Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA—the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa  are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans. What is fertility among species with internal fertilization? Fertility is their capacity  to produce offspring, either born from an egg or after carrying a pregnancy to term. In avian species fecundity, e.g., the potential for fertility is the most relevant in terms of gamete production and fertilization. In mammals, and particularly in eutherian species, fertility is the crucial capacity of bearing a partially hemi-allogeneic individual (the fetus) or parts of a decisive organ as the placenta. Some species are polytocous adding another level to fertility: prolificacy, imposing a grading of the capacity to fertilize and carry to term litters of various sizes.How does semen deposition in the female influence fertility? Semen entry in the female genital tract is a pre-requisite for internal fertilization, with spermatozoa still being the most relevant factor for fecundity in terms of numbers and normality of function. However, is there any other factor than spermatozoa affecting fertility? Yes, the seminal plasma (SP), because it modulates sperm viability and function but also affects the ability of spermatozoa to interact with the lining epithelium of the female genital tract and its secretions; it even serves as a carrier of signals for the female, their immune system in particular ,2,3,4. HSeminal plasma: what is it, and what roles does it play? The SP is a composite fluid built by the concerted mixture of secretions from the testis, epididymis and the accessory sexual glands, with distinct species differences that, together with emitted aliquots of the epidydimal sperm reserves, compose the ejaculate . The SP Is it just the SP proteome? The SP proteome appears to participate in this double signaling, triggering a transient inflammation and the changes in the expression of genes related to immune processes at the female internal genitalia ,29,30. TThis review attempts, by including our own results on various animal classes and model species, to critically summarize the current knowledge of the roles played by the SP components on ruling fertility and prolificacy in animal models and humans. The SP’s importance is hereby particularly discussed in relation to sperm survival, function and signaling to the female immune system towards fertility modulation, in vivo findings with major relevance for the development of diagnostic/prognostic biomarkers and the refinement of reproductive strategies. The review is comprehensive yet intending to avoid reiteration of the large volume of available literature elsewhere; nonetheless, it is written as comparative as we could.Semen, e.g., the ejaculate, is a complex suspension of spermatozoa bathing in a heterogeneous composite fluid, the so-called SP, built by contributions of the testis, the epididymis and/or the accessory sex glands; the latter conspicuously lead—in direct relation to variations in presence and gland size among animal classes and species—to differences in the volume of the ejaculate and SP composition ,45. In sCommon to boar, stallion and to some extent dog or man, the ejaculate is sequentially expelled in fractions usually classed as pre-sperm, sperm-rich (SRF) and post sperm-rich, clearly defined by the amounts of spermatozoa present and the volume and type of secretion building the SP of each fraction ,11,50. IEquines follow a similar pattern of ejaculation  as pigs.Differences in ejaculate building among species reflects on the SP composition. The SP is basically formed by secretions of epithelial cells and thus contains electrolytes, protein and steroid hormones, sugars and proteins/peptides, including enzymes . The relThe SP of most species contains protein compounds similar to those present in blood plasma, such as pre-albumin; albumin; α-, β- and γ-globulins; transferrin; enzymes and some immunoglobulins; complement factor; as well as differential amounts of cytokines and chemokines . ComprehIn mammals, SP proteome studies have dramatically expanded over the past decade, allowing for the identification of large and small proteins and peptides, which now account for the thousands available ,68,69,70The human SP has thousands of unique proteins, ~25% secretory from the accessory glands , and eitThe stallion displays not only a similar fractionated ejaculate as humans , but shoSus scrofa taxonomy) of other less abundant pig-specific SP proteins are present [Sus scrofa taxonomy were differentially expressed between the SRF and post-SRF. Eight of these, overexpressed in SRF, had been previously related to sperm membrane function, capacitation, the acrosome reaction and zona pellucida binding [Another species with a fractionated ejaculate, whose delivery sequence resembles humans, is the pig, our most commonly used animal biomedical model . Of inte present ,34, yet  present ,32,94,95 binding . A folloIt is thus evident that the proteins of the SP exert multiple functions, starting with their general influence as a vehicle aiding sperm survival when semen is highly extended ,87 or prInterestingly, the roles of the seminal fluid proteins mentioned for these mammals seem to be highly conserved across animal classes. The transfer of seminal proteins induces particular post-mating changes in female insects, inducing changes in behavior regarding feeding and decreased receptivity for re-mating, as well as of gene expression of the mating-dependent genes . Such ch1–3) were higher in the vesicular-dominated fractions [1–2, IFN-γ and IL-6, IL-10 and GM-CSF [The SP of most species contains differential amounts of a 5–20 kDa peptidomes with pro- and anti(or tolerance-related)-cytokines and chemokines . Both inractions ,41,106. d GM-CSF ,108,109.d GM-CSF ,95,110. d GM-CSF —play a rd GM-CSF ,116,117,d GM-CSF ,119,120.d GM-CSF . Leptin d GM-CSF , and affd GM-CSF ; their dd GM-CSF . Being pd GM-CSF ,125, andd GM-CSF . Their id GM-CSF . Notewor2 synthase in equines [In general, about 3% of proteins in SP are enzymes , with so equines ,131 and  equines , the PON equines ; or the  equines  also in Having reached this section in this review essay, it appears confirmed that the SP is evidently far from being a simple fluid, as it contains diverse molecules. Analysis of a tube containing a collected ejaculate that has been spun at relatively low speed/time to form a pellet of the suspended spermatozoa and eventual somatic cells revealed—using electron microscopy, for instance —a fluid These EVs are shed by the different organs that contribute their secretion to the seminal plasma, namely, the testes, ducti epididymis and the accessory sexual glands, explaining why they display diverse cell-derived membrane structures. These EVs sequentially interact with the spermatozoa  in theirSuch interactions with the female are well reviewed in reference , and theHowever, let us go back to the fact that the SP is built by several secretions and, moreover, the fact that spermatozoa are exposed to EVs during one of the most crucial periods once shed from the seminiferous epithelium: sperm maturation in the epididymis . During There is increasing evidence that the load of non-coding, smallRNA molecules in semen differentially contributes to reproduction, and that the dysregulation of their expression can explain many dysfunctions associated with infertility . Small RProcessing of the collected semen is of utmost importance since it can surely mix up the different allocations of the small RNAs being detected. Many small RNAs have been explored and detected in the male genital tract and semen, including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), YRNA and transfer RNA (tRNA)-derived small RNAs (tsRNAs) ,183,184.Differential analyses of the pig sperm transcriptome have shown the relevant associations with sperm quality  and fertWe have assumed that the fertility of a sire is not only ruled by the spermatozoa but also by the SP. However, can we separate these two intrinsic components of semen? One could state that this is what has been proven when IVF was established, putting most weight on spermatozoa, which included the removal of the native SP. IVF and intracytoplasmic sperm injection (ICSI) of ejaculated or epididymal spermatozoa, or even elongated spermatids that have never gone through sperm maturation, has resulted in fertilization, embryo development, pregnancies and births in humans  and animThe composition of SP and the differences among the ejaculate fractions has help define the possible influence on the fertility of the male ,96 via iDo we have evidence that the SP per se is, by inducing the changes of gene expression in the internal genitalia of the female we have already pointed out, able to mark differences in fertility when added separately from spermatozoa? The answer seems to be yes, as rates in early embryo survival ,173,208,Many of the above studies, and of many others being rather empirical in nature, however, show simple associations between the presence of SP and effects, with some of these complex in nature. The evidence gathered via association studies, at least regarding livestock fertility, show for proteins ,35,36,431 with native SP prior to insemination for their effects on both endometrial cytokines and on the development of pre-implantation embryos. It was rather evident that both SP and TGF-ß1 were able to influence endometrial cytokines, but only SP impacted pre-implantation pig embryo development, thus implying SP has a concerted action of various factors influencing embryo development by way of a modified gene expression by the female endometrium [Among the relevant genes overexpressed, SP-treated sows showed an overactivation of the transforming growth factor-ß (TGF-ß) signaling pathway, known as the pathways supporting the proliferation of Treg cells by regulating dendritic cell function  and thusometrium . FindingMost likely; this is indicated by the many SP proteins that have been associated with decreased motility and viability, and further to fertility ,94,217, On the down side, the methodologies to identify these markers are rather complicated and still only available at specialized laboratories , but we A parallel approach, complementing diagnostics for (in)fertility, is to identify molecules that enhance fertility in the semen of individuals already fertile. Yet, we need to remember that SP has been historically described as friend and foe to sperm survival. Being evolutionarily present, its roles for sperm function depend on what we consider. Spermatozoa are highly specialized cells, programmed for destabilization during their journey to the site of fertilization. They are covered by SP proteins whose adsorption to the plasma membrane prevent its destabilization and disruption, once gathered under the title of “decapacitation factors” , relevanSemen being processed, for any species, seems to benefit from the presence of SP for sperm survival and function, for instance when high-extension rates are applied ,250,251.In the end, would it be at all possible to use exogenous SP components to improve fertility after AI with extended, cooled, or frozen–thawed semen? Probably, the pertaining experimentation provides us with irrefutable proof that the associations seen between diverse molecules in SP and the fertility of the males go beyond that: associations. The reason for these words of caution resides in the fact that, despite many molecules, particularly proteins or antioxidative enzymes were presented as directly related to male or semen fertility, and their effect on semen has been elusive, sometimes arguing that the relation-to-effect seen was probably concerted, multifactorial and not simply related to one particular molecule. Moreover, the administration of such molecules, and particularly its resistance to degradation and its capacity to act onto living spermatozoa, are considered relevant hurdles to be explored. Hope is now focused on the growing knowledge on EVs and their functional cargo, mostly because they would be protected by the membrane-built EVs and because they would probably be the best delivered to living spermatozoa . There aHowever, we still need to determine the mechanisms ruling fertility for many of the molecules present in these SP-EVs, and the role they play on the immunoregulation of the female genital tract, modulating the capacity of the female to allow the presence and development of pregnancy to term, and for some species of interest, particularly for their value as livestock but also as suitable animal models for human reproduction.This review has critically summarized the current knowledge of the roles the SP and its particular components apparently at play in ruling fertility and prolificacy. Its focus has been to provide a comparative view, including humans, and considering suitable experimental animal models and livestock as well. The SP’s importance was particularly discussed in relation to sperm survival, function and signaling to the female immune system towards fertility modulation. Considering many in vivo findings with major relevance for the development of diagnostic/prognostic biomarkers and the refinement of reproductive strategies, particular interest has been put on the immunoregulation of the female genitalia, since it appears determinant for fertility in all species and even animal classes explored so far. Based on the available knowledge, it is expected that novel experimental research on SP EVs shall be of utmost relevance to advance not only our knowledge but our capacity to manipulate fertility.


