persisted [S. stercoralis even following parasite eradication. Many of these findings corroborate the proposed role of HTLV-1 in this co-infection. The presence of the retrovirus results in increased Treg cells and a downregulation of effector T-cells, likely contributing to increased parasite burden  mice [-/- mice [+ stem cells into NOD/Shi-scid/IL-2Rγc null (NOG) mice [The lack of a suitable small animal model has hindered understanding of the immuno- and neuropathogenesis of HTLV-1 in an in vivo system, due to the inefficient fusion of HTLV-1 envelope with murine cells. This shortcoming was overcome by the development of an infectious chimeric virus ,93, whicSG) mice ,100,101.-/- mice . Fairly -/- mice . With re-/- mice . For leuOG) mice . This moS. stercoralis, various mechanisms of innate and adaptive protective immunity have been studied in mouse models. In naïve mice, parasite-released soluble factors recruit neutrophils, eosinophils, and macrophages directly to the parasite. Neutrophils and eosinophils are then able to kill L3 through the release of myeloperoxidase and major basic protein, respectively. However, both of these granulocytes require complement factor C3b to kill the larvae. After the neutrophils die, they also release extracellular traps (NETs) consisting of a fibrous network of nuclear DNA. The worms are trapped within the NETs, preventing their movement and facilitating the attachment of slow-moving effector cells to the rapidly migrating worms [S. stercoralis is highly efficient and is dependent on CD4+ Th2 cells for the release of IL-4 and IL-5. B cells, in particular B-1 cells, are also required for adaptive immunity, resulting in the production of immunoglobulins necessary for neutrophil and macrophage mediated killing. Whereas neutrophils require complement and IgG to kill the larvae through antibody-dependent cellular cytotoxicity (ADCC), macrophages require IgM to kill the larvae independent of ADCC. While eosinophils do not act as effector cells in this response, their role as antigen presenting cells is vital to the production of antigen-specific IgM. Finally, mice infected with S. stercoralis developed alternatively activated macrophages within the peritoneal cavity after exposure to the cytokine IL-4. Alternatively, activated macrophages, but not classically activated macrophages, kill the larvae in collaboration with neutrophils and complement. Thus, the adaptive immune response kills through two antibody dependent mechanisms, IgG with neutrophils and IgM with macrophages, and one antibody independent mechanism consisting of macrophages, neutrophils, and complement [For ng worms ,106. Aftmplement ,106.S. stercoralis infection would develop in mice. To test this hypothesis, severe combined immunodeficiency (SCID) mice, which lack T and B cells, were first used to establish that mice are susceptible to infection with S. stercoralis. L3 of S. stercoralis were injected into SCID mice, yielding a small number of adult worms [S. stercoralis, L3 were injected into NSG mice, which have a profound deficiency in both innate and adaptive immunity. It was postulated that in the absence of a functional immune response, infection and hyperinfection with S. stercoralis would develop in NSG mice. While large numbers of adult worms and L1 consistently developed in the NSG mice, no signs of hyperinfection were detected. Treatment of NSG mice to eliminate residual neutrophils likewise did not result in hyperinfection, suggesting that the residual innate immune response was not responsible for controlling its development. Infected NSG mice were then treated with methylprednisolone acetate, a drug associated with the development of hyperinfection in humans, to determine if the drug would function in a similar manner in the infected NSG mice. Subsequently, S. stercoralis-infected NSG mice treated with methylprednisolone acetate displayed increased mortality rates, a greater than 10-fold increase in adults and L1, and the development of large numbers of L3a, consistent with a hyperinfection-like phenotype. Several hypotheses were generated to explain how hyperinfection developed in NSG mice in the presence of methylprednisolone acetate. One possibility is that the methylprednisolone acetate induced this phenomenon by eliminating a residual element of the immune response retained by the NSG mice. An alternative explanation was that the methylprednisolone acetate had a direct effect on the worms driving their development into L3a and thus the development of hyperinfection. This second explanation was further investigated by treating the NSG mice with methylprednisolone acetate-induced hyperinfection with an agonist to the parasite nuclear receptor Ss-DAF-12. Following this treatment, these NSG mice had significantly reduced worm burdens, opening the door to novel new treatments for this potentially fatal infection in humans [S. stercoralis [S. stercoralis cannot be studied in the Jird model. Since humanized immunodeficient mice are susceptible to infection with HTLV-1 [S. stercoralis, it may be possible to ask novel questions regarding the relationship between these two infections agents and the development of hyperinfection. Further studies should examine if S. stercoralis hyperinfection develops in NSG mice co-infected with HTLV-1. If it is possible to simulate this co-infection, the S. stercoralis infection could be further characterized to observe if L3a, with their characteristic size and morphology, develop in this model. Other directions include identifying shared and unique features in the hyperinfections induced by methylprednisolone acetate and HTLV-1. With these results, further research could determine if an antagonist to the parasite nuclear receptor Ss-DAF-12 may be effective at blocking hyperinfection induced by HTLV-1 in mice co-infected with S. stercoralis and HTLV-1.It is clear from these studies that the innate and adaptive immune responses in mice are highly efficient at controlling the infection. It was hypothesized that in the absence of a functional immune response, lt worms . To furtn humans . Methylprcoralis . Howeverh HTLV-1 ,110,111 S. stercoralis. The co-infection is well documented in several infected populations and has been characterized both through a number of case reports, a handful of cohort studies, and some mechanistic studies. Similar to the individual infections, the co-infection affects a variety of patient demographics and ages. It is also common for the co-infection to manifest many years after exposure to HTLV-1. Mechanistically, it is proposed that HTLV-1 alters the immune response to S. stercoralis by causing a Th2 to Th1 shift. This causes diminished IgE and decreased IL-4 and IL-5, resulting in difficulty mounting an effective anti-helminth response and predisposing to more severe infection. It is possible that IgG4 blocks IgE production, while IFN gamma decreases IL-4 and IgE. Treg cells may also have a role lowering eosinophil counts. When comparing co-infected patients to those with HTLV-1 alone, S. stercoralis appears to increase the proviral load of HTLV-1 through induction of clonal proliferation. Epidemiologic studies have suggested a potentially early onset of HTLV-1 induced ATLL. An intriguing possibility, needing considerably more study is that this may be mediated through activation of IL-2 promoter in infected cells, potentially enhancing clonal proliferation and also resulting increased amounts of soluble IL-2R. This may in turn have numerous effects on immune homeostasis. Case studies show that carriers of HTLV-1 with strongyloidiasis often present with additional infections, causing meningitis, gastrointestinal symptoms, or even respiratory distress. Furthermore, severe disseminated strongyloidiasis, which can infect systems other than the gastrointestinal system, and hyperinfection syndrome, which is often seen in HTLV-1 patients who receive corticosteroids [S. stercoralis infection in HTLV-1 carriers through PCR detection using stool samples [S. stercoralis. However, sometimes ivermectin is given for a longer course, or even combined with albendazole [HTLV-1 exhibits a wide variety of disease manifestations, although most infected individuals remain asymptomatic carriers. Even in carriers, the virus is a risk factor for the development of the disseminated hyperinfection of steroids  are more samples . Upon idendazole . AdditioS. stercoralis co-infection. The effects of S. stercoralis and HTLV-1 co-infection have been largely characterized in HTLV-1 carriers; relatively few studies have looked at presentations and outcomes of S. stercoralis in ATLL and HAM/TSP patients. One patient with ATLL and S. stercoralis experienced a severe response sepsis and organ failure following manifestation of symptoms [S. stercoralis co-infection, and the rest having ATLL alone [S. stercoralis. Given the short study and its performance prior to many of the newer therapeutic strategies for treating ATLL, it is difficult to determine the clinical significance of this difference in the current treatment setting. Subsequent work by this group focused on 11 ATLL patients with four being diagnosed with S. stercoralis and seven without the co-infection [S. stercoralis, particularly in the context of HAM/TSP. One study in particular observed low proviral