In vitro, this enhancer has been shown to bind SRF and ELK-1 host transcription factors, maintain chromatin openness and viral gene transcription, and induce aberrant host gene transcription near viral integration sites. However, the function of the viral enhancer in the context of early HTLV-1 infection events remains unknown. In this study, we generated a mutant Enhancer virus (mEnhancer) and evaluated its effects on HTLV-1-mediated in vitro immortalization, establishment of persistent infection with an in vivo rabbit model, and disease development in a humanized immune system (HIS) mouse model. The mEnhancer virus was able to establish persistent infection in rabbits, and there were no significant differences in proviral load or HTLV-1-specific antibody responses over a 25-week study. However, rabbits infected with the mEnhancer virus had significantly decreased sense and antisense viral gene expression at 12-weeks post-infection. HIS mice infected with wt or mEnhancer virus showed similar disease progression, proviral load, and viral gene expression. While mEnhancer virus was able to sufficiently immortalize primary T-lymphocytes in cell culture, the immortalized cells had an altered phenotype (CD8+ T-cells), decreased proviral load, decreased sense and anti-sense gene expression, and altered cell cycle progression compared to HTLV-1.wt immortalized cells (CD4+ T-cells). These results suggest that the HTLV-1 enhancer element alone does not determine persistence or disease development but plays a pivotal role in regulating viral gene expression.Human T-cell leukemia virus type 1 (HTLV-1) is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL) and chronic neurological disease. The disparity between silenced sense transcription versus constitutively active antisense (Hbz) transcription from the integrated provirus is not fully understood. The presence of an internal viral enhancer has recently been discovered in the Tax gene near the 3’ long terminal repeat (LTR) of HTLV-1.  An apprma (ATL) . The facThe regulation of viral gene expression from the integrated provirus is key to HTLV-1 persistence and the pathogenic outcomes of infection. Tax, the initial dominant transcript produced from the provirus, amplifies viral sense strand transcription and drives cellular transformation through interactions within major signaling pathways, including NF-κB and AP-1 . HTLV-1 in vitro that the HTLV-1 enhancer maintains chromatin openness, regulates viral gene transcription, and induces aberrant host gene transcription near viral integration sites. Given its potential contributions to frequent transcription driven from the 3’ LTR and constitutive Hbz expression, we aimed to characterize the role of the viral enhancer during early HTLV-1 infection events in vitro and in vivo. Our results showed that the viral enhancer is dispensable for persistence using a rabbit model of infection. However, disruption of the viral enhancer did significantly decrease both sense and antisense viral gene expression during infection. Using a HIS mouse disease model, loss of the viral enhancer did not affect HTLV-1-mediated disease progression. Absence of the viral enhancer did not affect immortalization efficiency in cell culture but did alter the immortalized cell phenotype to a predominance of CD8+ T-cells. Also, HTLV-1-immortalized T-cells lacking the viral enhancer had decreased proviral load, decreased sense and anti-sense gene expression, lower levels of cellular apoptosis, and altered cell cycle progression.Recently, an intragenic viral enhancer was discovered within the HTLV-1 provirus through a screen for transcriptional regulatory regions using proviral DNA-capture sequencing . The tra2 and air at 37°C.Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. Jurkat cells, an HTLV-1-negative, transformed human T-cell line, were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. Parental human 729.B control cells (lymphoblastoid B-cell line) and 729 HTLV-1 producer cell lines were cultured in Iscove’s DMEM  supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. Human PBMCs (hPBMCs) were isolated from whole blood freshly collected from healthy donors using Ficoll-Paque PLUS . Protocols for blood collection from human donors were approved by the Ohio State University Institutional Review Board. hPBMCs and early passage, HTLV-1-immortalized primary human T-cell lines (PBL) were cultured in RPMI 1640 supplemented with 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, and 10 U/mL recombinant human interleukin-2 . All cells were maintained in a humidified atmosphere of 5% CO(+) negative control (empty vector) was purchased from Invitrogen .The infectious HTLV-1 proviral clone ACH (HTLV-1.wt) has been previously characterized , 21. The5 cells in 2 mL media. Cells were co-transfected with 880 ng empty vector or proviral plasmid DNA, 100 ng LTR-1-Luc, and 20 ng TK-Renilla using Lipofectamine™ 2000 Transfection Reagent  at a 3:1 ratio of reagent (μL) to DNA (μg). Each condition was performed in duplicate. 72h post-transfection, cell supernatants were collected to measure HTLV-1 p19 Gag production using the RETRO-TEK HTLV p19 Antigen ELISA . Cells were collected by centrifugation for RNA extraction, RT, and subsequent quantitative PCR to detect Tax and Hbz gene expression . Cells were also collected for luciferase assay or immunoblotting . Luciferase assays were performed by lysing cell pellets in Passive Lysis Buffer  and following the manufacturer’s protocol for the Dual-Luciferase® Reporter Assay System . Firefly and Renilla luciferase relative light units were measured using the FilterMax F5 Multi-Mode Microplate Reader . Jurkat cells were plated in a 12-well dish at a density of 8 x 105 cells in 1 mL media. Cells were co-transfected with 2 μg total plasmid DNA  using Lipofectamine™ 2000 Transfection Reagent  at a 2:1 ratio of reagent (μL) to DNA (μg). 