N) inhibitor can inhibit the entire PA endonuclease activity. In this study, we screened the effectivity of PAN inhibitors from the FDA database through in silico methods and in vitro experiments. PAN and mutant PAN-I38T were chosen as virtual screening targets for overcoming drug resistance. Gel-based PA endonuclease analysis determined that the drug lifitegrast can effectively inhibit PAN and PAN-I38T, when the IC50 is 32.82 ± 1.34 μM and 26.81 ± 1.2 μM, respectively. Molecular docking calculation showed that lifitegrast interacted with the residues around PA or PA-I38 T’s active site, occupying the catalytic site pocket. Both PAN/PAN-I38T and lifitegrast can acquire good equilibrium in 100 ns molecular dynamic simulation. Because of these properties, lifitegrast, which can effectively inhibit PA endonuclease activity, was screened through in silico and in vitro research. This new research will be of significance in developing more effective and selective drugs for anti-influenza therapy.Drug repurposing can quickly and effectively identify novel drug repurposing opportunities. The PA endonuclease catalytic site has recently become regarded as an attractive target for the screening of anti-influenza drugs. PA N-terminal (PA N) contains an endonuclease active pocket and plays a crucial role in influenza virus polymerase activity. Drugs that abolish PAN endonuclease activity or disturb the assembly of RdRp can effectively inhibit the replication of the influenza virus [N endonuclease domain is highly conserved among different influenza virus subtypes, indicating that PAN is a promising broad-spectrum anti-influenza therapeutic target because of its ability to inhibit virus proliferation during the initial mRNA synthesis stage [Influenza is an infectious disease that causes 300,000 to 500,000 human deaths globally per year ,2. The Iis stage .N endonuclease inhibitor . Several classes of PAN endonuclease inhibitors researched included: 4-substituted 2,4-dioxobutanoic acids and 3,4-substituted 2,6-diketopiperazines [N endonuclease, was approved by the FDA as an inhibitor of influenza A and B after proving effective in clinical trials [The three classes of FDA-approved anti-influenza drugs are: neuraminidase inhibitor (oseltamivir and zanamivir), M2-ion channel inhibitor (adamantanes), and PAerazines , flutamierazines ,9,10, N-erazines , catechierazines , etc. Bal trials .N endonuclease and Baloxavir acid were disturbed after the Ile 38 mutated to Thr [N endonuclease domain is the primary mutant that leads to influenza’s resistance to Baloxavir acid. This resistance reduces the effectiveness of Baloxavir acid as an anti-influenza drug [A mutation in the influenza viral genome led to the influenza’s subsequent drug resistance. The hydrophobic interaction residues between PAd to Thr . The I38nza drug . Using tDrug repurposing research can effectively identify new drug repurposing opportunities, quickly expand the drug market, and reveal new commercially valuable uses for existing drugs . Some exN endonuclease inhibitors from the FDA-approved database through in silico methods and in vitro experiments. PAN inhibitors can inhibit the entire PAN endonuclease activity. Since the purpose of this study is to identify drugs that overcome resistance, we screened inhibitors that target both PAN and mutant PAN -I38T. Experimental tests have verified that the drugs lifitegrast and saquinavir have an inhibitory effect on PAN, while the drugs lifitegrast and conivaptan have an inhibitory effect on PAN-I38T. In addition, molecular docking shows the interaction mechanism between lifitegrast and the active site of PAN or PAN-I38T. Research suggests that lifitegrast may be a potential anti-flu drug. Finally, the method employed in this work could be utilized as a fast and viable strategy for accelerating research in the treatment of influenza.This research screened the effectivity of PAN endonuclease inhibitor structure, we performed the virtual screening protocol based on the PAN and mutant PAN-I38T structure. The residue Ile38 was not essential in either metal-ion binding or in catalytic activity. The I38 substitution does not block the endonuclease reaction but offers resistance to the inhibitor because the I38 side chain interacts with the Baloxavir acid. After the virtual screening, the compounds interacting with PAN or PAN-I38T were sorted by affinity score. The top-ranked compounds listed in N and PAN-I38T as hit compounds. Finally, six compounds, saquinavir, conivaptan, lifitegrast, rifaximin, dutasteride, and lurasidone, were used for further basic gel endonuclease determination. In comparison, three compounds, Teniposide [To develop the new PAniposide , Simepreniposide , and Nilniposide , were fiN or PAN-I38T, respectively. Next, we incubated different concentrations of compounds with PAN or PAN-I38T and then detected the cleavage of a substrate . Resistance develops when the hydrophobic interaction residues between PAN endonuclease and Baloxavir acid are disturbed after the Ile 38 mutates to Thr. Therefore, we used the mutant type of PAN-I38T as the virtual screening target to avoid drug resistance. We used the FDA database to effectively identify drug repurposing opportunities. Then, we discovered a potent inhibitor, lifitegrast, which differs from Baloxavir acid in that it has the ability to interact equally with both PAN and PAN-I38T.Researchers have persistently searched for a drug that could be used to treat all types of influenza viruses, including influenza A, influenza B, and influenza C, along with their mutants. In this study, we used PAN and PAN-I38T. Molecular simulation displayed that lifitegrast interacted with the active site of PAN or PAN-I38T. Further molecular dynamic simulation analysis suggested that the bindings between PAN/PAN-I38T and lifitegrast were stable.Gel-based endonuclease inhibitory assay showed that lifitegrast effectively inhibits the substrate cleavage of both PAHowever, there are still numerous challenges. Subsequent viral experiments in vivo need to be implemented in order to verify the reliability of experiments in vitro. Meanwhile, lifitegrast has been used for the treatment of dry eye, but further toxic experimentation is required before lifitegrast can be used as a treatment for the influenza virus. N endonuclease inhibitor from the FDA database through in silico and in vitro experiments. The findings suggest that lifitegrast may be a potential anti-flu treatment drug because it could solve the drug-resistant properties, which are superior to currently available flu treatments, such as Baloxavir acid. Finally, the method adopted in this work could be utilized as a fast and viable strategy for accelerating research on the treatment of influenza.In this research, we screened the effectivity of the PAN endonuclease protein crystal structure (PDB ID: 6FS6) and I38T mutant (PDB ID: 6FS7) in the PAN subunit were obtained from the RCSB Protein Data Bank database [The PAdatabase . One thodatabase . Virtualdatabase , with thCompounds saquinavir, conivaptan, lifitegrast, rifaximin, dutasteride, and lurasidone were purchased from Aladdin  or Yuanye Biotech . Substrate DNA was purchased from Takara Bio lic. N endonuclease domain from influenza A type virus (H1N1)  was cloned into the pET-28a vector, expressed in E. coli BL21 (DE3), and purified by IMAC. The positive clone was used for large-scale expression. Cells were expressed in LB medium at 37 °C for 4 h with 1 mM IPTG. Before the endonuclease activity assay, the protein sample was concentrated, and the buffer changed  by ultrafiltration. The protein concentration was measured by the BCA method. The PAN-I38T was cloned from the PAN through site-directed mutagenesis. The mutant PAN-I38T was expressed and purified the same as the wild PAN.The PAN endonuclease activity was detected through the digestion of a single-strand DNA substrate. In the digestion reaction, PAN or PAN-I38T was mixed with ssDNA in a digestion buffer that included 2 mM MnCl2. To determine the concentrate of PAN or PAN-I38T for endonuclease activity assay, 0–1.5 μM protein and 100 ng ssDNA were incubated at 37 °C for 60 min. After digestion, the samples were fractioned by 0.8% agarose gel and visible by a stain. Image J was used to determine the density of substrate ssDNA. Finally, 1.5 μM PAN or PAN-I38T were digested at 100 ng ssDNA in 10 μL volume for 60 min.The PAN or PAN-I38T and 100 ng ssDNA mixed with different concentrations of compounds were used for the experiment test. Samples without inhibitors were used as the no-compound control. Samples without protein were used as the substrate control. After digestion, the samples were loaded on the agarose gel for separation and were visible by a stain. The amount of remaining ssDNA was determined by image J. The IC50 value was calculated by GraphPad Prism 6.0 through non-Linear regression.Compounds screened from the FDA library analysis determined the inhibition of endonuclease activity. In total, 1.5 μM PAN and PAN-I38T with or without compounds was performed using GROMACS version 2018 with the CHARMM36 all-atom force field (28 March 2019) to investigate the inhibition mechanism [The MD simulation of PAechanism ,30. The echanism . Two addechanism , RMSD isechanism ,34,35.50 was calculated by GraphPad Prism 6.0 using non-linear regression. The data were represented as mean ± SD.The IC