load in a few patients manifesting HAM/TSP and proposed that helminths could inhibit HTLV-1 transcription and prevent progression to HAM/TSP [S. stercoralis.There are a number of important areas for future study of HTLV-1 and symptoms . As prevLL alone  and shownfection . Co-infenfection . Althoug HAM/TSP . They alS. stercoralis. As described above, mouse models are limited both in their ability to mimic the long-term pathogenesis of HTLV-1 infection and to probe the details of the host immune reaction to S. stercoralis. A particular problem is that the models of HTLV-1 infection in different mice carrying human CD4+ T-cells are often associated with a rapid progression from infection to a full-blown leukemic phenotype. This limits the ability to study the evolution of S. stercoralis infection due to deficits in the reconstituted human immune system before the onset of the florid HTLV-1 proliferative disease. Rabbits have been successfully infected with HTLV-1 and have been a long-standing model of in vivo viral replication ; hoiewed in ). Chroni monkeys . Whetherh HTLV-1  and S. srcoralis .S. stercoralis in the intestine, as this is the anatomic locus most likely to be the focus of these interactions. As the site of S. stercoralis invasion that is the hallmark of the hyperinfection syndrome associated with HTLV-1 co-infection and other immunosuppressed states, the intestine is likely the key element in regulating a transition to the hyperinfection state. As noted above, there is limited but very intriguing recent preliminary data from co-infected patients [Strongyloides-infected patients, or conversely duodenal biopsies from HTLV-1 infected patients. In fact, there is a striking lack of information about HTLV-1 infection of gut-associated lymphoid tissue. This is quite surprising considering that infection of the gut is the major route of HTLV-1 infection (through breast milk transmission). Furthermore, for HIV, the gut-associated lymphoid tissue with its high levels of CD4 T-cells is the major site of early viral replication [S. stercoralis co-infection of the intestine.Finally, as better animal models develop, it will be essential to examine potential pathogenic interactions of HTLV-1 and patients  showing lication . In factlication , there hlication , may offS. stercoralis co-infection will also increase providing both a new urgency as well as increased opportunities to study the pathogenic effects of co-infection. Overall, it is clear that HTLV-1-infected individuals are at an increased risk of developing severe disseminated strongyloidiasis. Therefore, further attention must be given to screening and treating these patients. Conversely, although data are less robust, there are both clinical studies as well as proposed mechanisms that would support an earlier onset of ATLL in co-infected patients. In order to develop new treatments that may lessen the disease burden for this growing patient population, further research is needed to truly determine the intricate role these microorganisms play as partners in the pathogenesis of HTLV-1 and S. stercoralis co-infection.As the prevalence of HTLV-1 increases in certain parts of the world ,124, it


Additionally, differential pulse voltammetric (DPV) analysis showed that the EPIG sensor showed the best selectivity for a tertiary mixture of UA, dopamine (DA), and ascorbic acid (AA) as compared to EPIP and EPIS.In this present work, an electrochemical sensor was developed for the sensing of uric acid (UA). The sensor was based on a carbon paste electrode (CPE) modified with electroactive polyimide (EPI) synthesized using aniline tetramer (ACAT) decorated with reduced nanoparticles (NPs) of Au, Pt, and Ag. The initial step involved the preparation and characterization of ACAT. Subsequently, the ACAT-based EPI synthesis was performed by chemical imidization of its precursors 4,4′-(4.4′-isopropylidene-diphenoxy) bis  BPADA and ACAT. Then, EPI was doped with distinctive particles of Ag, Pt and Au, and the doped EPIs were abbreviated as EPIS, EPIP and EPIG, respectively. Their structures were characterized by XRD, XPS, and TEM, and the electrochemical properties were determined by cyclic voltammetry and chronoamperometry. Among these evaluated sensors, EPI with Au NPs turned out the best with a sensitivity of 1.53 uA uM Generale 2 × 10− mol L−1 mol L−1) , and alomol L−1) . Therefomol L−1) . In genemol L−1) , fluoresmol L−1) , a singlmol L−1) , chemilumol L−1) , and dirmol L−1) .Among all these methods, the electrochemical technique is extensively used and has been proven to be an excellent method for the determination of UA due to its quick response, easy handling, specificity, low expenditure, and high sensitivity ,15. In aHowever, other potentially co-existing analytes in body fluids like AA and DA may interfere with the electrochemical detection of UA. The oxidation potential of UA, AA, and DA are too close to strongly overlap, which results in poor selectivity of electrodes. To address this concern, two techniques have been employed, i.e., polymer-modified electrodes and chemically-modified electrodes. Thus, the electrode can be modified and improved using conducting polymers (CPs), noble metal nanoparticles, organic/inorganic compounds, and ionic/polymeric film ,17. CondFor instance, Ramanavicius et al., recently developed a quartz-crystal microbalance sensor (EQCM) based on polypyrrole imprinted by uric acid (MIP(UA)-Ppy). Experimental measurements and theoretical calculations showed that the formation of a uric acid complex with MIP(UA)-Ppy is thermodynamically more favorable than that of complexation with NIP-Ppy. However, the sensitivity of the MIP(UA)-Ppy-based sensor to uric acid in a linear dependence interval was approximately 3.5 times higher than that of a NIP-Ppy-based sensor . GryglewDue to its superior environmental stability, ease of synthesis, low cost, and uniform conductive mechanism, PANI is a popular conjugated polymer. Doped PANI is often used in sensing applications as it shows high conductivity under acidic conditions. Generally, PANI doped with noble metal nanoparticles (NPs) enhances the electron transfer and improves the conductivity and stability. Therefore, it is used in electrochemical sensing ,23,24 as2S gas sensor based on an electroactive polymer containing aniline trimer in the main chain [As a model compound of PANI, oligoanilines have been studied enormously in the past decade. Consequently, oligoaniline-derived electroactive polymers (OAEPs) have gained considerable attention ,27,28,29in chain . Additioin chain . Some rein chain  of fullein chain , electroin chain ,41, biomin chain , electroin chain , and elein chain  and condin chain . Additioin chain ,52,53,543, H2PtCl6, and HAuCl4 for 6 h. The presence of metal particles that were reduced on the EPI surface was examined by XRD, TEM, and XPS. The electrochemical sensors constructed for UA sensing were investigated for various studies, such as electroanalytical, kinetic, sensitivity, selectivity, and differential-pulse voltammetry.To the best of our knowledge, OAEPs are generally use for electrochemical sensing of ascorbic acid ,56 but a4•3H2O), H2PtCl6•6H2O, hydrazine, and AgNO3 were all from Alfa Aesar. 1H-NMR and LCMS were performed on a Bruker AVANCE300 spectrometer and Bruker Daltonics IT mass spectrometer model Esquire 2000 , respectively. An FTIR spectrometer  and VoltaLab 50 (PST050) were used to perform FTIR and electrochemical experiments. The molecular weight was determined on a Waters GPC-150CV , and a JEOL-200FX TEM  was used for TEM study.All the chemicals purchased from Sigma-Aldrich , TCI  and Seechem . All the solvents were purchased from J.T. Baker , Riedel-de-haën  and Macron , with 99.99% purity. HAuCl2O (20 mL) and 1 N HCl (25 mL). After that, ammonium persulphate solution (2.28 g in 25 mL of 1 N HCl) was added slowly to the above-mentioned solution at 0 °C via a dropping funnel. Subsequently, after one hour of stirring at 0 °C the reaction was precipitated out in distilled water (700 mL). Finally, the vacuum filtration was used to obtain the precipitate followed by a washing with 400 mL of HCl (1 N), 100 mL of NH4OH (1.2 N), and distilled water. Lastly, the crude product of ACAT was dried in a vacuum at 50 °C and obtained as blue powder.As shown in Scheme I, aniline tetramer (ACAT) was prepared using a simple procedure . First, v/v) was added to the previous solution under stirring for 1 h. Subsequently, the reaction mixture was heated at 150 °C for 3 h to obtain the EPI solution. Finally, the precipitation was carried out in methanol and the precipitate washed with hot methanol. Lastly, the EPI was dried at 60 °C in a vacuum and collected in a good yield (80%) [The EPI preparation was a two-step process. First, 0.52 g of 4,4′-(4.4′-isopropylidene-diphenoxy) bis  and 0.379 g of ACAT were added to 16 g of DMAc in a two-neck-round bottom flask. The reaction mixture was stirred for 24 h at room temperature to produce electroactive polyamic acid (EPAA). Afterwards, the mixture of acetic anhydride and pyridine  .4OH containing 1 mL of hydrazine solution. The reaction mixture was stirred for 24 h, filtered, and washed with water until the pH became neutralized. At this time, a freeze-drying technique was used at −42 °C for 24 h, and EPI  was collected in