72h post-transfection, cells and supernatant were collected for dual luciferase assay and p19 Gag ELISA, respectively, as described above.HEK293T cells were plated in 6-well dishes at a density of 3 x 10® Cell Line Nucleofector® Kit V and Nucleofector™ 2b Device (program X-001) according to the manufacturer’s protocol . Seventy-two hours post-transfection, cells were placed under 1 mg/mL G418 selection . Approximately two weeks after G418 selection, cell supernatant was collected to measure HTLV-1 p19 Gag production by ELISA . Polyclonal cells with confirmed p19 production were subjected to single cell dilution. Subsequent single cell clones were plated at a density of 1 x 106 cells in 2 mL media and supernatant was collected after 24h for ELISA to obtain normalized p19 Gag values. 729 HTLV-1.mEnhancer clones with p19 Gag production comparable to established 729 HTLV-1.wt producer cells were used for in vitro immortalization assays and in vivo inoculation. Genomic DNA was isolated from single cell clones using the DNeasy Blood & Tissue Kit  and mEnhancer mutations were verified by PCR and Sanger sequencing .Stable producer cell lines were generated by nucleofecting 729.B cells with 2 μg HTLV-1.wt or HTLV-1.mEnhancer proviral plasmid DNA (contains neomycin resistance gene) using the Amaxa® TGX™ Precast Protein Gels  and transferred onto Amersham™ Protran® Western blotting nitrocellulose membranes . Membranes were blocked with 5% milk in PBS with 0.1% Tween-20 and incubated in the following primary antibodies: anti-Tax (rabbit anti-sera), anti-Hbz (rabbit anti-sera), and anti-β-actin . Membranes were incubated in appropriate anti-rabbit or -mouse IgG (H+L) HRP conjugate secondary antibodies . Pierce™ ECL Western Blotting Substrate  was used to develop the membranes and images were captured using an Amersham Imager 600 .Transfected HEK293T cells and 729 HTLV-1.wt or HTLV-1.mEnhancer producer cell clones were lysed in RIPA buffer with protease inhibitor cocktail . Total protein was quantitated using the Pierce™ BCA Protein Assay Kit  and FilterMax F5 Multi-Mode Microplate Reader . Protein was loaded in equal amounts on 4–20% Mini-PROTEAN6 freshly isolated hPBMCs were co-cultured with 1x 106 lethally irradiated (100 Gy) 729.B control cells, 729 HTLV-1.wt, or 729 HTLV-1.mEnhancer producer cells in 24-well dishes. 10 U/mL hIL-2 was supplied weekly with subsequent media changes. A portion of each irradiated producer cell line was maintained in culture to confirm cell death. Viable cells were enumerated at weekly intervals in triplicate wells for each condition by Trypan Blue exclusion . Cell supernatant was collected from enumerated wells for subsequent p19 Gag measurement by ELISA. Two different blood donors were used in our studies. Approximately equivalent numbers of cell lines were generated for both wt and mEnhancer conditions , however with different efficiencies between blood donors. A representative selection of both HTLV-1.wt and HTLV-1.mEnhancer immortalized cell lines from each blood donor was utilized in subsequent experiments.Approximately 2 x 10cscid Il2rgtm1WjlPrkd/SzJ mice  were purchased from The Jackson Laboratory . These mice lack mature T cells, B cells, and functional NK cells, and are deficient in cytokine signaling. Animals were housed in individually ventilated microisolation cages with corncob bedding and provided with commercial pelleted rodent chow and chlorinated reverse-osmosis–purified water without restriction. Cages containing autoclaved bedding were used for cage changes, which were performed in a ventilated biosafety cabinet. Mice were maintained in a room with constant temperature (20 ± 2°C) and relative humidity (50% ± 20%) under a 12:12-h light-dark cycle. The animal use protocol received prior approval by the Institutional Animal Care and Use Committee of The Ohio State University.Breeding pairs of NOD.Cg-4 to 1 × 105 CD34+ HUSC  in 50 μL PBS (pH 7.4) by using a sterile 26-gauge hypodermic needle. After recovery, pups were returned to their dams, allowed to mature normally, weaned at 21 d, and then housed in groups . At 10 wk after HUSC engraftment, mice were tested for the presence of human peripheral blood cells. Mice with at least 15% human CD45-positive lymphocytes were infected by intraperitoneal inoculation of 107 lethally irradiated (100 Gy) 729 HTLV-1.wt or 729 HTLV-1.mEnhancer producer cells; an aliquot of cells was maintained in culture to control for irradiation treatment. Animals were euthanized when they lost more than 20% of their body weight within 48 h.Shortly (24 to 48 h) after birth, pups were removed temporarily from the dam and treated with whole-body irradiation at 1 Gy . Each mouse then was injected into the liver with 3 × 10® AQueous One Solution Cell Proliferation Assays (MTS)  were performed on HTLV-1.wt and HTLV-1.mEnhancer newly immortalized cell lines according to the manufacturer’s protocol. Briefly, cells were counted and plated at 2000 cells/well in 96-well round-bottom plates on day 0 and monitored over a 4-day time course. CellTiter 96® AQueous One Solution reagent was added to each well, agitated slightly, and incubated at 37°C, 