Therefore, this 10-year surveillance of the infection showed stable HTLV-1/2 prevalence overall but a trend toward increased prevalence among the younger and more educated donors despite Brazilian policies to control sexually transmitted infections being in place for more than 10 years.It is unknown whether HTLV-1/2 prevalence has been stable or changing with time in Brazil. We present a 10-year (2007–2016) analysis of HTLV-1/2 infection in first-time blood donors from four blood banks in Brazil. The Brazilian blood centers participating in this multicenter Recipient Epidemiology and Donor Evaluation Study (REDS) are located in Recife in the Northeast and in São Paulo, Rio de Janeiro and Belo Horizonte located in the Southeast of the country. A previous REDS study using the same database from 2007 to 2009 showed that the prevalence per 100,000 donors was 222 in Recife, 83 in Belo Horizonte and 101 in São Paulo. From 2007 to 2016, HTLV-1/2 prevalence was calculated by year, blood center and birth cohort. Covariates included age, gender, schooling, self-reported skin color and type of donation. From 1,092,174 first-blood donations, in the general analysis, HTLV-1/2 infection predominated in females, donors over 50 years of age, black skin color and less educated. The average prevalence was 228 per 100,000 donors in Recife, 222 in Rio de Janeiro, 104 in Belo Horizonte and 103 in São Paulo. In the 10-year analysis, HTLV-1/2 prevalence was stable, but a trend was observed toward an increase in HTLV-1/2 infection among younger people ( Screening for HTLV-1/2 was introduced in Brazilian blood banks in 1993. In the routine of blood banks, all candidates for blood donation undergo pre-donation screening, which includes a questionnaire with their health history and a brief physical examination followed by serological and molecular tests for infections susceptible to transmission by blood. All serological results are recorded in databases in each blood center under the recommendation of the Ministry of Health, Brazil. Ongoing monitoring of infectious diseases markers in the blood donor population places blood banks in a privileged position to observe infection trends in the population , funded by the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH) . Since 2We present here the results of a 10-year collaborative study (2007–2016) of HTLV-1/2 infection screening in first-time donors, utilizing the REDS database covering four Brazilian blood centers that have been participating in the REDS project during this period.This study has an observational design; the database was set up by the REDS program in a study carried out by four large Brazilian blood centers located in state capitals Recife (Pernambuco State), Rio de Janeiro (Rio de Janeiro State), Belo Horizonte (Minas Gerais State), and São Paulo (São Paulo State). The database contains consolidated information recorded by computer systems in the participant blood banks, encompassing all steps of the donation process, from the donor registration at the time of donation to the release of the serological results.We studied all eligible, first-time blood donors from January 1st, 2007, to December 31, 2016 at the four participating Brazilian blood centers. To be eligible, the candidate had to be approved as a blood donor, according to the standard operating procedures of the blood centers, which comprise the clinical screening and blood collection. There were no other exclusion criteria. Donors were classified as community or replacement donors according to their declaration at registration as to whether they came to donate as a voluntary, community donor or in the name of a specific patient (to “replace” the blood he might need).The serum samples used in the present study were tested using the immunoenzymatic assay (EIA) for HTLV-1/2 detection .The reactive samples were sent to the central laboratory of this study, in São Paulo, for the second EIA using a kit that was different from the one used in the assay in the serological screening of the blood bank where the first sample came from . A positive result was defined as a serum testing reactive in two different EIA assays. The subset of reactive samples in the first EIA and non-reactive or borderline in the second assay, performed in São Paulo, were retested at the blood bank of origin using a Western blot (WB) MP Diagnostics HTLV Blot. Western blot results were considered positive when they showed reactivity to gag (p24 or p19) and env (GD21) proteins. Reactive samples for rgp46I and rgp46II were considered HTLV-1, and HTLV-2, respectively. The absence of bands characterized the WB as negative. The presence of isolated bands characterized indeterminate WB result. If the sample was positive in WB assay, the donor was considered reactive for HTLV-1/2.Statistical analysis was performed using the R statistics version 4.0.5 program . The characteristics of the HTLV-1/2-infected blood donor included year of donation, age, gender, education, self-reported skin color, type of donation. The analysis considered the comparison of these characteristics and of the prevalence among the four participant blood centers.The prevalence of HTLV-1/2 and 95% confidence intervals (CI) was calculated for first-time donors per year. For cases in which the result of the second test was classified as indeterminate WB, the probability of a positive result was applied to impute cases as either positive or negative. We define a positive ratio as the number of positive results in the first and second tests divided by the total number of units tested in the first test. We applied this positive ratio to define the positivity of the samples that were undefined by the WB. The prevalence was calculated as the number of positive HTLV1/2 result after the imputation divided by the number of donors, multiplied by 100,000. The comparison of prevalence between the years 2007 and 2016 was performed using the comparison of proportions test. To assess trends in the prevalence over the years, the univariate Poisson model was used. The frequency was considered to estimate the prevalence and, to analyze the birth cohort effect, the donors were stratified into five birth cohort groups: (1) born before 1980, (2) born between 1980 and 1984, (3) born between 1985 and 1989, (4) born between 1990 and 1994, and (5) born after 1995.The association between qualitative variates was evaluated using the Chi-Square test. The level of significance considered was 5%.The study protocols were approved by the National Research Ethics Commission of the Ministry of Health in Brazil (Number: 13236), as part of the International REDS Program, and by the local ethical committees in each blood center. In addition, it was also approved by the Institutional Review Board (IRB) of the University of California San Francisco, USA (Number: IRB# H5866-28606 and IRB# 11-05195).From 2007 to 2016, a total of 1,092,174 first-time blood donations were received in the four participating centers. São Paulo represents the largest and more populated Brazilian capital. Therefore, the majority of the donations came from São Paulo, representing 36.5% of analyzed donations. p < 0.001; In order to disclose variations and possible trends over the period regarding the HTLV-1/2 prevalence in relation to gender, age, schooling, skin color, and type of donation, we presented the 10-year data and compared the HTLV-1/2 prevalence of 2007 and 2016 regarding the covariates .The analysis by gender showed that HTLV-1/2 prevalence was higher among women , althougp = 0.014) and between 20 and 29 years of age  and for the highest level of education  compared to black skin color (p = 0.023) during the 10-year period (Regarding the self-declared skin color, black skin color predominated in the general analysis (r period .Regarding the type of donation, replacement donors had higher HTLV-1/2-seroprevalence when compared to community donors . Both tyIn The analysis of a 10-year HTLV-1/2 surveillance in four blood centers in Brazil representing the Northeastern and Southeastern regions, which are the most populous in the country, showed that overall HTLV-1/2 prevalence has been stable from 2007 to 2016. However, during the period, there was an increase in HTLV-1/2 seropositivity among male, white skin color, more educated people and younger aged. The same trend was detected in the four blood centers . This ocWhen we compared our results encompassing data from 2007 to 2016 with those published by REDS with data from 2007 to 2009 regarding the same blood banks and using the same diagnostic algorithm presented here, it is evident that the prevalence barely changed . Based oHTLV-1/2 prevalence has been falling in other HTLV-1/2-endemic countries. A study carried out from 2000 to 2006 with data from blood donors in Nagasaki, an endemic area for HTLV-1/2 in Japan, demonstrated a reduction in the prevalence of HTLV-1/2 among people born after 1987 and the persistence of high prevalence among those born before 1960. The reasons for this declining trend in the prevalence of HTLV-1/2 include social changes over the years, such as decreased duration of breastfeeding, increased frequency of artificial feeding, migrations and common use of condoms by younger people in endemic areas .p = 0.049; Regarding gender, the overall prevalence in females was higher, which is a feature of HTLV-1/2 infection already known in Brazil and worldwide , 11. TheThe distribution of donors by age group was similar in the four capitals and the HTLV-1/2 prevalence increased with age, which is another known epidemiological feature of HTLV-1/2 infection and previously attributed to a birth cohort effect , 14. We The age group analysis during the 10-years period showed a trend toward an increase in prevalence of people under the age of 29, which suggests recent transmission by sex . InjectiEducational status varied among the centers, with São Paulo and Rio de Janeiro presenting a higher prevalence of HTLV-1/2-positive donors with more years of schooling . This reSocio-economical vulnerability has largely been correlated with HTLV-1/2 infection –24, someRegarding the type of donor, we found a higher prevalence of replacement donors when compared to community donors among the HTLV-1/2-positive individuals. Donors of replacement have been reported to have a higher prevalence of several viral markers, including HTLV-1/2 infection .Our study has limitations. We confirm the higher prevalence of HTLV-1/2 in Recife and Rio de Janeiro compared to São Paulo and Belo Horizonte , which cIn conclusion, STIs have been considered a serious public health problem in Brazil due to their widespread occurrence, lack of symptoms and concurrent transmission of HIV. We believe that HTLV-1/2 infection deserves more attention within STIs prevention efforts since the current profile of the infected population includes younger people, indicating an increase in recent infection.dropbox.com [https://www.dropbox.com/sh/1dg5gx8s3snrb2d/AAB-c457wuFs6s6tg5igV-tRa?dl=0].The datasets for this study can be found in The studies involving human participants were reviewed and approved by National Research Ethics Commission of the Ministry of Health in Brazil number: 13236. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.ES and BC conceived the study. ES, BC, PL, AC-P, CA-N, LA, and MR contributed to the design and implementation of the research. CM and IG processed the data and performed the analysis. IG designed the figures. DG, AC-P, EM, CM, IG, and FP contributed to the analysis of the results and to the writing of the manuscript. DG, AC-P, EM, and CM drafted the manuscript. ES, BC, PL, AC-P, CA-N, LA, MR, PB, and SG supervised the project and the data collection. All authors contributed to and approved the manuscript.This study was supported by the National Institutes of Health, National Heart, Lung, and Blood Institute by Grant No. HHSN268201100007I.