form of blue powder. Similarly, EPIS, EPIP, and EPIG were prepared by immersing 0.1 g EPI  in 19 mL of 0.1 mM AgNO3, H2PtCl6•6H2O and HAuCl4•3H2O for 6 h, respectively. Later, the EPIS, EPIP, and EPIG powders were collected by centrifugal filtration, washed with an excess amount of distilled H2O and dried at 50 °C for 24 h. Finally, the as-prepared EPI decorated with distinctive metallic particles was collected as a fine blue powder. The synthetic route for EPI decorated with Ag/Pt/Au particles is shown in The standard procedure for the preparation of EPIS, EPIP, and EPIG was as follows: Firstly, an EPI  was prepared by dispersing 0.1 g of as-prepared EPI in 10 mL of 1.0 M NHThe redox behavior of the as-prepared EPI, EPIS, EPIP, and EPIG was determined by spin-coating these electroactive materials onto an indium titanium oxide (ITO) followed by performing a series of CV studies. Firstly, different solutions were prepared by dissolving 0.05 g of respective sample in 5 mL of DMAc under magnetic stirring for 6 h. After this, these solutions were spin-coated on an ITO and dried by heating at 120 °C for 1 h. Cyclic voltammetry (CV) of as-prepared ITOs was performed on a AutoLab PGSTAT 204 .1H-NMR spectrum of ACAT reveals the signal at 5.50 ppm indicating the NH2 protons. Moreover, the signals around 7.3–6.8 ppm show the splitting of the aromatic protons. The FTIR spectrum of ACAT in −1 corresponding to the NH2 groups, while the vibrational bands for quinoid and benzenoid ring appears at 1600 and 1500 cm−1, respectively. The peak at 830 cm−1 may be correlated to the substitution pattern of a 1,4-disubstituted benzene ring. Moreover, m/z) [er, m/z) ,64.−1. In addition, the peaks observed at 1596 and 1503 cm−1 indicated the presence of quinoid and benzenoid rings. On the other hand, EPI showed typical asymmetric and symmetric carbonyl group absorption bands at 1715 and 1675 cm−1. Moreover, the absorption band of the carboxylic acid group completely disappeared, which indicated the complete conversion of EPAA to EPI.As shown in Various characterization tools were used for the determination of structure and morphology of the as-prepared materials. The XRD patterns clearly indicates that EPIS, EPIP, and EPIG had a crystalline nature as shown in 5/2 Ag 3d3/2 was found to be 371.5 eV and 365.6 eV, respectively, which were comparable to the values reported in the literature for bulk Ag crystals  [7/2 and 4f5/2, respectively, corresponding to the zero-oxidation state [7/2 and Au 4f5/2 was observed as doublets at 81.5 and 85.3 eV, respectively, which clearly indicates the reduction of Au3+ into Au0, as the binding energy of Au 4f7/2 for Au+3 is 86.5 eV [The XPS analysis was employed to determine the composition of all the given samples as shown in  374 eV) . Similar 86.5 eV .−2. However, the doping of EPI with respective metal nanoparticles resulted in an increase in current density. For example, the current density for EPIS 5c was 225 A cm−2, which was 2.3 times higher than that for EPI, while the response for EPIP 5d increased by four times with a current density of 400 A cm−2. Subsequently, the highest response was observed for EPIG with a current density of about 600 A cm−2, as shown in The cyclic voltammograms (CV) of all EPIs are shown in −2 to 8.6 µA cm−2. Furthermore, the peak potential shifted towards the positive side with the increasing scan rate. p) and square root of the scan rates (ν1/2), and the regression equation was Ip (μA) = 11.147 ν1/2(VS−1)1/2 + 0.562 with a correlation coefficient of R2 = 0.9958. Thus, it indicated that the UA oxidation process proceeds via a diffusion-controlled electron transfer reaction, which is generally observed in systems when the kinetics are fast. Additionally, to investigate the mechanism of UA electrocatalysis on an EPIG, a graph was plotted between the peak potential (Ep) and logν as shown in p was proportional to logν by the following linear equation Ep = 0.1112 log (ν) + 0.5346, R2 = 0.97548, which indicates that the reaction is irreversible. Hence, an anodic peak potential can be evidenced by [a is the number of electrons transferred; and T is the room temperature. The Tafel slope value calculated from To study the nature and kinetics of the electrochemical reaction, the effect of the scan rate on CV was determined on a CPE modified with EPIG was used in 0.1 M PBS (pH = 7) towards the oxidation of 50 μM UA as evident in enced by (1)Ep=2.−1. As observed, the bare CPE 7a showed the smallest response with a current density of 12.73 uA cm−2 at a more positive potential of 459 mV, while, the current density for the CPE modified with EPI, EPIS, EPIP, and EPIG was found to be 28.10, 29.67, 32.68, and 37.39 uA cm−2, respectively. It may be conclude that UA sensitivity decreases in the following order EPIG > EPIP > EPIS > EPI.CV and amperometric studies were used to determine the electrocatalytic activity of the respective material for the detection of UA. −1, which is 1.84-fold, 3.56-fold, 4.5-fold, and 6.12-fold higher than those of a CPE modified with EPIP, EPIS, EPI, and a bare CPE, respectively, under the same analytical conditions as shown in The quantitative detection of UA with bare CPE and the EPI, EPIS, EPIP, and EPIG modified CPE was investigated by chronoamperometric measurements as shown in −1) and having a good correlation coefficient (R2 = 0.9951). On the other hand, at a higher concentration of UA, the relationship between current density, and UA concentration was determined by the following equation [At a constant potential of 340 mV and a lower concentration (0–50 µM), all the electrochemical sensors showed a linear relationship between current response and UA, as shown in equation :j = jmaxmax = current maxima at higher UA concentration). Based on the data, the previous equation yielded jmax = 15.33 A cm−2, b = 92.59 M, and a correlation coefficient of R2 = 0.9968. However, it is not easy to apply the above equation for a current vs concentration plot. Thus, we generated a linear relationship by taking the log of the abscissa of the calibration curve. So, the sensitivity values calculated for a CPE modified with an EPIG, EPIP, EPIS, EPI, and a bare CPE was 0.153, 8.3 × 10−2, 4.7 × 10−2, 3.4 × 10−2, and 2.5 × 10−2 A M−1, respectively. In addition, the current density increased with the successive addition of UA, while the steady state was achieved after 2 s. These results indicated that, an EPIG is a highly sensitive sensor with a rapid response, wide linear dynamic range (LDR) and low limit of detection (LOD)  .Differential pulse voltammograms (DPVs) were used  to deter.M−1 UA), low LOD  and wide LDA (5–50 µM UA) at a lower oxidation potential of 310 mV. Similarly, the DPV results clearly indicated the excellent selectivity of EPIG as compared to other constructed sensors. Hence, the EPIG electrochemical sensor developed for UA detection showed reliable results. Considering the good selectivity, simple preparation, low cost, and selectivity of the modified sensor of UA the practical application value of this system will be later evaluated using real samples.In conclusion, we fabricated stable and sensitive UA sensors based on EPI decorated with reduced nanoparticles of Ag, Pt, and Au, i.e., EPIS, EPIP, and EPIG, respectively. The characterization of the as-prepared materials via XRD, XPS, CV, and TEM demonstrated the metallic nanoparticles were successfully reduced upon the electroactive segment of the EPI. The redox capability of the UA sensors based on CV studies decreased in the following order EPIG > EPIP > EPIS > EPI > ITO, which implied that EPIG may reveal the highest sensitivity. The amperometric data showed that the UA sensor EPIG exhibited higher sensitivity (0.153 A


Here, we summarize current knowledge on the roles of Wnt signaling components including ligands, receptors/co-receptors and soluble modulators in adult hippocampal neurogenesis. Also, we review the data suggesting distinctive roles for canonical and non-canonical Wnt signaling cascades in regulating different stages of neurogenesis. Finally, we discuss the evidence linking the dysfunction of Wnt signaling to the decline of neurogenesis observed in aging and Alzheimer’s disease.Neurogenesis persists during adulthood in the dentate gyrus of the hippocampus. Signals provided by the local hippocampal microenvironment support neural stem cell proliferation, differentiation, and maturation of newborn neurons into functional dentate granule cells, that integrate into the neural circuit and contribute to hippocampal function. Increasing evidence indicates that Wnt signaling regulates multiple aspects of adult hippocampal neurogenesis. Wnt ligands bind to Frizzled receptors and co-receptors to activate the canonical Wnt/β-catenin signaling pathway, or the non-canonical β-catenin-independent signaling cascades Wnt/Ca The subgranular zone (SGZ) of the hippocampal dentate gyrus is one of the neurogenic niches of the adult brain where the generation of new neurons persist during adulthood. Compelling evidence indicate that this process is conserved in mammals including humans . New neuAlthough the role of adult hippocampal neurogenesis has been challenging to determine in humans, increasing evidence in rodents and non-human primates indicate that adult-born neurons contribute to the structural