5% CO2 for 2 hours. Absorbance at 490 nm was collected on a FilterMax F5 Multi-Mode Microplate Reader . Proliferation in each cell line was measured in triplicate wells at each time point.Cell Titer 96g) for cell phenotype, apoptosis, or cell cycle analysis via flow cytometry. Collected cells were stained using fluorescein isothiocyanate (FITC)-conjugated anti-human CD3 and phycoerythrin (PE)-conjugated anti-human CD4 or CD8 antibodies  and analyzed for cell phenotype by flow cytometry using a Guava® easyCyte™ Benchtop Flow Cytometer. The purity of the isolated CD4+ and CD8+ T-cell population by positive antibody selection was determined to be between 90-95% using flow cytometry. Percentages of CD4+ and CD8+ T-cells were determined within the CD3+ T-cell gate and were normalized to 100. Alternatively, collected cells were stained using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions to measure the level of cellular apoptosis. For cell cycle analysis, 1 x 105 HTLV-1.wt and HTLV-1.mEnhancer immortalized PBLs were seeded in 96-well round-bottom plates. Cells were synchronized by serum starvation for 2 hr. Twenty-four hours later, cells were fixed in 70% ice-cold ethanol for 3 h and stained using the Guava® Cell Cycle Reagent  according to the manufacturer’s protocol. Data were acquired using the Guava CellCycle program (CytoSoft Version 1.3) and the Guava® easyCyte™ Benchtop Flow Cytometer.Immortalized PBLs (HTLV-1.wt or mEnhancer) were collected by slow centrifugation  729.B control cells or 729 HTLV-1 producer cells (wt or mEnhancer) were inoculated into the lateral ear vein. Blood was drawn via the central auricular artery at Weeks 0 (pre-inoculation), 4, 8, 12, 16, 20, and 25 (study endpoint). Plasma was collected and rabbit PBMCs (rPBMCs) were isolated using Ficoll-Paque™ PREMIUM . rPBMCs or plasma were assessed for proviral load, HTLV-1 gene expression, and HTLV-1-specific antibody response, as described below. Sanger sequencing of the viral enhancer region was performed at week 25 to monitor for viral reversions. All animal procedures were performed in accordance with a protocol approved by University Laboratory Animal Resources (ULAR) of The Ohio State University.Fourteen-week-old, male, specific pathogen-free New Zealand White  rabbits were obtained from Charles River Laboratories . After a 2-week acclimatization period, 1 x 10® Green Supermix  and 300 nM of each sense and antisense primer . For Env detection only, 500 ng of RNA was reverse transcribed, and 2 μL cDNA was pre-amplified using the SsoAdvanced™ PreAmp Supermix . The pre-amplification assay pool included primers for Env and human GAPDH (hGAPDH). The final reaction volume was 50 μL with 50 nM of each primer. The cycling protocol was as follows: 95°C for 3 min followed by 12 cycles of 95°C for 15 s and 58°C for 4 min. Products were diluted 1:5 in TE buffer  for detection of Env and 1:50 for detection of high abundance targets , according to the manufacturer’s protocol. Approximately 8.8 μL diluted, pre-amplified products were used per qPCR reaction. Reactions were carried out in 96-well plates on the CFX96 Touch Real-Time PCR Detection System . The reaction conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. Primer and probe sequences are listed in ® Green Supermix  and 300 nM each of 5’ primer (#20) and 3’ primer (#19). The reaction conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. Total copy number was calculated using a standard curve generated by duplicate log10 dilutions of ACHneo plasmid DNA. Proviral copies per 100 hPBMCs was calculated based on the approximation that 6 pg human DNA is equivalent to 1 cell.Genomic DNA and RNA were isolated from cell lines using the AllPrep DNA/RNA Mini Kit  according to manufacturer’s instructions. RNA samples were subjected to on-column DNase digestion using an RNase-Free DNase . RNA concentrations were measured using the ND-1000 Nanodrop spectrophotometer , and 250 ng was used for cDNA synthesis using the SuperScript IV First-Strand Synthesis System . 2 μL cDNA was used per qPCR reaction with iQ™ SYBR10 dilutions of plasmid DNA to generate a standard curve. Copy numbers were normalized appropriately to 1 x 106 rabbit or human GAPDH. Samples and standards were run in duplicate with no-RT and no-template controls included on each plate. To determine proviral load, 250 ng genomic DNA was used for qPCR with a primer and probe set specific to HTLV-1 gag/pol. The 20 μL final reaction volume included iQ™ Supermix , 300 nM each of 5’ primer (#20) and 3’ primer (#19), and 100 nM probe (TMP-3). The reaction conditions were as follows: 94°C for 3 min followed by 45 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 40 s. Total copy number was calculated using a standard curve generated by duplicate log10 dilutions of ACHneo plasmid DNA. Proviral copies per cell were determined by estimating that 1 μg rPBMC DNA is equivalent to 134,600 cells  according to manufacturer’s instructions. RNA samples were subjected to on-column DNase digestion, and 250 ng of RNA was used for cDNA synthesis, as described above. For rabbit samples only, 10 μL cDNA was pre-amplified using the SsoAdvanced™ PreAmp Supermix . The pre-amplification assay pool included primers for Hbz, gag/pol, and rabbit GAPDH (rGAPDH). The final reaction volume was 50 μL with 50 nM of each primer. The cycling protocol was as follows: 95°C for 3 min followed by 12 cycles of 95°C for 15 s and 58°C for 4 min. Products were diluted 1:5 in TE buffer  for detection of viral genes and 1:50 