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Many putative uremic toxins—like indoxyl sulfate, p-cresol sulfate, kynurenic acid, uric acid, and CMPF—are organic anions. Both inter-organ and inter-organismal communication are involved. For example, the gut microbiome is the main source of indole, which, after modification by liver drug metabolizing enzymes (DMEs), becomes indoxyl sulfate. Various organic anion transporters —often termed “drug transporters”—mediate movement of uremic toxins through cells and organs. In the kidney proximal tubule, critical roles for OAT1 and OAT3 in regulating levels of protein-bound uremic toxins have been established using knock-out mice. OATs are important in maintaining residual tubular function in chronic kidney disease (CKD); as CKD progresses, intestinal transporters like ABCG2, which extrude urate and other organic anions into the gut lumen, seem to help restore homeostasis. Uremic toxins like indoxyl sulfate also regulate signaling and metabolism, potentially affecting gene expression in extra-renal tissues as well as the kidney. Focusing on the history and evolving story of indoxyl sulfate, we discuss how uremic toxins appear to be part of an extensive “remote sensing and signaling” network—involving so-called drug transporters and drug metabolizing enzymes which modulate metabolism and signaling. This systems biology view of uremic toxins is leading to a new appreciation of uremia as partly due to disordered remote sensing and signaling mechanisms–resulting from, and causing, aberrant inter-organ  and inter-organismal  communication. There is a new appreciation of the role of the kidney in organ cross-talk and inter- organismal communication  mediated by small organic molecules. Many of these molecules are organic anions transported by renal e.g., OAT1) and non-renal transporters (OATP1B1) and altered by Phase 1 and Phase 2 drug metabolizing enzymes (DMEs); many of these molecules also have well-described roles in metabolism, signaling, and modulation of redox state  relevant to CKD. The RSST is a general systems biology theory of how transporters and enzymes help optimize remote communication by small organic molecules between organs  and organisms .At least 500 transporters, enzymes, and regulatory proteins  participate in this remote sensing and signaling (RSS) system. Many of the proteins in the RSS system have a broad substrate specificity and are well-known in the pharmaceutical field for their involvement in the absorption, distribution, metabolism and excretion (ADME) of drugs such as NSAIDs, statins, antibiotics and antivirals. Thus, certain SLC and ABC transporters and enzymes in the RSS system are often referred to as drug transporters and drug metabolizing enzymes (DMEs).in vitro and in vivo studies in model organisms as well as human genome wide association studies (GWAS) that the proteins of the RSS system interact with a wide range of metabolites, signaling molecules, antioxidants, nutrients, and gut microbe products. These small molecules participate in key physiological and biochemical pathways, including those involving bile acids, the citric acid cycle, fatty acid oxidation, cellular redox state, and a wide variety of signaling events.However, while well-known for their pharmaceutical roles, homologs of many of the genes encoding these proteins are found in mice, fish, flies, and other organisms. This strongly suggests an important role for these proteins in the physiology of very diverse organisms. Furthermore, it is now clear from It is through the movement, modification, and action of these small organic molecules between organs and body fluids—as well as between organisms—that remote sensing and signaling is believed to occur. Thus, the transporters, enzymes, and regulatory proteins of the RSS system—a network of over 500 proteins differentially expressed across tissue and cell type—help maintain homeostasis and also help restore homeostasis in the context of injury to the kidney and other organs. Among the small molecules regulated by the RSS system are many protein-bound uremic solutes and uremic toxins accumulating in CKD that are taken up by the proximal tubule. One of the best-studied of these uremic toxins is indoxyl sulfate.indigo and indican—have a long and rich history. Indoxyl sulfate is a small organic molecule—an organic anion—that is considered to be one of the most important protein-bound uremic toxins originating in the gut. Early in the twentieth century the indigo-related molecule, indoxyl sulfate, was recognized as a solute capable of binding to plasma albumin  on the basolateral surface of proximal renal tubular cells . In progInterest in the importance of “protein-bound uremic solutes” was stimulated when it became clear that patients with end-stage renal disease (ESRD) exhibited a high propensity to cardiac death during the first 3 years of hemodialysis . Most ofuremic toxins or uremic retention solutes.During the period dating from 1960 through the early 2000's when chronic renal failure was treated by thrice-weekly hemodialysis, only modest attention was given to the nature of the toxins removed by hemodialysis . In the signaling molecules as well as potential toxins. An effect of uremic plasma on gene expression was demonstrated in normal human renal tubular cells , polyamines, and CMPF (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid). Although some are organic cations , a large number are, like indoxyl sulfate, small organic anions. In recent years, several lines of evidence have led to the view that indoxyl sulfate and some other uremic solutes function as ar cells . In humaar cells  consisteHowever, more than 1,500 genes remained dysregulated when cells were incubated with post-dialysis plasma , consistsecrete fluid. It was argued that secretion of fluid must have been preceded by the transport of some solute into the tubular lumen that created an osmotic driving force. Grantham observed that addition of p-aminohippurate, which is now known to be taken up by OATs in the proximal tubule, to the bathing medium stimulated fluid transport. Further, it was observed that plasma from ESRD patients, added to the bathing medium, caused fluid secretion in perfused rabbit renal tubules, which in retrospect, suggests that some molecule(s) in uremic serum acted as a “signal” to induce tubular secretion of fluid. Much of the “signal” was most likely the osmotic effect of solute transported into the tubular lumen. But with a growing appreciation of the importance of these small organic molecules in signaling, it is possible that specific signaling mechanisms within the tubular cells also help explain the phenomenon. Nevertheless, these older studies suggested that, despite poor glomerular filtration, or possibly because of it, the OATs in the proximal tubule contribute to the elimination of indoxyl sulfate and other uremic solutes. As Grantham wrote, “under conditions of markedly reduced or complete cessation of glomerular filtration, mammalian proximal tubules could secrete fluid and hippurate and similar substances… elimination of some of the potentially toxic products normally excreted by the kidneys… would serve a useful survival function”  and other transporters in the proximal tubule in chronic kidney disease (CKD). Renal transport not only regulates the elimination of uremic toxins but also plays an important role in setting the systemic and proximal tubule cell levels of these small organic molecules. In a sense, this renewed attention on proximal tubule handling of solutes has led to the “rediscovery” of renal tubule microperfusion studies performed in the 1970's , 13. Graunction” . This miunction” , 16. Impunction” .in vivo transporters of indoxyl sulfate and other protein-bound uremic toxins  in plasma. Renal excretion is facilitated by OATs on the basolateral (blood side) of proximal tubular cells that shift the equilibrium toward free solute that is readily transported into the tubular lumen. In this way, renal tubular secretion serves to remove suspect uremic toxins. A recent study  demonstrxenobiotic response element (XRE)”—implying that it functions to inactivate foreign molecules–it has become increasingly apparent that binding of the AHR-ARNT to the “xenobiotic response element” also mediates responses to endogenous stimuli presented through binding to the aryl hydrocarbon receptor. Indeed, it seems likely that the XRE, which was originally perhaps best known for mediating the response to exogenous 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, has responded to small endogenous solutes long before “modern society” produced TCDD as a side product in organic synthesis and burning of organic materials. The ARNT complex leads to the expression of “drug” metabolizing enzymes (DMEs), SLC transporters, and ABC transporters. Other nuclear receptors, such as PXR (pregnane X receptor) and HNF4a   in the cytoplasm . The com4 alpha)  also regA number of studies have now identified AHR as an important target of indoxyl sulfate. Indoxyl sulfate, bound to AHR, can activate a great number of genes, including cytochrome P450 enzymes, as well as genes involved macrophage-dependent inflammation . Today, Recent data indicates that free (unbound) indoxyl sulfate (and other aryl hydrocarbons) can bind to the EGF receptor of cells lacking OATs and induce a signaling cascade that results in ARNT translocation and initiation of gene transcription . This prSo, the answer to the hypothetical question Homer Smith might have posed is that, while the renal clearance by OATs and other transporters serves to prevent excessive buildup of uremic toxins, unbound uremic solutes may be important signaling molecules, metabolites, and antioxidants acting upon remote organs  or remote organisms  and uremia are complex syndromes involving altered inter-organ and inter-organismal remote communication affecting metabolism, signaling, and redox potential—among many other biochemical and cellular processes. In the past decade, indoxyl sulfate has been the uremic toxin at or near the center of research in this field, and the mechanism of its action as a signaling molecule in the proximal tubule is even more complex than we have described— involving not only OAT1 and AHR, but also EGF receptor, oxidative state, and a microRNA . Furtherin vivo it follows the gut microbe-gut-liver-kidney axis and requires the participation of “hepatic drug metabolizing enzymes” (Phase 1 for hydroxylation and Phase 2 for sulfation) and drug “transporters” like the renal OATs  communication in the body in that, as described above, nal OATs . In othenal OATs . Many ofAlthough the Remote Sensing and Signaling Theory was originally formulated over a decade ago to synthesize a growing amount of data related to the biological role of “drug” transporters that did not