and functional plasticity of the hippocampus , and to 2+ , and the multiprotein complex composed of the scaffolding protein Axin, APC, and the enzymes casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3-β) . In consesterase , increasesterase . IP3 andesterase , proteinesterase  or the pesterase .In the central nervous system, Wnt signaling pathways play pivotal roles during development, controlling cell division, differentiation, polarity, migration, and synaptogenesis . In the Compelling evidence indicate that components of the Wnt signaling pathway play multiple roles during adult neurogenesis. As will be discussed, the data also suggest that canonical and non-canonical Wnt signaling cascades regulate different stages of neurogenesis: Wnt/β-catenin signaling regulates proliferation and fate commitment, while non-canonical Wnt signaling controls the differentiation and development of newborn neurons. In this section, we summarize current knowledge on the role of Wnt signaling components and pathways in controlling different stages of adult hippocampal neurogenesis .Wnts are secreted lipid-modified glycoproteins that act as autocrine and paracrine signaling molecules . Wnts arin vivo showed that general blockade of Wnt signaling with a dominant negative of Wnt1 ligand, which non-autonomously blocks Wnt signaling, almost completely eliminated the generation of new neurons in adult rat hippocampus . In undifferentiated NSCs, Sox2 and the histone deacetylase HDAC1 repressor protein are associated with the Sox/LEF site in the Neurod1 promoter. In differentiated neurons β-catenin, along with acetylated histone H3 and methylated histone H3 at lysine 4, which are related with transcriptional activation, are present in the Neurod1 promoter  . AD is tInterestingly, we and others have determined that hippocampal neurogenesis is stimulated in AD mouse models through physiological , and phaConsidering the crucial role of the Wnt signaling in the regulation of neurogenesis, it might be possible that the dysregulation of this signaling pathway may contribute to neurogenesis deficits observed in AD. Of note, overexpression of Wnt3 restored neurogenesis in the hippocampus of the 5xFAD mouse model of AD , that exAndrographis paniculata  and Ethosuximide (ETH), which inactivate GSK-3β through the PI3K/Akt pathway, also promoted neurogenesis in AD models . TogetheCurcuma longa), was also suggested to induced neurogenesis through the activation of Wnt/β-catenin signaling pathway. Curcumin encapsulated in PLGA nanoparticles induced NSC proliferation and neuronal differentiation in the hippocampus of an Aβ-induced rat model of AD, and reduced the cognitive deficits (Xanthoceras sorbifolia Bunge, ameliorated the cognitive impairment and concomitantly increased NSCs proliferation and neuronal differentiation in APPswe/PS1ΔE9 mice (Curcumin, a natural polyphenol compound derived from turmeric (deficits . CurcumiΔE9 mice . InteresΔE9 mice . MoreoveThe reviewed studies indicate that the Wnt signaling plays multiple roles in adult hippocampal neurogenesis including NPCs proliferation, fate-commitment, development and maturation of newborn neurons. Evidences suggest a stage-specific expression of particular receptors that might activate different Wnt signaling cascades to control the progression of neurogenesis. Although the role of the canonical Wnt co-receptor LRP6 support this notion, the role of other co-receptors that control the activation of non-canonical Wnt signaling remains to be elucidated. The identification of Wnt co-receptors involved in adult neurogenesis is a critical issue that should be addressed to gain a more comprehensive understanding of how canonical and non-canonical Wnt signaling are regulated during adult neurogenesis. In addition, it will be interesting to further study the downstream signaling components and effectors involved in the regulation of adult hippocampal neurogenesis by non-canonical Wnt signaling.Several studies indicate that Wnt proteins released by hippocampal astrocytes and progenitor cells are crucial components of the SGZ niche. In addition, endogenous Wnt inhibitors are also components of the neurogenic microenvironment that dynamically regulate Wnt-mediated neurogenesis under physiological conditions. Considering the increasing number of Wnt regulators identified to date, it will be interesting to further investigate the contribution of these molecules to the dynamic control of neurogenesis.In agreement with the critical roles of Wnt signaling in adult neurogenesis, evidence indicates that Wnt signaling is associated with the age-dependent decline in neurogenesis. Concomitantly with the decrease in the generation of new neurons, in normal aging there is a reduction in the expression of Wnt proteins, an increase in the expression of Wnt inhibitors, and a decrease in canonical Wnt signaling activity in the dentate gyrus. Wnt dysfunction might also underlie the impairment of neurogenesis observed in AD. Interestingly, genetic and pharmacological activation of Wnt signaling was shown to restore adult hippocampal neurogenesis, and also to improve cognitive performance in animal models of AD. Although it is not yet known how neurogenesis contribute to hippocampal function in humans, compelling evidence in animal models suggest that adult-born neurons are important for learning and memory, cognitive flexibility and mood regulation. In addition, recent findings support that neurogenesis impairment contributes to cognitive decline in aging and AD. Therefore, a better understanding on the molecular mechanisms involved in the regulation of neurogenesis may have important therapeutic implications. The reviewed evidence suggests that stimulation of Wnt signaling emerges as an attractive strategy to enhance endogenous neurogenesis and improve hippocampal-dependent cognitive function.SBA, DV-B, and MDM wrote and revised the manuscript. LV-N, wrote, drafted, and edited the manuscript. All authors approved the final version as submitted.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.


Educational differences in female cohort fertility vary strongly across high-income countries and over time, but knowledge about how educational fertility differentials play out at the sub-national regional level is limited. Examining these sub-national regional patterns might improve our understanding of national patterns, as regionally varying contextual conditions may affect fertility. This study provides for the first time for a large number of European countries a comprehensive account of educational differences in the cohort fertility rate (CFR) at the sub-national regional level. We harmonise data from population registers, censuses, and large-sample surveys for 15 countries to measure women’s completed fertility by educational level and region of residence at the end of the reproductive lifespan. In order to explore associations between educational differences in CFRs and levels of economic development, we link our data to regional GDP per capita. Empirical Bayesian estimation is used to reduce uncertainty in the regional fertility estimates. We document an overall negative gradient between the CFR and level of education, and notable regional variation in the gradient. The steepness of the gradient is inversely related to the economic development level. It is steepest in the least developed regions and close to zero in the most developed regions. This tendency is observed within countries as well as across all regions of all countries. Our findings underline the variability of educational gradients in women’s fertility, suggest that higher levels of development may be associated with less negative gradients, and call for more in-depth sub-national-level fertility analyses by education. Research on variation in fertility in contemporary societies often focuses on the relationship between education and fertility  at the regional level in contemporary Europe. We assess (1) whether there is sub-national regional variation in the educational gradient in CFRs, and (2) whether this variation is systematic by regional level of development. Based on our theoretical considerations, we expect to find that negative educational fertility gradients are more common among women living in less economically developed regions, and that these gradients might be low or even positive in more developed regions. In our analyses, we investigate the cohort fertility of women born in the late 1960s and early 1970s in 15 European countries. These cohorts are of interest at least for two reasons. First, they provide an up-to-date cohort perspective to fertility in Europe, i.e. they recently completed their childbearing. Second, for them, we witness in some European countries substantial shifts towards less negative or even slightly positive gradients in cohort fertility by educational attainment. For these women, we are able to measure number of children, level of education, and region of residence at the end of the reproductive career.Women may move across regions over their life course—before, during, and after having (any) children. While sub-national migration and family formation are often closely interrelated life-course steps, long-distance moves are less common at higher parities and higher reproductive ages  classification