for detection of high abundance targets . Approximately 8.6 μL diluted, pre-amplified products were used per qPCR reaction with iQ™ Supermix , 300 nM of each sense and antisense primer, and 100 nM probe . The reaction conditions were 95°C for 3 min followed by 45 cycles of 95°C for 15 s and 57.5°C for 30 s. Total copy numbers of each gene target in cell lines, rPBMCs, and HIS mice were determined by log00 cells . Analysiin vitro immortalization assays, as well as in infected rabbits and HIS mice. Genomic DNA was isolated from producer and PBL cell lines using the DNeasy Blood & Tissue Kit . Genomic DNA was isolated from rPBMCs at the study endpoint (Week 25) and hPBMCs collected from mouse spleen at the time of sacrifice  using the AllPrep DNA/RNA Mini Kit . The mEnhancer region was amplified by PCR using GoTaq® Flexi DNA Polymerase  and the following flanking primers: 1F and 7R. The PCR conditions were as follows: 95°C for 2 min followed by 95°C for 30 s, 52.2°C for 30 s, and 72°C for 1 min (35 cycles), and a final extension of 72°C for 5 min. PCR products were run on a 1.5% TAE agarose gel with ethidium bromide. The amplified fragment was gel extracted using the QIAquick Gel Extraction Kit  and Sanger sequencing was performed with individual reactions containing forward or reverse primer (OSUCCC Genomics Shared Resource). Sequencing data was analyzed using SnapGene® 5.1.5 software .Sequencing was performed to verify enhancer mutations in 729 HTLV-1.mEnhancer producer cells and to screen for mutation reversions in PBL cell lines from The HTLV-specific antibody response was measured using the Avioq HTLV-I/II Microelisa System . The manufacturer’s protocol was modified by substituting the provided HRP-conjugated goat anti-human IgG with an HRP-conjugated goat anti-rabbit IgG . Rabbit plasma was diluted 1:50 with Sample Diluent (supplied by the manufacturer) to obtain absorbance values within the range of the standard curve. Absorbance was measured at 450 nm using the FilterMax F5 Multi-Mode Microplate Reader .Data were analyzed using the appropriate statistical test (denoted in figure legend) with GraphPad Prism 9.0 software .in vitro immortalization as well as in vivo persistence and pathogenesis, we introduced mutations into the SRF/ELK-1 binding sites using the established wild-type (wt) HTLV-1 molecular clone ACH (HTLV-1.wt) to generate HTLV-1.mEnhancer (A novel HTLV-1 enhancer was recently identified within the HTLV-1 provirus through a screen for nucleosome-free regions (NFR), which generally harbor transcriptional regulatory elements due to their accessibility to transcription factors . Matsuo tax (sense) and (hbz) (antisense) mRNA transcript levels in transfected HEK293T cells. Copy numbers of viral genes were normalized to 1 x 106 human gapdh (hgapdh) copies in each sample. While there was no difference in the copy number of tax between the HTLV-1.wt and HTLV-1.mEnhancer proviral clones, HTLV-1.mEnhancer showed a slight (however not significant) reduction in hbz . The 729 HTLV-1.mEnhancer cells had significantly lower p19 Gag in the culture supernatant  copies in each sample. Tax was undetectable in most rabbits from both the wt and mutant groups, and it was previously demonstrated that tax mRNA expression peaks as early as 1-2 weeks post-infection . To assess viral transcription levels according to the proviral copies per cell, gag/pol copy number was also normalized to the respective proviral load in each rabbit. Rabbits infected with HTLV-1.wt and HTLV-1.mEnhancer showed similar trends in the levels of gag/pol transcript per proviral copy number over the course of time, with the only statistically significant difference at Week 12 ; therefore, hbz copy number was normalized to proviral load in each animal. The level of hbz copies per proviral copy number was significantly lower in the HTLV-1.mEnhancer condition, but by Week 25, the expression was comparable to the level of HTLV-1.wt infected rabbits  mice, which were previously shown to be susceptible to lymphoproliferative disease induced by infectious HTLV-1 molecular clones . Sub-letTo determine the effect of the mEnhancer on HTLV-1-mediated immortalization of primary cells, we co-cultured human PBMCs freshly isolated from healthy donors with lethally irradiated 729 HTLV-1.wt or 729 HTLV-1.mEnhancer producer cells in two independent co-culture experiments. 10 U/ml human IL-2 was supplied weekly to the culture with media changes. Over time, both HTLV-1.wt and HTLV.1-mEnhancer viruses demonstrated the capacity to immortalize T-cells. At 10 weeks, cells from Donor 1 infected with HTLV-1.wt showed rapid proliferation as individual wells began to grow out from the co-culture as an immortalized, polyclonal cell population. Cells infected with HTLV-1.mEnhancer exhibited the typical cell clumping that is observed with immortalized PBL lines but grew at a much slower rate in culture , one major difference is the short-term expression from transient transfections in HEK293T and Jurkat cells compared to the long-term cumulative effects from an intact viral genome in 729 cells.Recently identified DNA elements within the HTLV-1 provirus that have potential roles in the regulation of sense versus antisense viral gene expression, as well as the surrounding host genome, include the viral enhancer  and the ications –52. A susistence . Given tin vitro . The resgag/pol and hbz mRNA expression in rPBMCs infected with HTLV-1.mEnhancer compared to HTLV-1.wt. Inoculating HIS mice with either wt or mEnhancer virus led