conform to the view that their primary role was in drug absorption, distribution, metabolism, and excretion (ADME), the applicability of the RSST to CKD and uremia has become increasingly apparent –36. A smWith the intense recent attention given to the mechanisms by which uremic toxins and solutes affect cell and organ function, there is a rapidly growing appreciation of CKD and uremia as a multi-organ disorder involving altered metabolism and signaling mediated by small organic molecules—implying the involvement of specific metabolic and signaling pathways definable at the cellular and molecular levels–as well as a renewed appreciation of the importance of residual renal function which in large part represents the activity of proximal tubule transporters of protein-bound molecules .Given the emphasis in the Remote Sensing and Signaling Theory on the role of multi-specific transporters and enzymes, as well as their regulators  in remote inter-organ and inter-organismal small molecule communication (including gut microbe-host), the theory serves as a basis for understanding disordered small molecule communication in disease settings like CKD and uremia . In thisThe growing understanding of the mechanistic basis of indoxyl sulfate action serves as a good example of the need to think beyond toxins and examine effects upon specific signaling and metabolic pathways. While nephrologists have been conditioned to think in terms of uremic “toxins,” this traditional view is challenged by a number of observations. First, most uremic toxins and uremic solutes are present in the body in the absence of kidney dysfunction. In addition to the OATs, there are transporters of these small molecules in many non-renal tissues. Crucially, some of these solutes, such as kynurenine and polyamines, are known to participate in major biological pathways independent of kidney disease. One possibility, consistent with a growing amount of biochemical and molecular data, is that so-called uremic toxins, while harmful when in excess in the setting of kidney failure, might have other important “non-toxic” roles in normal biology, including metabolism, signaling, regulating redox state, and gut microbiome population dynamics. For example, there is evidence that indoxyl sulfate, at physiologic plasma concentrations, acts as a superoxide radical scavenger . IndoxylIf we consider uremia as a disturbance of remote sensing and signaling, we might say that CKD alters the normal functioning of a highly-regulated transporter and “drug” metabolizing enzyme network involved in sensing and signaling to maintain homeostasis of small organic molecules that have “high informational content” with respect to remote inter-organ and inter-organismal communication. The body's attempt to restore homeostasis in the face of the perturbations caused by uremic syndrome and ongoing progression of kidney disease then depends upon the homeostasis-promoting properties of several interacting systems: the remote sensing and signaling system, the neuroendocrine system, the growth factor-cytokine system, and the autonomic nervous systems.If indoxyl sulfate is a key small molecule in a larger “remote sensing and signaling system” involved in inter-organ crosstalk within the body and inter-organismal communication mediated by transporters and “drug” metabolizing enzymes, it seems tempting to ask, what is the central conductor? For uremic toxins and uremic solutes derived from gut microbes, a plausible candidate for this role might be the gut microbiome–with its vast number of enzymes and signaling pathways. The small molecules produced by gut microbes, while perhaps of greatest current interest from the viewpoint of inter-organismal communication and kidney disease, do not, however, include the multitudes of endogenous metabolites and signaling molecules generated largely independent of the gut microbes and handled by “drug” transporters, “drug” metabolizing enzymes, and related proteins .The Remote Sensing and Signaling Theory is a general theory that attempts to explain how multi-specific, oligo-specific, and monospecific transporters and enzymes work together, along with regulatory proteins  to achieve optimal levels of hundreds if not thousands of small molecules in different body tissues  and body fluids . In While the systems pathophysiology of CKD is a new field, we conclude by again reminding the reader that the history of molecules related to indoxyl sulfate is very old. In the thirteenth century, Marco Polo described many caravans traveling from the Middle East and India that contained cloths dyed with the precious indigo. An important feature of indigo or indican was tight binding of the dye to cloth, and even in recent times, indigo serves as the dye that gives jeans their distinctive color. Once more we note that many such organic dyes played an important role in furthering our early understanding of pathology and physiology, including that involving protein-bound uremic solutes.Here we have provided a glimpse of the physiological and pathophysiological history of a close relative of a highly valued dye employed to dye fabrics 6,000 years ago—from a solute found to accumulate in the serum of patients with renal disease, to a protein-bound uremic toxin, to a ligand of the renal “drug” transporters OAT1 and OAT3, to one of many signaling molecules participating in a “remote sensing and signaling system” that can now be conceptualized as a network of hundreds of proteins.Viewing indoxyl sulfate and other uremic toxins through the multiscale systems biology perspective of the Remote Sensing and Signaling Theory suggests, as discussed elsewhere , a wide The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.JL and SN contributed equally in the authorship of this submission. Both authors contributed to the article and approved the submitted version.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.


Plant cell signaling is an intensive research topic in which reductionist can be achieved when we investigate the systems of model plants . To contArabidopsis thaliana is the most utilized system for revealing the in-depth mechanisms of cell signaling in this Special Issue. Krogman et al. investigated the concentration of cytosolic calcium ion ([Ca2+]cyt) in different root cells of A. thaliana that express GCaMP3 for producing a modified version of a genetically encoded calcium indicator [2+]cyt that are elevated by extracellular ATP (eATP) and ADP (eADP) in the root of A. thaliana [2+]cyt in root cells in response to both eATP and eADP, which might be a reactive oxygen species (ROS)-activated Ca2+ channel in the plasma membrane. The mechanism that regulates the size of Arabidopsis root meristem was investigated by Hashem et al. [Arabidopsis mTERF27 when faced with salt stress [A. thaliana. They have been found to participate in the development, defense, and stress response in plants. Jarratt-Barnham et al. indicated that the heterotetrameric complexes of CNGC subunits act differently from the homotetramers and that cyclic nucleotide-gated channel-like proteins ma bey involved in the regulation of the complex formation [Among the model plants, m ion (Ca+cyt) in m et al.  with an m et al. , it was t stress . It was CitGF14s, that codes for phosphorylated proteins in Citrus sinensis in response to biotic and abiotic stresses [C. sinensis. A receptor for brassinosteroid signaling, SlBRI1, was modified in a phosphorylation site of serine 1040 and then tested for its functions in tomatoes [In plant research, the exploration of cell signaling pathways is crucial for breeding stress-tolerant crops. Min et al. studied the role of clade A Type 2C protein phosphatases in rice OsPP2C09) with an emphasis on the relationship with abscisic acid (ABA) signaling under osmotic stress . The aut with an stresses . The auttomatoes . Wang ettomatoes . The CDKA. thaliana. Shibuya et al. identified an FKF1 homolog SlFKF1 in tomatoes and confirmed that the expression of SlFKF1 at a low level was associated with late flowering, increased leaflets, and low concentrations of lycopene [A. thaliana to play a role in defense responses. Additionally, a MAPK in Lotus japonicus, LjMPK6, was known to be involved in regulating symbiosis between legume and rhizobia. Yan et al. identified a type 2C protein phosphatase, LjPP2C, and confirmed that it is required for dephosphorylating LjMPK6 to regulate the nodule development in L. japonicas [In cell signaling, the knowledge obtained from model plants could be applied in crops to accelerate the progress of research and breeding programs. FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) has been recognized as a blue-light receptor that plays a role in the promotion of flowering in lycopene . A mitogaponicas . Chaulagaponicas . The sig2+) are the second most abundant cation in living cells and they serve as a signal of the adenylate status. Kleczkowski and Igamberdiev refined the signaling pathway of Mg2+, particularly the adenylate kinase (AK) equilibrium and its involvement in the adenylate status [2+ signaling and is an allosteric effector in cellular metabolism for energy homeostasis in cells.Magnesium ions (Mge status . It was ROS are a universal regulatory element in plant cells and participate in an array of signaling pathways. Breygina and Klimenko reviewed the role of ROS and the underlying mechanism involving ion transport systems during plant sexual reproduction . They foOrobanche and Striga focusing on the activities of proteins with nucleotide-binding and leucine-rich repeat domains, NLR proteins, that encoded by resistance genes of the host [Parasitic plants absorb water and nutrients from hosts and could cause substantial losses of yield. Hu et al. summarized the virulence mechanisms in obligate root parasites of the genera the host . This prThe communication between the nucleus, organelles, and cellular compartments is a critical system for plant cells to face changing environments, including stresses. Mielecki et al. contributed a review for a better understanding of retrograde signaling in stress responses, such as its involvement in the induction of cell death and in the biogenesis of organelles . The autTheoretically, cell signaling affects virtually every aspect of plant cell structure and function, and thus to scientists, unveiling the global network and the underlying machinery is always a great challenge. This Special Issue only presents a snapshot of this intensive field of plant biology and we believe that with the rapid development of advanced technology such as integrative multi-omics and CRISPR genome editing, more and more critical networks of plant cell signaling will be unraveled in the near future.