by Eurostat , a sub-rIn register data, information on the region of residence is derived from registers on the place of dwelling. In survey data, it is self-reported. In census data, it is either self-reported or obtained from registers and corrected, where necessary, based on self-reports. We measure regional development using GDP (purchasing power standardised gross domestic product per capita) in 2011 extracted from the Eurostat database Eurostat . We alsoThe measurement of education is based on registers in the register data and self-reports in other data. We distinguish between low, medium, and high educational attainment following the International Standard Classification of Education (ISCED) UNESCO . High reFertility is measured as the mean total number of children per woman, corresponding to the cohort fertility rate. This number includes all children women have ever given birth to and is derived from self-reports in census or survey data and information on registered births in register data. In France, the Netherlands, and Norway, children given for adoption are linked to their adoptive parents instead of their biological parents. The country-specific mean age at the measurement of fertility was at least 42 in all cases except Belgium, where it was 41. Thus, completed fertility is particularly in Belgium slightly underestimated. Prior research indicates, however, that changes in the educational gradient of women are very small past this age  and as covariate log of GDP at the regional level. This regression describes how the educational gradient in fertility varies across levels of GDP across all countries and regions. The second approach is a regression model that has the same outcome and covariate, but in addition country fixed effects. This model effectively estimates for each country the within-country association between the educational difference in fertility and log of GDP and provides an average of these within-country associations. The results from this model describe how, on average, the educational gradient in fertility relates to log of GDP within countries.Our data include both regions with large and with small numbers of observations: in 9 out of the 15 countries, full population data are not available  educated women in more developed regions suggests that the overall negative educational gradients in cohort fertility at the country level are more strongly driven by women living in less economically prosperous sub-national regions. Such tendency is most clearly evident in Central and Eastern Europe, where country-level negative educational gradients in cohort fertility remain strong. Given that the shares of women with high educational attainment are particularly large in well-developed regions, these findings may help to explain why overall fertility has been relatively high in well-developed regions in Europe in recent years. A longitudinal approach covering successive birth cohorts and information on place of residence over the life course would be useful to complement these descriptive findings.https://fertility.shinyapps.io/cfr_edu_region/; the cohort fertility rates in digital format and the code used to generate the interactive map: https://github.com/DemogrFertility/cfr_edu_region.Additional material: an interactive map showing women’s cohort fertility rate by level of education at the sub-national regional level in 15 European countries:


In this review, we focus on the role of post-translational modifications in regulating nNOS and nNOS-phosphorylating protein kinases and communication among themselves.Reactive oxygen species (ROS) are not only harmful to cell survival but also essential to cell signaling through cysteine-based redox switches. In fact, ROS triggers the potential activation of mitogen-activated protein kinases (MAPKs). The 90 kDa ribosomal S6 kinase 1 (RSK1), one of the downstream mediators of the MAPK pathway, is implicated in various cellular processes through phosphorylating different substrates. As such, RSK1 associates with and phosphorylates neuronal nitric oxide (NO) synthase (nNOS) at Ser847, leading to a decrease in NO generation. In addition, the RSK1 activity is sensitive to inhibition by reversible cysteine-based redox modification of its Cys223 during oxidative stress. Aside from oxidative stress, nitrosative stress also contributes to cysteine-based redox modification. Thus, the protein kinases such as Ca N-terminal kinase (JNK)-1/2/3, and p38-MAPK  are most extensively studied. ERK1/2 activation controls cell proliferation. Meanwhile, the JNKs and p38 are responsive to stress stimuli and play roles in the inflammatory and the apoptotic responses, respectively [The mitogen-activated protein kinases (MAPKs) are a highly conserved family of serine/threonine kinases that play a central role in the range of fundamental cellular processes like cell growth, proliferation, death, and differentiation . Among tectively .N-terminal kinase domain (NTKD) at Ser 221 [RSKs contain two functional protein kinase domains, and in mammals, four expressed homologs (RSK1–4) have been identified. RSKs are activated by ERK1/2  and 3-ph Ser 221 , respect Ser 221 .Nitric oxide (NO) is a ubiquitous gaseous biologically active messenger . NeuronaS-nitrosylation, the addition of an NO to the thiol side chain of cysteine residues [l-type Ca2+ channel and Ras, an upstream activator of MAPK [Physiological functions of NO in the CNS include regulation of neurotransmission, neuroprotection, and neurotoxicity . Initialresidues . As is t of MAPK ,18,19. W of MAPK ,21.2−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), and causes oxidative stress by disturbing the redox balance in vivo [2O2 by superoxide dismutase and catalase but reacts with NO produced by NOS to produce a more reactive peroxynitrite (ONOO−) and hydroxyl radical. Excessive ROS can induce oxidative damage in cell constituents and promote several degenerative diseases and aging [In addition to NO, reactive oxygen species (ROS) are constantly produced by several normal cellular events, with a major source being aerobic respiration. ROS are highly reactive molecular groups derived from oxygen molecules such as superoxide  is abundant in cells, and sulfenic acid and S-nitrosylation are converted to S-glutathionylation, suggesting that S-glutathionylation is a common feature in redox signaling [In the function of ROS as a signal molecule, the highly nucleophilic cysteine thiol plays a major role in maintaining an appropriate redox state in vivo . Proteinsylation ,30,31,32sylation , energy sylation ,35, phossylation ,37,38, asylation . The thisylation . The revignaling .2O2 mimics oxidative stress and leads to activation of MAPK pathways [S-glutathionylation at Cys223 [S-glutathionylation. Thus, the protein kinases such as Ca2+/calmodulin (CaM)-dependent protein kinase I (CaMKI) and II (CaMKII) that phosphorylate nNOS [S-glutathionylation. This article gives an overview of the role of S-glutathionylation in regulating NOS and NOS-phosphorylating protein kinases and in relating neuronal diseases.Several cellular stimuli that induce ROS production can activate MAPK pathways in multiple cell types ,43. Morepathways ,45. We ht Cys223 . These at Cys223 ,21. We ht Cys223 . In otheate nNOS ,49 wouldS-glutathionylation depends on the acid dissociation constant (pKa) of cysteine thiol. In other words, reactivity increases in a microbasic environment (low pKa) where arginine, histidine, and lysine are located nearby. Under oxidative stress, S-glutathionylation at these cysteine sites is caused by some reactions, such as thiol-disulfide exchange with GSSG (pathway 1), the reaction of oxidant-induced protein sulfenylation (P-SOH) with GSH (pathway 2), the reaction of NO-induced S-nitrosylation protein (P-SNO) with GSH (pathway 3), and reaction with S-nitrosoglutathione (pathway 4) (S-glutathionylation formation under mimicking oxidative (pathway 2) or nitrosative (pathway 3) stress [S-glutathionylation is an established modification as a response against ROS/ reactive nitrogen species (RNS) and S-nitrosylation and S-glutathionylation may occur at the same site. In fact, we have found that CaMK I activity is inhibited by either S-nitrosylation or S-glutathionylation at Cys179 [S-nitrosylation of Cys6 residues and is also sensitive to inhibition by S-glutathionylation of the residues besides Cys6 [One of the key characteristics of the redox switches is the change of the redox potential. The main endogenous biomolecule defining the redox potential is the redox couple formed by GSH and its oxidized form, glutathione disulfide (GSSG), due to the high reversibility of this system and high concentration of GSH in tissues . Other ithway 4) . The cel) stress . That ist Cys179 ,54. MeanS-glutathionylation plays an important role in protecting cysteine residues from irreversible oxidation during oxidative stress. Meanwhile, it can change the structure, function, and activities of modified proteins which affects signal transduction. Both the forward- (S-glutathionylation) and reverse- (deglutathionylation) reactions are involved in the pivotal redox-dependent cell signaling cascades. Grx is an essential thioltransferase whose primary role is to reduces S-glutathionylation. Grx 1 insufficiency results in increased levels of