to the development of lymphoproliferative disease induced by HTLV-1 infection. There were no significant differences in proviral load or tax and hbz mRNA levels between the HTLV-1.wt and HTLV-1.mEnhancer groups. Given that the mice recapitulate disease onset and rapid progression within a compact time frame following initial infection (5 weeks), this may not be the appropriate model to study the long-term effects of the enhancer element on viral gene expression and persistence. While there are limitations to both rabbit and HIS mouse animal models utilized in this study, each allows for the evaluation of numerous aspects of HTLV-1 biology, including transmission, viral replication, persistence, and the immune response in rabbits as well as HTLV-1-mediated disease development and pathology in HIS mice. Although we have previously examined viral replication kinetics in infected rabbits in a 12-week timeframe, we extended the current study to 25 weeks to monitor the increasing trends in proviral load and HTLV-1-specific antibody response that were observed in both the HTLV-1.wt and HTLV-1.mEnhancer conditions. Our data showed that each of these features of persistent HTLV-1 infection reached their highest levels by the endpoint and confirmed that HTLV-1.mEnhancer behaves similarly to the wt virus in a long-term in vivo study. Although we detected a decrease in gag/pol and hbz transcripts in rabbits at week 12, we failed to detect a difference between the different animal groups at later time points. This suggests that while the viral enhancer may contribute to Hbz expression, there are alternative factors (both from the virus and the host) which regulate viral mRNA levels within host cells.Inoculation of NZW rabbits with 729 HTLV-1.wt or 729 HTLV-1.mEnhancer producers led to the establishment of persistent infection, marked by increases in proviral load and HTLV-1-specific antibody response over time in animals from either condition. While viral gene expression was variable in individual rabbits and the mean copy numbers of Gag/pol and Hbz fluctuated between time points, our results are characteristic of working with outbred animals and consistent with previous studies , 24, 37.in vitro immortalization assays showed that mutation of the viral enhancer did not hinder the ability of HTLV-1 to immortalize T-cells, since both HTLV-1.wt- and HTLV-1.mEnhancer-infected primary human lymphocytes showed similar efficiencies of immortalization. Both conditions had accumulation of p19 Gag in the culture supernatant and sufficiently immortalized target T-cells. Somewhat surprisingly, the cells immortalized by HTLV-1.mEnhancer had an altered immortalization phenotype and were predominantly CD8+ T-cells. Although HTLV-1 can infect both CD4+ and CD8+ T-cells, immortalized cells in vitro, infected cells in patients with HTLV-1, and patients with HTLV-1-mediated disease predominantly carry infected CD4+ T-cells  was also lower in immortalized cell lines in the enhancer mutant condition compared to wt. Interestingly, PBLs which lacked the viral enhancer element consistently had 1-4 proviral integrations per cell compared to 10-40-fold more integrations in HTLV-1.wt immortalized cells. In the co-culture immortalization assay, the HTLV-1.mEnhancer infected cells produced lower levels of p19 Gag compared to HTLV-1.wt infected cells, and the wild-type cell lines had far more integrated proviruses. This is likely due to re-infection of the wild-type PBL cell lines, which produce sufficient levels of viral transcripts compared to the enhancer mutant PBL cell lines that had decreased levels of viral transcription. Although it would appear that enhanced viral transcription is favorable for immortalized cells in vitro, the HTLV-1.wt immortalized PBLs had higher levels of apoptosis than the HTLV-1.mEnhancer immortalized PBLs. Tax has been shown to induce rapid cell senescence driven by the transcriptional activity of NF-κB  cells, as well as the human T lymphoblastic cell line SupT1 , 57. HowA relationship between SRF/ELK-1 and HTLV-1, outside the context of the viral enhancer, has been demonstrated by previous studies . SRF andin vivo, but loss of this element does not affect establishment of persistent infection in rabbits or the induction of lymphoproliferative disease in HIS mice. Although abrogation of the viral enhancer does not affect the capacity of HTLV-1 to immortalize primary T-cell in culture, it does significantly alter the immortalization phenotype and viral gene expression. Other in vitro work has demonstrated that enhancer mutations abrogate the binding of SRF/ELK-1 and reduce host transcription near viral integration sites in HTLV-1-infected cells . We are grateful to Dr. Lianbo Yu from the Department of Biomedical Informatics at The Ohio State University for aid in the statistical analyses for our in vivo studies. We also thank members of the Panfil and Green laboratories for valuable discussions.