Background: High hydrostatic pressure (HHP) processing is a non-thermal method proposed as an alternative to Holder pasteurization (HoP) for the sterilization of human breast milk (BM). HHP preserves numerous milk bioactive factors that are degraded by HoP, but no data are available for milk apelin and glucagon-like peptide 1 (GLP-1), two hormones implicated in the control of glucose metabolism directly and via the gut–brain axis. This study aims to determine the effects of HoP and HHP processing on apelin and GLP-1 concentrations in BM and to test the effect of oral treatments with HoP- and HHP-BM on intestinal contractions and glucose metabolism in adult mice. Methods: Mice were treated by daily oral gavages with HoP- or HHP-BM during one week before intestinal contractions, and glucose tolerance was assessed. mRNA expression of enteric neuronal enzymes known to control intestinal contraction was measured. Results: HoP-BM displayed a reduced concentration of apelin and GLP-1, whereas HHP processing preserved these hormones close to their initial levels in raw milk. Chronic HHP-BM administration to mice increased ileal mRNA nNos expression level leading to a decrease in gut contraction associated with improved glucose tolerance. Conclusion: In comparison to HoP, HPP processing of BM preserves both apelin and GLP-1 and improves glucose tolerance by acting on gut contractions. This study reinforces previous findings demonstrating that HHP processing provides BM with a higher biological value than BM treated by HoP. Human breast milk (BM) donated to human milk banks (HMBs) is the sole diet for preterm infants when maternal BM is not available. Pasteurization of donated BM is an essential step to inactivate pathogens to ensure microbial safety for preterm babies as, due to immature gut, these infants are at high risk of developing diseases such as necrotizing enterocolitis and sepsis . The mosMore than twenty peptide hormones have been characterized in BM ,5. It waHere, we investigate the effect of HoP- and HHP-treatments of BM on the concentration of apelin and GLP-1 and test in vivo the effect of chronic (7 days) oral administration of HoP- and HHP-BM on intestinal contractions and glucose metabolism in adult mice.−1, number of cycles = 4 cycles, duration of each cycle = 5 min and a latency time with normal pressure between each cycle of 5 min. Samples were stored at −80 °C until analysis.Frozen BM samples from 11 donors were provided by the regional HMB . Donors provided written, informed consent for the use of their milk for this research purpose. After thawing of milk samples, 8 different batches of BM were created under sterile conditions by mixing various volumes (from 10 to 30 mL) of all BM samples, primarily in order to homogenize BM composition between batches. Three aliquots of BM were prepared for each batch: one fraction was stored at −80 °C without any other treatment (raw milk sample (RM)); one fraction was subjected to HoP according to the standard pasteurization protocol (62.5 °C for 30 min) in our regional HMB; the last fraction was subjected to HHP processing as previously described . BrieflyApelin and GLP-1 were quantified using commercial ELISA kits  in raw milk (RM), HoP- and HHP-BM samples using whole milk.ad libitum. Oral gavage of HoP- or HHP-BM (100 μL) was performed daily during 7 days before experiments.Nine-week-old male C57BL/6J mice  were housed in controlled environment . Food and water were proposed After a 10 h of fasting, an OGTT (3 g of glucose/kg of body weight) was performed in mice at day 7. Glycaemia was recorded from −30 to 120 min, and insulin and glucagon levels were measured and analyzed at −30, and +15 min as previously described .Mice were euthanized under fed conditions. Duodenum, jejunum, ileum and colon, were washed and incubated with an oxygenated Krebs-Ringer solution, pH 7.4, for 20 min at 37 °C. Intestinal fragments were attached to an isotonic transducer . The lever is adjusted to have a load of 1 g (10 mN). Each intestinal fragment was immersed in an organ bath containing an oxygenated Krebs-Ringer solution maintained at 37 °C. After a recovery period of 15 min, 100 μL of Krebs-Ringer (vehicle) solution or BM (HoP- or HHP-BM) was put directly in the organ bath and data were collected for 15 min. After attaching the intestinal segments, basal contractions were recorded for 15 min. For acute treatments, 100 μL of Krebs-Ringer (vehicle) solution or BM (HoP- or HHP-BM) were added to the medium, and contractions were recorded for 15 min. Contraction amplitudes and frequencies are presented as percentages relative to the basal response (before the injection of vehicle or milk) . For chrHomogenization of tissues, total RNA extraction, reverse transcription and real-time PCR were performed as previously described in detail . The seqt-test, one-way ANOVA, or two-way ANOVA test according to sample normality assessment results. Variations were considered as significant when p value was <0.05.Results are presented as mean ± SEM. GraphPad Prism 7.0. software  was used to analyse the data. Outliers were detected via a Grubb’s test. A D’Agostino-Pearson test was used to evaluate the normality of variables. Statistical differences were then tested by paired Compared to raw milk (RM) and HHP-BM, HoP treatment reduced apelin  oral gavage of mice with HHP- or HoP-BM on intestinal contractions, no significant variation was observed concerning the contraction of the duodenum A, the jeThe impact of BM treatments on glucose tolerance and gut motility was not associated with variations of body weight in the different experimental groups .Milk hormones are non-nutritive bioactive compounds that exert beneficial short- and long-term health effects as well as possible long-term metabolic programming in newborns, as suggested by animal studies . Here, wIn line with this hypothesis, the second objective of our study was to test in vivo the effect of a chronic gavage with HoP- and HHP-BM on intestinal contractions and glucose metabolism in adult mice. First, in a preliminary ex vivo study, we discovered that the addition of HHP-BM in the incubation medium of mouse duodenum significantly reduced the amplitude of contractions. This first rapid effect probably implies the action of milk apelin, which is now considered an enterosyne . Indeed,nNos mRNA level in the ileum? GLP-1 is known to increase the activity of nNOS in myenteric neurons leading to NO release that inhibits the mechanical activity of the intestine [Several questions remain to be resolved with future experiments. First, could the effects of HHP-BM observed in our study be due to apelin alone or GLP-1 alone or in combination with other enterosynes? This point could be raised by assessing the levels of other molecules  present in HHP-BM that could act on enteric neurons or other cellular targets in the digestive tract or in other organs after intestinal absorption. Second, which factors are implicated in the increase in ntestine . Whetherntestine . FinallyTo conclude, we have discovered that HHP protocol preserves both apelin and GLP-1 levels in human milk, demonstrating the potential clinical importance of this original sterilization method. Based on our observations, we postulate that HHP-treatment of BM could improve glucose homeostasis and the gut–brain axis activity in preterm newborns.


Thus, calcium–cAMP signaling must be tightly regulated, e.g., by agents that catabolize cAMP or inhibit its production , and by proteins that bind calcium in the cytosol . Many genetic or inflammatory insults early in life weaken the regulation of calcium–cAMP signaling and are associated with increased risk of schizophrenia . Age-related loss of regulatory proteins which result in elevated calcium–cAMP signaling over a long lifespan can additionally drive tau phosphorylation, amyloid pathology, and neurodegeneration, especially when protective calcium binding proteins are lost from the cytosol. Thus, the “genie” we need for our remarkable cognitive abilities may make us vulnerable to cognitive disorders when we lose essential regulation.Neurons in the association cortices are particularly vulnerable in cognitive disorders such as schizophrenia and Alzheimer’s disease, while those in primary visual cortex remain relatively resilient. This review proposes that the special molecular mechanisms needed for higher cognitive operations confer vulnerability to dysfunction, atrophy, and neurodegeneration when regulation is lost due to genetic and/or environmental insults. Accumulating data suggest that higher cortical circuits rely on magnified levels of calcium  near the postsynaptic density to promote the persistent firing needed to maintain, manipulate, and store information without “bottom-up” sensory stimulation. For example, dendritic spines in the primate dorsolateral prefrontal cortex (dlPFC) express the molecular machinery for feedforward, cAMP–PKA–calcium signaling. PKA can drive internal calcium release and promote calcium flow through NMDAR and calcium channels, while in turn, calcium activates adenylyl cyclases to produce more cAMP–PKA signaling. Excessive levels of cAMP–calcium signaling can have a number of detrimental effects: for example, opening nearby K The primate cortex performs an extraordinary number and range of operations decoding sensory events, integrating sensory inputs with previous experience, and representing information in higher networks independent of sensory stimulation to create our “mental sketch pad” and generate goals for action. We are learning that the cortical networks that subserve these varied functions also have differing circuit architectures and unique molecular needs. For example, the newly evolved dorsolateral prefrontal cortex (dlPFC) contains microcircuits in deep layer III with extensive recurrent excitation that generate the persistent neuronal firing needed to maintain mental representations in working memory. Emerging data suggest that the molecular mechanisms needed to sustain persistent firing in dlPFC are very different from those required to accurately decode a sensory stimulus in primary visual cortex (V1). Most importantly, the molecular signaling events needed to sustain mental representations also appear to render these circuits especially vulnerable to dysfunction when errors emerge from genetic and/or environmental insults. In particular, we propose that the magnification of calcium signaling needed for the persistent firing of dlPFC microcircuits confers vulnerability to atrophy and degeneration when regulation is lost, e.g., by inflammation. The current review describes the differences in neurotransmission and neuromodulation between newly evolved vs. primary sensory cortices, and then speculates on how these disparities may underlie the susceptibility of dlPFC circuits in mental disorders.The hierarchical