S-glutathionylation, implicated in many pathological events, including cardiovascular [S-glutathionylation, lower GSH:GSSG ratio might attenuate Grx activity, mimicking Grx deficiency. In contrast, Grx 1 plays a primary proinflammatory role in microglia [S-glutathionylation proteins have been experimentally verified. Thus, S-glutathionylation is thought to participate in the protection and progression of pathological consequences, requiring examination of cell-type specific manipulation of Grx1 content or activity.vascular  and Parkvascular  diseasesicroglia  or an anicroglia . More thS-glutathionylation detection [S-glutathionylated protein is blocked with an alkylating agent and then recovered with a resin having an affinity for the thiol obtained by reducing S-glutathionylation. However, it is important to ensure the selectivity of de-S-glutathionylation by reductive treatment. Bioinformatics has also predicted the site of protein cysteine residues that are S-glutathionylated [The direct and indirect methods are employed to identify the modified molecules. As a direct method, technically advanced proteomics have facilitated high-level enhanced sensitivity of etection . There aonylated .S-glutathionylation  is unstable, and excessive oxidation results in irreversible modification of sulfinylation to sulfonylation. Sulfenic acid can be also further oxidized to form disulfide bonds, nylation . TherefoS-glutathionylation of eNOS was reported in the vascular wall of hypertensive rats [S-glutathionylation of eNOS occurs on Cys689 and Cys908, which are critical to maintaining the normal function of the enzyme [S-glutathionylation triggers eNOS uncoupling, switching the protein activity from an NO producing enzyme towards a NOX activity producing O2−, causing the pathophysiology of cardiovascular and renal diseases [S-glutathionylation of eNOS is implicated in the pathogenesis of necrotizing enterocolitis (NEC). The eNOS-derived NO is inhibited by S-glutathionylation of eNOS, which can be reversed by Grx1. Grx1 deficient mice increase the severity of experimental NEC in association with S-glutathionylation of eNOS [S-glutathionylation may contribute to endothelial dysfunction in various pathological conditions.The ive rats  and endoive rats  or decreive rats . The S-ge enzyme . Consequdiseases ,65,66,67S-nitrosylation [S-nitrosylation [S-nitrosylated in resting rat hippocampal neurons and the enzyme undergoes de-nitrosylation during the process of rat brain ischemia/reperfusion [The zinc-tetrathionate motif present in either nNOS or eNOS is related to enzyme activity. The Cys96 and Cys101, which comprise the eNOS zinc-tetrathiolate complex, have been identified as the key sites of eNOS sylation ,70. Cys3sylation ,72. In aerfusion . Intereserfusion .S-glutathionylation-sensitive site to regulate the activity of RSK1 [S-glutathionylation at Cys223 appears to be dominant over activation of the kinase by its phosphorylation at Ser221 . It was recently demonstrated that a large portion of cysteine thiols are oxidized to persulfidated thiols such as cysteine perthiosulfenic acid (CysSSOH) and perthio-glutathion (CysSSSG) [S-glutathionylation and S-nitrosylation. In fact, RSK1was inactivated by its polysulfidation at Cys223 residue (our unpublished observation). The RSK1 inactivation by S-glutathionylation, S-oxidation, and polysulfidation at Cys223 might play important roles in regulating NOS signaling  . QuantitCysSSSG) ,82. In rCysSSSG) . Thus, pignaling .Multifunctional CaMKs are activated by the phosphorylation of a crucial threonine residue either by itself (CaMKII) or by upstream kinases, CaMK kinases (CaMKKs) (CaMKI and CaMKIV). Multifunctional CaMKs, present in most mammalian tissues, can phosphorylate many downstream targets, thereby regulating numerous cellular functions. Aside from canonical post-translational modifications, cysteine-based redox switches in CaMKs affect their enzyme activities . It was S-glutathionylation, S-nitrosylation, or S-oxidation of Cys179 in the activation loop [2+/CaM to nNOS [S-glutathionylation, S-nitrosylation, S-oxidation, or polysulfidation at Cys179 might play important roles in regulating NOS signaling , Cys290  also induce autonomous activity [S-nitrosylation of CaMKIIδ at Cys273, which is not conserved in CaMKIIα, to Ca2+/CaM exposure strongly suppresses kinase activation [S-nitrosylation of CaMKIIα in the hippocampal slices [S-nitrosylation of Cys6 inhibits CaMKIIα activity in ATP competitive fashion. Furthermore, we showed that S-glutathionylation induced by diamide/GSH also inhibits CaMKIIα activity, but it is not via the site-specific modification at Cys6 [S-glutathionylation  is an autosomal dominant disease that causes degeneration of medium spiny γ-aminobutyric acid neurons, main symptoms are involuntary movements and psychiatric symptoms/dementia caused by the addition of polyglutamine to the  nucleus . Further nucleus ,108. S-g+ influx . The exp+ influx . Thus, tS-glutathionylated CaMKI at Cys179 and phospho-nNOS at Ser847/S-glutathionylated RSK1 at Cys223 and S-nitrosylated CaMKII at Cys6/30. In more detail, the CaMKI activity is inhibited via its S-glutathionylation, S-nitrosylation, S-oxidation, or polysulfidation of Cys179. The CaMKII activity is inhibited via its polysulfidation and S-nitrosylation at Cys6 but neither its S-glutathionylation nor S-oxidation at Cys6. The RSK1 activity is inhibited via its S-glutathionylation, S-oxidation, and polysulfidation at Cys223 but not by its S-nitrosylation (S-glutathionylation or S-oxidation. The CaMKIV activity is inhibited via its polysulfidation and S-oxidation at Cys198 but neither its S-glutathionylation nor its S-nitrosylation. CaMKIV is localized predominantly in the nucleus and cannot phosphorylate nNOS which is not present in the nucleus and might not be sensitive to nitrosative stress. However, it is not clear how nNOS-phosphorylating protein kinases as CaMKI, CaMKII, and RSK1 are regulated by the specific type of redox-sensitive Cys modification in pathophysiological response in cells. We have shown that NO can promote the nicotine-induced activation of MAPK in PC12 cells [S-glutathionylated proteins were increased in cell lysates, suggesting S-glutathionylation might not be involved in NO-induced promotion of MAPK activation by nicotine [2O2 production which induced oxidation of and inhibition of protein Tyr phosphatases, leading to enhanced EGF receptor activation [2O2 production in A431 and HaCaT cells [2+-signals including CaMKs and NOS activations to modulate diverse neuronal processes.We propose here the phosphorylation- and NOS-signaling are mutually regulated via phospho-nNOS at Ser741/sylation . Thus, C12 cells ,21 (Figu12 cells . In the nicotine . The tartivation . The calaT cells . It appeaT cells . Thus, o


The human T-lymphotropic viruses (HTLVs) are causative agents of severe diseases including adult T-cell leukemia. Similar to human immunodeficiency viruses (HIVs), the viral protease (PR) plays a crucial role in the viral life-cycle via the processing of the viral polyproteins. Thus, it is a potential target of anti-retroviral therapies. In this study, we performed in vitro comparative analysis of human T-cell leukemia virus type 1, 2, and 3  proteases. Amino acid preferences of S4 to S1′ subsites were studied by using a series of synthetic oligopeptide substrates representing the natural and modified cleavage site sequences of the proteases. Biochemical characteristics of the different PRs were also determined, including catalytic efficiencies and dependence of activity on pH, temperature, and ionic strength. We investigated the effects of different HIV-1 PR inhibitors  on enzyme activities, and inhibitory potentials of IB-268 and IB-269 inhibitors that were previously designed against HTLV-1 PR. Comparative biochemical analysis of HTLV-1, -2, and -3 PRs may help understand the characteristic similarities and differences between these enzymes in order to estimate the potential of the appearance of drug-resistance against specific HTLV-1 PR inhibitors. Human T-cell leukemia virus type 2 and type 3 (HTLV-2 and HTLV-3) belong to the delta group of retroviruses, together with HTLV-1, HTLV-4, their simian counterparts , and bovine leukemia virus (BLV) [Based on the 2015 report from the European Centre for Disease Prevention and Control . The datHTLV-1 is associated with adult T-cell leukemia, tropical spastic paraparesis/HTLV-I-associated myelopathy , or other diseases, such as HTLV-1 associated uveitis or infective dermatitis ,7. The eThe life-cycle of the HTLVs is very similar, even though, in the case of HTLV-3, it is barely understood. They prefer different molecules for entry. HTLV-1 requires the presence of heparan sulfate proteoglycans (HSPGs), neuropilin 1 (NRP-1), and glucose transporter type 1 (GLUT-1) on the surface of activated CD4+ T-cells. HTLV-2 requires NRP-1 and GLUT-1 on CD8+ T-cells, while HTLV-3 also targets CD4+ and CD8+ T-cells and its specific receptors are not known . HTLV viIt is common in all retroviruses that the viral protease (PR) has an essential role in the viral life-cycle by processing the Gag and Gag-Pol polyproteins by limited proteolysis, which results in the release of functional proteins, such as matrix (MA), capsid (CA), nucleocapsid (NC), reverse transcriptase (RT), and integrase (IN). The retroviral PRs, including those of HTLVs, are active as homodimers. The dimerization is the prerequisite for enzyme activity ,22. TheyCurrently, there is no standard ATLL therapy, but usually chemotherapy combined with interferon-alpha/zidovudine is used. There are also some potential molecules, which are used in the therapy or are in clinical trials, especially purine analogs, histone deacetylase inhibitors, or monoclonal antibodies . Only soThe inhibitors that have been developed to target Human immunodeficiency virus type 1 (HIV-1) PR were also considered to be potentially applicable in the treatment of HTLV-1 infection. While HIV-1 protease inhibitors (PIs) are effective inhibitors of HIV-1 PR , they arOur previous studies showed that the oligopeptides representing natural cleavage sites of retroviral proteases are effective substrates for HTLV-1 PR, but HTLV-1 PR shows substantially narrower specificity compared to that of HIV-1 PR . In thisAll materials were purchased from Sigma-Aldrich . Otherwise, it is indicated.The coding sequence of stabilized (C2A) HTLV-1 PR cloned into the pET11a expression plasmid was prepared previously . The seqE. coli cells  using heat-shock at 42 °C for 90 s. The transformed cells were cultured in Luria-Bertani (LB) medium supplemented with ampicillin and grown at 37 °C while shaking until the optical density measured at 600 nm wavelength reached a value between 0.6–0.8. The protein expression was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), which was followed by incubation at 37 °C for 3 h while continuously shaking.The expression plasmids were transformed into BL21(DE3) g  for 20 min at 4 °C. Cell pellets were lysed in buffer A , 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.2). The suspended cells were disrupted by sonication , which was followed by centrifugation at 25,000× g for 20 min at 4 °C  to prepare a total cell lysate (step 1). To isolate the proteins from inclusion bodies, the cell pellets containing HTLV PRs were first dissolved in buffer B  (step 2). The latter step was not applied previously in the purification of HTLV-1 PR [After protein expression, the cells were harvested by centrifuging the suspensions at 4000× TLV-1 PR . After aAfter solubilization, the proteins were purified by a reversed-phase high performance liquid chromatography (RP-HPLC)-based method. The purification was performed by Äkta Purifier instrument  using the POROS R2 column . For separation, an increasing water/acetonitrile gradient (0–100%) was used in the presence of 0.05% trifluoroacetic acid (TFA) at a 1-mL/min flow rate. The purities of the eluted fractions were determined by SDS-PAGE (using 14% or 16% polyacrylamide gels). The fractions of highest purity (>90%) were applied in downstream steps.Folding of the enzymes were ensured by dialysis overnight at 4 °C against buffer E  (PIPES), 1 mM EDTA, 100 mM NaCl, 10 v/v% glycerol, 0.5 v/v% NP-40, 5 mM DTT, pH 7.0).The oligopeptides representing the naturally occurring matrix/capsid (MA/CA), capsid/nucleocapsid (CA/NC), trans-frame protein/protease (TF1/PR), and PR/p1 cleavage sites of HTLV-1, -2, and -3 PRs were used as substrates. The oligopeptide substrates representing the natural sequences of HTLV PRs were ordered from BioBasic, while the oligopeptide substrates representing naturally occurring cleavage sites of various viruses ,37, or tThe IB-268 (KTKVL-r-VVQPK) and IB-269 (APQVL-r-PVMHP) reduced peptide bond (-r-)-containing inhibitors, which were synthesized by Dr. Ivo Blaha . The DMP-323, which is a tight-binding inhibitor of HIV-1 PR , and oth3PO4, 4 M NaCl, 10 v/v% glycerol, 10 mM DTT, pH 5.6), 5 µL of HTLV-1, HTLV-2, or HTLV-3 PR , and 0.5–5 µL oligopeptide substrate representing HTLV PR natural cleavage sites . The final volume of the reaction mixture was set to 20 µL with distilled water if it was necessary. The reactions were initiated by the addition of the enzyme, were incubated at 37 °C for 0.5–4 h, and stopped by the addition of 180 µL 1 v/v% TFA. For substrates representing natural cleavage sites of retroviruses other than HTLV-1, -2, and -3 PRs , Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), Mason-Pfizer monkey virus (MPMV), and murine leukemia virus (MuLV) and BLV) in a longer incubation time (24 h) was also applied. The cleavage products were separated by using a Nova-Pack C18 RP-HPLC column  on HPLC (Merck-Hitachi LaChrom). Water-acetonitrile gradient (0–100%) was used for separation in the presence of 0.05% TFA at 2 mL/min flow rate. The kinetic parameters were determined by fitting the data—obtained at less than 20% substrate hydrolysis—to the Michaelis–Menten equation. The Gaussian equation was used to determine pH optimum and exponential growth equation for determination of optimal NaCl concentration, while linear regression was applied to plot the dependence of enzyme activity on temperature. Prism8 software  was used for evaluation. The kcat/KM catalytic constants were calculated based on the active enzyme concentration, which was determined by active site titration using the method described previously [For kinetic measurements, we applied 10 µL 2× incubation buffer . The reaction mixtures contained 10 µL buffer, 5 µL enzyme, and 5 µL substrate. The cleavage reactions were initiated by the addition of the enzyme, and incubated and analyzed as described above in the To compare the amino acid preferences of HTLV-2, and HTLV-3 PRs to those of HTLV-1 PR, the oligopeptides representing the wild-type and the P4, P3, P2, P1, and P1′ variants of HTLV-1 CA/NC cleavage site (KTKVL*VVQPK) were applied as substrates. To study whether S5 and S4 sites contribute to substrate recognition, shortened variants representing P4-P5′ and P3-P5′ residues of the same HTLV-1 CA/NC natural cleavage site were also used. The enzyme reactions were performed as described above in the protease activity assays section. The reaction mixtures contained 10 µL 2× incubation buffer, 5 µL HTLV-1, HTLV-2, or HTLV-3 PR , and 5 µL of substrate , in a 20-µL final volume. The reactions were initiated by the addition of the enzyme, followed by incubation at 37 °C for 0.5–4 h, prior to HPLC-based separation of 180 µL of 1 v/v% TFA added to the mixtures. The relative activities were determined in the case of all substrates. The activity measured on the wild-type KTKVL*VVQPK substrate was considered to be 100% in the case of each enzyme.The reaction mixtures were prepared to contain 4.8 µL of substrate , 0.2 µL of inhibitor, 5 µL of the enzyme, and 10 µL of a 2× incubation buffer. The substrates representing HTLV-2 and HTLV-3 MA/CA cleavage sites were used for HTLV-2 and HTLV-3 PRs, respectively. In the case of HIV-1 PIs, we applied 100 µM inhibitor stocks, while 0.5–100 µM stocks of IB-268 and IB-269 were used for screening. The inhibitors were dissolved in DMSO. Therefore, 0.2 µL of DMSO was added to the control samples. The IB-269 inhibitor  was applied to determine the amount of the active enzyme.The coding sequences of HTLV-2 and HTLV-3 PRs containing an eight residue-long N-terminal linker region  were cloned into pMALc2x vectors, which enabled expression of the proteins fused to an N-terminal maltose-binding protein (MBP) tag. The cloning procedure included overlap extension PCR, digestion by EcoRI and BamHI restriction endonucleases, and ligation. It was followed by site-directed mutagenesis of HTLV-2 PR  and HTLV-3 PR  coding sequences using a QuikChange II mutagenesis kit , according to the manufacturer’s instructions. All applied primers are shown in E. coli cells using heat-shock at 42 °C. The recombinant HTLV-2 and HTLV-3 PRs were expressed fused to an N-terminal MBP fusion tag (MBP-HTLV-2 and HTLV-3). The protein expression was induced by the addition of 1 mM IPTG, and then the cells were lysed by sonication in buffer A. The cell lysates were loaded onto 16% polyacrylamide gel. After SDS-PAGE, proteins were transferred onto a nitrocellulose membrane  and the proteins were detected by a Western blot based on the protocol described previously [The expression constructs were transformed into BL21 (DE3) E. coli cells, and each enzyme was purified from inclusion bodies. The extraction of the proteins from the inclusion bodies was the first phase of a purification procedure. The same conditions were applied for each enzyme. The solubilization and purification of HTLV PRs was performed based on a slight modification of the protocol described previously for HTLV-1 PR [HTLV PRs were expressed in BL21 (DE3) TLV-1 PR . We intrAfter solubilization, the proteins were purified by an RP-HPLC method. The eluted fractions were collected and the purity of fractions was assessed by SDS-PAGE . The ~37First, we tested the effects of different reaction conditions on enzyme activity and determined the dependence of HTLV-2 and HTLV-3 PR activities on temperature, pH, and ionic strength. Later, the optimal conditions—that were found previously to be optimal for HTLV-1 PR ,43—were For cleavage reactions, the substrates and cleavage products were separated by an RP-HPLC-based method, which is a representative chromatogram shown in Both HTLV-2 and HTLV-3 PRs showed elevated activity at increasing temperatures, the highest activities were measured at ~40 °C, and >50% activity was measured only at