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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Giardia duodenalis genetic diversity and assess the epidemiologic scenario of subclinical infections in different Brazilian biogeographic regions. Cross-sectional surveys (n = 1334 subjects) were conducted in four municipalities in order to obtain fecal samples and socioenvironmental data. Microscopy of non-diarrheal feces and nucleotide sequencing of a β-giardin gene fragment were performed. From a total of 51 samples that could be sequenced, 27 (52.9%) β-giardin sequences were characterized as assemblage A and 24 (47.1%) as assemblage B. In the Amazon, assemblage B was the most frequently detected, predominantly BIII, and with two novel sub-assemblages. Assemblage A predominated in the extra-Amazon region, with five novel sub-assemblages. Prevalence reached 17.8% (64/360) in the Amazon, 8.8% (48/544) in the Atlantic Forest, 7.4% (22/299) in Cerrado and 2.3% (3/131) in the Semiarid. People living in poverty and extreme poverty presented significantly higher positivity rates. In conclusion, subclinical giardiasis is endemic in Brazilian communities in different biogeographic regions, presenting high genetic diversity and a heterogeneous genotypic distribution.Human infections with gut protozoan parasites are neglected and not targeted by specific control initiatives, leading to a knowledge gap concerning their regional diversity and epidemiology. The present study aims to explore  Giardia duodenalis is a cosmopolitan, flagellated gut protozoan parasite with higher prevalence in developing countries, mainly in regions with poor sanitation and inappropriate drinking water supply [G. duodenalis trophozoites inhabit the small intestine and, during the course of infection, a variable proportion of cells encyst and are shed in feces. Cysts can persist in the environment and contaminate water and food, constituting the parasite infective stage. In industrialized regions, G. duodenalis causes outbreaks of diarrheal diseases which have water contaminated with fecal material as their main source [r supply . G. duodn source ,3.G. duodenalis as an etiologic agent of diarrheal diseases in children living in African and Asian developing countries in a 3-year prospective, age-stratified, multicentric and matched case–control study, demonstrating that G. duodenalis more frequently infected controls than children aged 12–59 months with diarrhea [G. duodenalis positivity rates were found among controls when compared with children with diarrhea [G. duodenalis—through complex pathophysiological mechanisms—impacts the absorption of nutrients and affects both the nutritional status and physical development of children in a process that does not depend on the presence of diarrhea [The Global Enteric Multicenter Study (GEMS) assessed the role of diarrhea . Similardiarrhea ,7. Thesediarrhea ,9. Therediarrhea ,11.G. duodenalis infects a wide range of mammalian species and presents considerable intraspecific genetic variation, with eight distinct assemblages (A to H). Assemblages A and B were identified mainly in humans, C and D in wild and domestic canines, E in ruminants and domestic pigs, F in cats, G in mice and rats and H in seals [in seals ,13. Giarin seals . The degin seals ,16. In Rin seals .Brazil occupies most of the South American continent and has great biogeographic and climatic diversity, with specific rainfall regimes with great variation, from rain forests to semiarid, associated with different water management strategies, creating regional scenarios for the epidemiology of water-borne infections.G. duodenalis infection in an urban Amazonian area and three extra-Amazonian Brazilian biogeographic regions.Infections with gut protozoan parasites are not targeted by specific intestinal parasite control strategies, which are based on the mass administration of anthelmintic drugs . This han = 1334; n = 844) and height (n = 742) were obtained from individuals between 0–14 years of age. Weight was obtained with a portable electronic scale, to the nearest 100 g. Children were barefoot and with minimum clothing. Infants aged less than 12 months were weighed in their mothers’ arms. Height or length was measured using an anthropometer to the nearest 0.1 cm. The nutritional parameters height-for-age Z-scores (HAZ) and weight-for-age Z-scores (WAZ) were calculated with the Nutrition module of the Epi Info™ v.3.5.1 software to verify the presence of protein–energy malnutrition characterized by stunting (HAZ < −2) and low weight (WAZ < −2). Extreme poverty was defined when monthly family per capita income was below BRL 125, which corresponds to USD 25 (considering the exchange rate of USD 1 = BRL 5). Poverty was defined by monthly family per capita income between BRL 125 and BRL 250 (USD 25–50). The researchers gathered information about the site of defecation, i.e., if the family had a latrine inside the house or if members of the family practice open defecation in the peridomestic environment. The final destination of the feces was also characterized, being adequate when the feces went to closed septic tanks and inadequate when they were deposited in the ground, in rudimentary holes or directly into a waterbody.Cross-sectional surveys , usually from inhabitants who lived in the homes of positive subjects. Thus, a total of 179 fecal samples were submitted for PCR. Genomic DNA was extracted from 200 µL of the sedimented fecal material using the ZR Fungal/Bacterial DNA MiniPrep™ kit . PCR was performed using the Platinum Taq DNA Polymerase kit  with a final volume of 50 μL, and targeted a 753 bp region of the β-giardin locus of G. duodenalis, as described [2, 0.05 mM dNTP, 10 pmol of each primer, 2.5 U of Taq polymerase and ~40 ng of template DNA. Amplification parameters included an initial denaturation at 94 °C for 5 min followed by 35 cycles of amplification comprising denaturation (94 °C for 30 s), annealing (65 °C for 30 s) and extension (72 °C for 30 s), and a final extension at 72 °C for 5 min. The PCR products were purified with polyethylene glycol (PEG). Capillary electrophoresis was performed in an ABI3730 automated DNA sequencer (Applied Biosystems) in PDTIS/Fiocruz Genomic Platform RPT01A.After parasitological examinations, 137 escribed . The PCRhttps://www.ncbi.nlm.nih.gov accessed on 20 August 2021) was used to verify similarity with G. duodenalis sequences. All sequences generated were deposited in the GenBank database under the accession numbers MW679411-MW679461. To determine G. duodenalis assemblages (genotypes), an alignment was performed with 55 G. duodenalis orthologous reference sequences retrieved from GenBank in BioEdit v.7.2.5 software. Sequences with degenerate bases were not included. Further details of reference strains can be found in www.fluxusengineering.com) [G. duodenalis, diversity indexes were determined for each population pair using Arlequin v.3.5.2.2 software (http://cmpg.unibe.ch/software/arlequin35 accessed on 20 August 2021) [The sequences were edited and analyzed using the BioEdit v.7.2.5 software . The Basing.com) . The DNAing.com) . To evalst 2021) . The popG. duodenalis positivity rates were described in different groups defined by sociodemographic characteristics and nutritional status. Prevalence ratios (PRs) and their respective 95% confidence intervals (CIs) were calculated. The statistical significance of the differences between the positivity rates was assessed by Fisher’s exact test. Associations were considered statistically significant when p < 0.05. Statistical analyses were performed with Epi Info 2000® .The study was approved by the Research Ethics Committee (license CAAE 12125713.5.0000.5248) of the Oswaldo Cruz Institute, Fiocruz.Of the 179 fecal samples submitted to PCR/sequencing, 51 were successfully genotyped using the β-giardin locus. Overlapping peaks were not observed in the nucleotide sequences. Twenty-seven (52.9%) sequences were characterized as assemblage A and twenty-four (47.1%) as assemblage B .G. duodenalis sequences were grouped by assemblages, as expected. Assemblages A and B presented a star-like shape, including the sequences obtained in this study as a central and dominant haplotype (except the novel haplotypes).The MJ haplotype network  showed tG. duodenalis sequences were grouped by assemblages. The main difference between the two trees was in the NJ tree: assemblage D shared a common ancestor with assemblages B and E. Concerning the molecular diversity indexes, in general, assemblage B revealed greater intraspecific diversity when compared with assemblage A (H = 0.921 ± 0.028 vs H = 0.854 ± 0.029)  . In Braz± 0.029) .Entamoeba histolytica/E. dispar and with Entamoeba coli). The overall positivity rate was 137/1334 (10.3%). The frequency was significantly higher in Bagre, in the Amazon region, reaching 64/360 (17.8%) and lower in São João do Piauí, in the Caatinga (3/131 (2.3%)). In Bagre, Teresina and Cachoeiras de Macacu, the age groups of 3 to 6 years old and 7 to 15 years old had the highest rates of positivity, but infants and toddlers up to 2 years old were also frequently infected in Bagre and Cachoeiras de Macacu. Giardiasis was significantly more frequent among people living in poor and extremely poor families in Bagre, Teresina and Cachoeiras de Macacu. People living in scenarios of inappropriate disposal of feces and open defecation also presented significantly higher positivity. Individuals positive for Entamoeba histolytica/E. dispar or E. coli were infected with G. duodenalis at significantly higher frequencies.G. duodenalis in different Brazilian biogeographic regions. Regarding the detection frequencies of the main assemblages—even considering the relatively small proportion of positive samples in which the partial sequencing of the β-giardin gene was achieved—some regional differences could be observed. It is important to note that the large amount of bacterial DNA in the fecal samples, as well as inhibitory substances in the material, usually interfered negatively in the success of PCR amplification.The present study explored regional differences in the genotypic composition and epidemiologic profile of G. duodenalis subclinical infection. In São Paulo (southeastern Brazil), a predominance of assemblage A was reported in low-income families [G. duodenalis assemblage B infections is higher, with greater shedding of cysts and, consequently, a greater potential for spreading [In the Amazon region, there was a predominance of assemblage B, and in the extra-Amazonian area, assemblage A was more frequently detected. A similar genotypic profile was described in a previous work when we compared another Amazon region (the Rio Negro basin) with different extra-Amazonian regions ,26. A hifamilies ; this prfamilies . In Rio families ,31. A prpreading . Outsidepreading ,34.G. duodenalis assemblages A and B. However, prospective studies with large sample sizes and quantitative assessments of the parasite load, response to treatment and the clinical and nutritional impact of giardiasis have not yet been carried out in order to assess clear clinical and epidemiological differences between the main genotypes of G. duodenalis. The present study did not include subjects with diarrhea, and infections were identified in subclinical conditions. In Saudi Arabia, children infected with assemblage B were predominantly symptomatic, whereas asymptomatic participants harbored assemblages AI and AII [Taken together, these data may point to putative differences in the epidemiologic profile of  and AII . Convers and AII .G. duodenalis assemblages A