organization of the primate cortex has been appreciated for decades by neuroanatomists –3. More A hierarchy in cortical processing is particularly relevant to our understanding of mental disorders, as there is a general correlation between circuits with longer timescales, and vulnerability in cognitive disorders. For example, there is a near perfect correspondence between the cortical hierarchy and the pattern and sequence of tau pathology in Alzheimer’s disease (AD), something noted by primate neuroanatomists and neuropathologists for decades –15. SimiThere are important differences in neuronal morphology, circuit architecture, and physiological properties across the cortical hierarchy , 21. TheA comparison of the glutamate receptors mediating neurotransmission in primate V1 vs. dlPFC shows striking differences , where VNeurons in monkey area V1 that respond to visual stimuli have a great reliance on AMPAR transmission, where even low doses of AMPAR blockers such as CNQX markedly reduce stimulus-related firing  Fig. 2a. In contNeurons in the dlPFC have a very different function than those in V1: they must generate and sustain mental representations without sensory stimulation. Neurons with this capability to maintain persistent firing across the delay period in a working memory task are termed “delay cells.” Persistent firing of delay cells is thought to arise from extensive recurrent excitatory circuits mediated by NMDAR  Fig. 2c, a findi2+) block within the NMDAR pore, permitting NMDAR actions  channels to reduce firing, the genie in the bottle [There are also gradients in neuromodulators and signaling mechanisms across the cortical hierarchy. There are gradients in modulatory receptors , catechoe bottle . These p+ channels and spur cAMP–PKA signaling [The traditional neuromodulatory role of cAMP signaling is to strengthen synaptic connectivity, through both presynaptic and postsynaptic actions. For example, in rodent hippocampus, elevated calcium–cAMP–PKA signaling in spines can ultimately lead to phosphorylation of CREB, and transcriptomic events that strengthen existing synapses or even make new ones , 56. As ignaling , and eleignaling . LTP in ignaling –62 that cAMP–PKA signaling can also produce transient increases in synaptic strength by increasing transmitter release from the presynaptic terminal, e.g., in chromaffin cells, where PKA phosphorylation primes vesicles for release  Fig. 3a3a. ImmunIn contrast to V1 where phosphodiesterase 4 (PDE4A) is focused in presynaptic terminals, cAMP-related proteins are concentrated in spines in primate layer III dlPFC, where they are positioned to magnify internal calcium release from the SER Fig. . The SEROne can see the molecular machinery for cAMP–PKA drive on calcium release from the spine apparatus in layer III dlPFC spines. For example, the anchoring protein, AKAP6, anchors PKA to the SER, and can be seen on the spine apparatus in dlPFC Fig. . The phov3.1 (CACNA1G), the ATPase plasma membrane calcium transporter (ATP2B4), and the “master stress” signaling peptide, PACAP (ADCYAP1), which drives cAMP and calcium signaling through both Gs and Gq-coupled receptors, . While the location and physiological contributions of these proteins, as well as other calcium channels, are still unknown, the data are consistent with an enrichment of calcium–cAMP-related signaling in dlPFC. Interestingly, a duplication in VPAC2 is associated with schizophrenia [Transcriptomic analyses of layer III pyramidal cells in rhesus monkey also show an enrichment of genes encoding for calcium channels/transporters and cAMP-activating proteins in dlPFC compared to LIP . dlPFC pophrenia , as disc+ channels provides negative feedback to prevent seizures, as well as a mechanism to dynamically alter the strength of synaptic inputs onto dlPFC pyramidal cells, which can serve to gate network inputs, and alter dlPFC function based on arousal state [+ channels to weaken connectivity and reduce firing, thus producing a narrow inverted-U dose–response.The dendritic spines of layer III dlPFC primate neurons express a large number of receptors that increase  or decrease  feedforward calcium–cAMP signaling, often in close proximity to the spine apparatus , 70–74. al state . We hypo+ channels [+ signaling leads to removal of spines and dendrites [Both NE α1-AR and DA D1R have an inverted-U dose/response on dlPFC persistent firing and working memory function through activation of calcium–cAMP signaling in spines Fig. : moderatchannels , 74, 75.channels , 77. Howchannels , 78, 79 channels , switchichannels . With chendrites –85, a fiendrites . Thus, t+ mechanisms are tightly regulated, as they weaken connectivity, but also because their feedforward nature can rapidly generate high levels of cytosolic calcium and cAMP, which can have multiple toxic actions (see below). Regulatory proteins include those that directly bind calcium , as well as those that regulate cAMP signaling. There are high levels of calbindin in layer III pyramidal cells in young monkey [It is essential that these powerful calcium–cAMP–PKA–Kg monkey  and humag monkey  dlPFC, cg monkey , 89, e.gg monkey , consisti/o signaling: both α2A-AR [+ channel signaling. α2A-AR are of interest as they protect PFC spines and working memory from chronic stress [There are also receptors that inhibit cAMP production via Gh α2A-AR  . NMDAR  subtype  Rare de  in DLG2 , a PSD p in DLG2 .v1.2 encoded by CACNA1C is a risk factor for multiple mental disorders, and the risk variant of this gene increases channel expression in human brain [CACNB2, and the t-type subunit CACNA1I. The locations and physiological actions of these channels are not yet known in nonhuman primate dlPFC, but the risk allele in CACNA1C is associated with inefficient dlPFC activation in humans [de novo mutations in the internal calcium channel, RyR types 3 or 2, are associated with increased risk of adult-onset [CACNA1C), or those that alter internal calcium release , confer risk for mental illness.GWAS studies have also found links between risk of schizophrenia and alterations in genes encoding for calcium channels , 110. Than brain , and incan brain . There an humans . It is alt-onset  or childlt-onset  schizoph2 increases cAMP signaling and increases risk of schizophrenia [2 stimulation influences dlPFC physiology, and whether excessive VPAC2 stimulation reduces dlPFC neuronal firing by opening K+ channels in spines.Genetic mutations that increase cAMP signaling are also associated with schizophrenia. A rare copy number variant that duplicates the gene encoding for VPACophrenia , 114. ThGRM3) and DISC1, which normally serves to anchor PDE4s to the spine apparatus.There are also important genetic links with schizophrenia risk and proteins already known to inhibit cAMP signaling in dlPFC spines, including mGluR3  GCPII [GRM3, and (2) kynurenic acid (KYNA) [While calcium dysregulation drives inflammatory cascades, the converse is also true, where inflammation can dysregulate cAMP–calcium signaling, thus creating a vicious cycle that can propel atrophy. Inflammatory cascades can induce a number of changes that can weaken dlPFC function and mimic the effects of genetic insults. For example, MK2 inflammatory signaling inactivates and unanchors PDE4, so that it may no longer be correctly positioned to inhibit cAMP drive on calcium release , 137, po1) GCPII –140  , which bd (KYNA) , 143 andd (KYNA) , and mayd (KYNA) , supportin utero may sensitize inflammatory signaling and increase risk of later mental illness, e.g., by exacerbating the pruning of dendritic spines later in adolescence [An extensive literature indicates that environmental insults lescence . Animal lescence  and complescence  in the blescence . Thus, glescence , or envilescence , may lealescence , suggestWhile critical genetic and perinatal insults often express as disease relatively early in life, more subtle genetic changes and/or environmental insults in adulthood may only manifest as their consequences build over a long lifetime. Activation of inflammatory and stress signaling pathways play a major role in age-related cognitive disorders, e.g., where psychological stress  and infl+ channels [+ channels and reduces neuronal firing [Age-related deficits in dlPFC and medial temporal cortical cognitive functions can be seen in rodents, monkeys and humans (reviewed in ), with dchannels  , then expanding more generally in pyramidal cells of the association cortices, with primary sensory cortices only afflicted at end-stage disease , 15, 176+ channels that reduce neuronal firing, mitochondrial changes that drive inflammation and loss of synapses, and activation of kinases that induce tau pathology and degeneration. Therapeutic strategies that help to restore calcium regulation may protect higher cortical circuits and reduce the risk of cognitive disorders.In summary, evidence from multiple sources indicates that elevated calcium signaling occurs in higher cortical circuits where it may be needed to maintain depolarization of the PSD to support the persistent firing that underlies much of higher cognitive functioning, and to dynamically gate network connections. In particular, the magnification of internal calcium release by cAMP signaling requires tight regulation, given its feedforward signaling. Thus, the “genie” that sparks our remarkable cognitive abilities also renders us vulnerable to cognitive disorders when regulatory proteins are unanchored or lost, and the “genie” can no longer be put back in the “bottle.” Dysregulated calcium–cAMP signaling can lead to a number of detrimental actions, including excessive opening of K