a temperature ≥35 °C c,d. In aThe activities of both HTLV-2 and HTLV-3 PRs were found to be boosted by high ionic strength and showed the highest activity at the highest salt concentration we tested (2 M NaCl) a,b. ThisThe dependence of enzyme activity on pH was studied in a 4.5–8.0 range. We found that pH optimum of HTLV-1 (pH 6.11 ± 0.03) and HTLV-2 (pH 6.14 ± 0.06) PRs are highly similar, while optimal pH for HTLV-3 PR (pH 5.56 ± 0.04) is slightly lower . Our resWe performed activity measurements to determine catalytic efficiencies of HTLV PRs, using oligopeptides representing natural HTLV cleavage site sequences  . The HTLcat/KM values were determined previously for HTLV-1 PR on substrates representing HTLV-1 cleavage sites [cat/KM was obtained by an HTLV-1 PR TF1/PR substrate. Accordingly, we observed the lowest catalytic efficiency with this substrate for HTLV-2 and HTLV-3 PRs among the tested HTLV-1 cleavage sites.Of the tested substrates, the highest kge sites . Here, wAll tested substrates were processed by HTLV-2 PR, the highest catalytic efficiency was obtained on HTLV-2 TF1/PR and PR/P1 substrates. The HTLV-1 and HTLV-2 CA/NC cleavage site sequences differ only in the P5′ residue, which is Lys in the case of HTLV-1 and Arg in HTLV-2. Therefore, only HTLV-1 CA/NC substrate was applied while HTLV-2 CA/NC was omitted from the analysis.cat/KM was obtained for the HTLV-1 PR/P1 cleavage site, which was the most preferred substrate of HTLV-1 PR as well.HTLV-3 PR also cleaved all the tested oligopeptides, but, interestingly, the highest kWe decided to perform activity measurements with substrates representing HIV-1, EIAV, RSV, MMTV, MPMV, BLV, and MuLV PR cleavage sites. However, HTLV-1 PR was found previously to cleave some of these substrates . We obseFor the comparative analysis of amino acid preferences, activity measurements were performed to determine relative activities of HTLV PRs using series of oligopeptide substrates. Each substrate was modified from the wild-type HTLV-1 CA/NC (KTKVL*VVQPK) substrate by shortening its length  or by modifying single positions  .We found that each HTLV PR showed lower activity on shortened substrates as compared to the wild-type (KTKVL*VVQPK). The shortest substrates (P3-P5′) were not processed or only negligible hydrolysis was detected . In agreThe mutations at the P4 position were well tolerated by each HTLV PR. This is in agreement with the relatively larger hydrophobic S4 cavity of HTLV-1 PR ,44,53. TBased on the relative activities obtained for the tested P3 variants, various residues can be accommodated at the S3 subsite. The amino acid preferences were similar, but only HTLV-1 PR accepted P3-Phe and P3-Val mutants as substrates, and showed higher preference for a P3-Ser residue as compared to P3-Asp. In the case of HTLV-1 PR, the P3-Ser variant was the only substrate for which higher relative activity was observed than for the wild-type.The P2-Ile mutant was found to be the most preferred substrate of the tested P2 variants, and the relative activity obtained for HTLV-2 and HTLV-3 PRs was higher as compared to the wild-type substrate . SimilarTyr and Phe were tolerated in the P1 position only by HTLV-1 PR. These variants were found previously to be good substrates of this enzyme . HTLV-3 In agreement with our previous results , variantWe tested the inhibitory potentials of different PIs including approved therapeutic HIV-1 PIs  and DMP-323, which is a tight-binding inhibitor of HIV-1 PR. All tested PIs showed only moderate inhibitory effect against HTLV-2 and HTLV-3 PRs, and we observed <50% loss of activity even when using the inhibitors in a 1-µM final concentration a. The obRitonavir has already been tested as a potential inhibitor against HTLV-1 PR. This HIV-1 PI was found to have anti-leukemic activity against ATLL cells ex vivo , but theWe found that darunavir and indinavir showed the highest inhibition of the studied inhibitors in the case of each protease, but the most remarkable inhibition was observed for indinavir, which caused a ~50% decrease of HTLV-1 PR. In all other cases, more moderate inhibition was observed, which is in agreement with the results of previous studies revealing that the HIV-1 PIs are not effective against HTLV-1 PR .i value for HTLV-1 PR has already been determined previously for IB-268 (298 nM) and for IB-269 (465 nM) [i values obtained for HTLV-2 and HTLV-3 PRs were lower as compared to those determined for HTLV-1 PR .Inhibition studies were also performed to study the inhibitory potential of IB-268 (KTKVL-r-VVQPK) and IB-269 (APQVL-r-PVMHP) HTLV-1 PIs. The K(465 nM) . These i(465 nM) . We repeTLV-1 PR c. IB-268The proteases of HTLV-1, HTLV-2, and HTLV-3 viruses show no high sequence identity . HTLV-1 We prepared mutant HTLV-2 and HTLV-3 PRs, which contained mutations that were introduced previously to HTLV-1 PR to study its self-processing capability . The ratIn the case of HIV-1 and HIV-2 PRs, the I50 residue—corresponding to A59 of HTLV PRs—is suggested to have a role in flap mobility. A59I mutation in HTLV-1 PR was found previously to cause undetectable activity .F67 is considered to be part of the S4 subsite and its change to Gln was also not tolerated by HTLV-1 PR .HTLV-2 and HTLV-3 PRs were expressed as fusion proteins containing an N-terminal MBP tag. There was an 8-residue-long linker sequence between the tag and the enzyme in both cases in which the sequence represented the N-terminal flanking sequences of the proteases (P8-P1 residues). This linker enabled the proteases to process themselves from the fusion protein. This sequence was SRSRHLDT in the HTLV-2 PR, and LTSPRTIL in the HTLV-3 PR a. The seThe results showed a similar effect of mutations on autoproteolysis in the case of each enzyme. Differences were observed in the case of mutations at the 37th and 57th positions . MutatioIt was found previously that HTLV-1 PR bearing the individual L57G mutation was a very inefficient enzyme and did not hydrolyze the CA/NC cleavage site peptide (KTKVL*VVQPK). Therefore, its ability for self-processing was not studied . In agreIn this study, we aimed the characterization of HTLV-2 and HTLV-3 PRs and the comparison of their main biochemical features and specificities with those of HTLV-1 PR.E. coli cells and, after their extraction from inclusion bodies, they were purified by an RP-HPLC-based method. First, we investigated the effects of different reaction conditions on enzyme activity to determine optimal conditions. We found that similarly to HTLV-1 PR and numerous retroviral PRs, the optimal temperature for HTLV-2 and HTLV-3 PRs is close to 37 °C, and their activity is boosted by high ionic strength (even by 2 M NaCl) (The enzymes were expressed in BL21(DE3)  M NaCl) . Optimal M NaCl) , but eacFor the comparison of the substrate specificities of HTLV PRs, we used series of oligopeptides representing naturally occurring cleavage sites of HTLV PRs  and variIn contrast to the differences in their specificity, our results imply that the size of a substrate-binding site is similar in each HTLV PR. As compared to the decapeptide, we observed lower cleavage efficiency for the N-terminally shortened nonapeptides and octapeptides, lacking P5 and P5-P4 residues, respectively . This rei was found to be between 12 and 14 nM, respectively. This was similar to the inhibition constant determined previously for BLV PR [The inhibition profiling of the three HTLV PRs revealed that those HIV-1 PIs, which were found previously to be inefficient against HTLV-1 PR ,34,41 har BLV PR . Ritonavr BLV PR . Thus, tr BLV PR .To assess the tolerance of HTLV-2 and HTLV-3 PRs toward mutations likely appear in the development of resistance towards PIs. Protease mutants were also designed to contain HIV-1-like residues in the active site, at 37th, 57th, 59th, and 67th positions, which correspond to the 30th, 48th, 50th, and 58th positions in HIV-1 PR. Being part of the ligand-binding sites, they are prone to mutations in drug resistance. The effect of mutations on the enzyme folding and activity were studied by investigating self-processing of MBP-fused enzymes containing a natural cleavage site sequence prior to the protease. None of the single mutant HTLV-1 PRs were found to retain their ability for autoproteolysis upon single point mutations  , but, inRetroviruses can replicate by two fundamentally different mechanisms. The replication can occur by forming exogenous virions, and infection of target cells, like in the case of HIV, but also in the integrated DNA form, by forcing the infected cells to divide, as seen in the case of HTLV-1. The first method involves the action of two error-prone enzymes, known as RNA polymerase II and RT, resulting in accumulation of mutations that may contribute to a much better mutation tolerance of the viruses. This enables the quick development of resistance against any kind of drugs. Our previous studies suggested that HTLV-1 PR is much more sensitive toward binding site mutations as compared to HIV-1 PR . AlthougIn summary, our result provides important information about biochemical characteristics of HTLV-2 and HTLV-3 PRs, which may aid design of specific inhibitors, as identification of an effective treatment to control HTLV-1 infection and to treat HTLV-1-related diseases is still an ongoing challenge.