Human T cell Leukemia virus type 1 (HTLV-I) is etiologically linked to adult T cell leukemia/lymphoma (ATL) and an inflammatory neurodegenerative disease called HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The exact genetic or epigenetic events and/or environmental factors that influence the development of ATL, or HAM/TSP diseases are largely unknown. The tumor suppressor gene, Fragile Histidine Triad Diadenosine Triphosphatase (FHIT), is frequently lost in cancer through epigenetic modifications and/or deletion. FHIT is a tumor suppressor acting as genome caretaker by regulating cellular DNA repair. Indeed, FHIT loss leads to replicative stress and accumulation of double DNA strand breaks. Therefore, loss of FHIT expression plays a key role in cellular transformation.Here, we studied over 400 samples from HTLV-I-infected individuals with ATL, TSP/HAM, or asymptomatic carriers (AC) for FHIT loss and expression. We examined the epigenetic status of FHIT through methylation specific PCR and bisulfite sequencing; and correlated these results to FHIT expression in patient samples.We found that epigenetic alteration of FHIT is specifically found in chronic and acute ATL but is absent in asymptomatic HTLV-I carriers and TSP/HAM patients’ samples. Furthermore, the extent of FHIT methylation in ATL patients was quantitatively comparable in virus-infected and virus non-infected cells. We also found that longitudinal HTLV-I carriers that progressed to smoldering ATL and descendants of ATL patients harbor FHIT methylation.These results suggest that germinal epigenetic mutation of FHIT represents a preexisting mark predisposing to the development of ATL diseases. These findings have important clinical implications as patients with acute ATL are rarely cured. Our study suggests an alternative strategy to the current “wait and see approach” in that early screening of HTLV-I-infected individuals for germinal epimutation of FHIT and early treatment may offer significant clinical benefits.The online version contains supplementary material available at 10.1186/s12943-021-01370-2. The HTLV-I virus is etiologically linked to a neurodegenerative disease, HTLV-I-associated myelopathy or tropical spastic paraparesis (TSP/HAM), and adult T-cell leukemia/lymphoma (ATL), a fatal T-cell leukemia stemming from infection with the HTLV-I virus , 2. The Studies using different approaches, such as genome-wide sequencing and transcriptome and methylome analyses have revealed numerous cellular genes altered in ATL samples . Risk faAmong the genes reported to be altered in a genome-wide methylome analysis, we selected FHIT as a potential candidate for an ATL predisposition factor because of its essential role in preserving genome integrity and its frequent inactivation in human cancers. The FHIT gene is found at 3p14. 2, a region with enhanced replication stress. This location contains the FRA3B gene, the most common fragile site in the genome that is involved in cancer induced chromosome rearrangements . Due to This retrospective cross-sectional study was carried out using PBMCs collected and immediately frozen. All samples used here are part of previous studies, for which these samples were collected after informed consent and approved by appropriate institutional review boards. According to the revised Shimoyama classification , 124 patGenomic DNA (gDNA) was extracted from samples using DNAzol (ThermoFisher), and treated with Proteinase K, according to the manufacturer’s instructions. gDNA was treated with bisulfite using the MethylCode Bisfulfite Conversion Kit (ThermoFisher) and equal amounts of bisulfite DNA were used in methylation specific PCR (MSPCR) reactions. Equal amounts of PCR product were loaded onto TBE gels and visualized with SYBR green I nucleic acid stain (Molecular Probes). Primers used are described in Suppl PBMC were isolated by ficoll centrifugation and RNA were extracted by TRIzol (ThermoFisher) lysis reagent, treated with TURBO DNAse (ThermoFisher), and used for RNA-to-cDNA amplification (Applied Biosystems). RNA expression was determined using iTaq Universal SYBR Green Supermix (Biorad) or iTaq Universal Probes Supermix (Biorad) with the StepOnePlus real-time instrument (AppliedBiosystems). Primers are described in Supplemental p-values and confidence intervals are provided in Supplemental 2  = chi-square statistic value, p = p value). One-way ANOVA statistics were determined as needed and were performed using the Social Science Statistical calculator using the One-Way ANOVA Calculator, including turkey HSD. For expression data, significance was determined using a two-tailed T-test for two independent means with the formula t((N1–1) + (N2–11)) = the t statistic, with appropriate p-values. Pearson’s correlation coefficient and corresponding p-values were performed using the Social Science Statistical calculator.Odd risk ratios and chi-square statistics were determined for different HTLV-I diseases  against HD, AC, or TSP patients for FHIT methylation. Odd risk ratios and their corresponding Cell sorting was performed by magnetic bead separation or cell sorting by FACS. For magnetic bead sorting, PBMCs were washed in PBS containing 2% FBS and incubated with pre-washed Dynabeads anti-CD25 Fig.  or anti-An international collaborative effort was organized to collect over 400 samples from HTLV-I-infected individuals. To ensure genetic diversity of the tested population, samples originated from Asia, Africa, Europe, South and North America. We performed a comprehensive analysis on FHIT gene methylation and expression in uncultured peripheral blood mononuclear cells (PBMC) isolated from healthy volunteer donors (HD), HTLV-I infected asymptomatic carriers (AC), and HTLV-I infected individuals diagnosed with tropical spastic paraparesis/HTLV- I-associated myelopathy patients (TSP/HAM) and adult T-cell leukemia (ATL). Extracted genomic DNA was subject to bisulfite treatment and amplified using methylation-specific polymerase chain reaction (MSPCR) primers located in intron1 of the FHIT gene , AC (n = 89), TSP/HAM (n = 136) and ATL (n = 198) patient samples. To ensure genetic background diversity and worldwide representation in our analysis, we obtained patient samples that originated from South America, Japan, Africa, Caribbean islands, Europe, and the United Sates  with increasing methylation in advanced ATL diseases . On the ATL disease progression is associated with the expansion of tumor cells through cellular replication resulting in higher proviral loads . In turnTo demonstrate this hypothesis, we decided to investigate the methylation status of FHIT in ATL patients’ cells not infected with HTLV-I. Circulating ATL cells are phenotypically characterized by markers CD3+/CD4+/CD25+; and CD3+/CD4+/CD25- cells are generally HTLV-I negative , 40. Thrn = 14). We obtained gDNA from 10 patients that progressed to smoldering ATL. DNA was collected during the asymptomatic carrier stage of these patients and FHIT methylation analyzed and compared to samples from patients with smoldering ATL  and the exceptionally long incubation period to acquire ATL disease (over 20 years) . A recenFinally, several studies have implied that ATL disease may cluster in families , 7. A syATL diseases have a dismal prognosis with median survival times of 8.3 and 10.6 months and a 4-year overall survival rates of 11 and 16% for the acute and chronic ATL, respectively . To dateOur study identifies germinal FHIT methylation as a signature of ATL disease development that is present in more than 80% of HTLV-I-infected patients with acute or chronic ATL. A recent study has shown that the transcriptomic profile of North American ATL is distinct from Japanese ATL . The facThis is also the first study to address the expression of FHIT in HTLV-I infection and ATL disease. Studies have shown that loss of FHIT expression can direct a cell into a more genetically favorable cancer inducing phenotype. Given the long latency period between HTLV-I infection and the development of ATL disease, the loss of FHIT early on would be extremely beneficial. Besides it roles as a genome caretaker, FHIT has known roles in apoptosis, attenuating NF-κB and AKT/survivin signaling pathways, Ras/RhoGTPase, a repressor of the β-catenin pathway, interacts with the SUMO-1 conjugating enzyme, Ubc9, and plays a role in the cytoskeleton , 58–60. In summary, we have identified the tumor suppressor gene, FHIT, as being highly disrupted in ATL disease. The majority of acute and chronic ATL patient’s cells carry methylated FHIT, which is present in tumorigenic and non-tumorigenic cells, and leads to loss of FHIT expression. This suggests that FHIT may be a predisposition marker for identification of HTLV-I infected individuals that will progress to ATL disease.Additional file 1: Supplemental 1. Development of MSPCR and BGS analysis for examination of FHIT methylation. (A) MSPCR was used to determine the FHIT methylation status in patient samples. A standard was derived by mixing bisulfite treated DNA from a patient with UM FHIT with bisulfite treated DNA from a patient with M FHIT. Ratios of 100:0, 75:25, 50:50, 25:75, and 0:100 were used in MSPCR for UM and M PCR, respectively. (B) Example of BSG sequencing of an UM and M patient sample. Arrows point to distinct CpG islands in the same DNA sequence of FHIT; demonstrating UM (C to T) or M (C remains) in bisulfite treated DNA. Supplemental 2. Primers used in FHIT study. Accuprime (Invitrogen) or Q-solution (Qiagen) was used for FHIT, miR-124a, CDKN1A, and CDKN2A PCRs. Two PCRS were carried out for FHIT, SHP1, SYK, CDKN1A, and CDKN2A. BGS PCR conditions were as follow: FHIT BGS: 95–30″, TD: 61–51–1′, 72–1′ (Touchdown). SHP1 BGS: 95–30″, 64–1′, 72–1′ (35-40c). SYK BGS: 95–30″, 49–30″, 72–30″ (40c). CDKN1A BGS: 95–30″, TD: 55–45-1′, 72–1′ (Touchdown). CDKN2A BGS: 95–30″, TD: 61–51–1′, 72–1′ (Touchdown). miR-124a BGS: 95–30′, 54–40″, 72–40″ (40c). Supplemental 3. Statistical analysis of HTLV-I diseases for FHIT methylation. Odd risk ratios and chi-square statistics were determined for different HTLV-I diseases  against HD, AC, or TSP patients for FHIT methylation. Odd risk ratios and chi-square statistics were determined for different HTLV-I diseases  against HD, AC, or TSP patients for FHIT methylation. Chi-square results were determined using X2  = chi-square statistic value, p = p value). Supplemental 4. Geographical distribution of patient samples. Pie diagrams were used to illustrate the geographical distribution of ATL , TSP/HAM, and ACs. Continent of origin , South America (S.Amer.), and Europe) was determined from providers. If the continent of origin was not known at the time, the samples are marked as “unknown”. Acute ATL (n = 124), chronic ATL (n = 44), smoldering ATL (n = 20), lymphoma ATL (n = 20), TSP/HAM (n = 136), and asymptomatic (AC), HTLV-I carriers (n = 89). Supplemental 5. FHIT methylation in viral lymphomas. (A) Representation of BGS of lymphoma-type ATL patients. U vs M alleles are noted by white and black boxes, respectively. Horizontal rows represent sequencing of a single colony. (B) FHIT methylation status in viral lymphomas. HTLV-I, ATL-lymphoma (n = 10), Kaposi’s sarcoma herpesvirus (human herpesevirus-8) (KSHV) associated lymphoma (n = 13), Epstein-barr (EBV) associated Hodgkin’s lymphoma (HL) (n = 18), and hepatitis C virus (HCV) associated lymphomas (n = 15). Results were determined by MSPCR of FHIT. Supplemental 6. Deletional Analysis of the FHIT gene in ATL patients. (A) Chart representing UM (or very weak M) ATL patients for deletional analysis of the FHIT gene. One patient carried a deletion in exon 5. (B) Representation of the PCR bands for exon 4, 5, and 8. In some patients, exon 4 was also amplified. Genomic DNA was used to amplify different FHIT exons with the following primers: FHIT Exon 4: F-GACTAGGAATCAGAAATGAATAATTA, R-GCATGTCAGTCAGGTAACAGGTAAGC; FHIT Exon 5: F-GCTGTTTTATTGTCCACGTGGAAGCT, R-CTCAGCTATGGTAGTGAAAAGGTCAA; and FHIT Exon 8: F-GATGCACTGTCATTTCAAAGCACTGG, R-CATATCTCCATGCAAATATTTACTGTC.Additional file 2: Supplemental Table 1.