and B, the genetic variation between them is well established. G. duodenalis complete genomes revealed substantial phylogenetic divergence between the two main genotypes infecting humans and demonstrate that: (i) the average amino-acid identity in 4300 orthologous proteins is not superior to 78% and (ii) the full-genome-derived similarity between enzootic assemblage E and assemblages A and B at the level of amino-acid identity is 90% and 81%, respectively [Despite the lack of robust data on the clinical and epidemiological differences between ectively . Our phyG. duodenalis infection, has the lowest human development index and the highest proportion of people living in poverty and extreme poverty. Historically, in the Amazon, the process of demographic concentration of populations of Amerindian descent has favored the spread of parasitic and fecal-borne diseases. The Amazon is the region of the country with the highest prevalence of soil-transmitted helminthiases, incidence of diarrheal diseases and diarrhea-related mortality. The riverside character of Bagre should also be considered, with its close proximity and greater contact of the population with waterbodies in a region of abundant rainfall and poor sanitation, favoring the spread of water-borne infections, which contrasts with the semiarid climate and very low rainfall in São João do Piauí, where the prevalence of giardiasis was lower. Intermediate positivity rates were observed in Teresina and Cachoeiras de Macacu, in the Cerrado and Atlantic Forest biogeographic regions, respectively. In addition, our data demonstrated that G. duodenalis positivity is strongly influenced by the living conditions of the studied subjects, as the positivity rate was higher among children within the poorest families in Bagre, Teresina and Cachoeiras de Macacu and with inadequate destination of feces in Teresina. The detected association of giardiasis with other gut protozoan parasites, such as E. histolytica/E. dispar and Entamoeba coli, reinforces the vulnerability of the poorest families to fecal-borne infections.The comparison of some socio-environmental characteristics of the four regions assessed in the present study reveals that Bagre in the Amazon region, the locality with higher prevalence of In conclusion, subclinical giardiasis, identified in subjects without diarrhea, is frequent in resource-poor Brazilian communities and with a heterogeneous geographic genotype distribution and prevalence. The data suggest the need to improve control strategies, including better access to diagnosis and treatment.


Purpose: 1) to explore the test-retest reliability of a new device for measuring ankle inversion proprioception during walking, i.e., the Ankle Inversion Discrimination Apparatus—Walking (AIDAW) in individuals with or without Chronic Ankle instability (CAI); 2) to assess its discriminant validity in differentiating individuals with or without CAI; 3) to investigate its convergent validity by examining its association with Cumberland Ankle Instability Tool (CAIT) and the Y Balance Test (YBT).Methods: For test-retest reliability, 15 participants with CAI and 15 non-CAI healthy controls were recruited. Participants completed the AIDAW test twice with a 7-day interval. The area under the receiver operating curve (AUC) was obtained as the AIDAW score. The intraclass correlation coefficient (ICC) and MDC90 were calculated. For the validity study, another 20 individuals with CAI and 20 non-CAI healthy controls were involved. The AIDAW scores were analyzed by an independent samples t-test, and the optimal cutoff value of AIDAW scores to best distinguish individuals with CAI was calculated by Youden’s index. Spearman or Pearson correlation analysis was used to analyze the correlation between AIDAW proprioceptive scores and the CAIT and final YBT scores.Results: For test-retest reliability, the ICC values for the CAI, non-CAI, and the whole group were 0.755, 0.757, and 0.761 respectively. The MDC90 of the CAI and non-CAI group was 0.04 and 0.05. Regarding discriminant validity, the AIDAW proprioceptive discrimination scores in the CAI group were significantly lower than those in the non-CAI group (p = 0.003); and the cutoff score for distinguishing CAI from the non-CAI participants was 0.759. For convergent validity, the AIDAW scores were significantly correlated with the functional balance YBT final scores (p = 0.001) and the CAIT scores (p = 0.009).Conclusion: The AIDAW is a reliable and valid device for evaluating ankle inversion proprioception during walking in individuals with and without CAI. AIDAW can be used as a clinical assessment tool to discriminate CAI from non-CAI individuals and to monitor effects of rehabilitation. The AIDAW proprioceptive discrimination scores were significantly and positively correlated with YBT and CAIT scores. The ankle-foot complex is the only part of the human body that contacts the ground in most daily and sports activities, with this causing a variety of ankle injuries . LateralProprioception is the ability to perceive and integrate the position and movement sense of the body parts to determine their status in space , and it Indeed, In addition to landing, CAI individuals may also have difficulties in walking on uneven ground, as reflected by ankle function and symptoms questionnaires . TherefoSelf-reported instability and balance deficit are common conditions in CAI individuals, which are related to the severity of CAI . The CAIAccordingly, we developed a novel device for measuring ankle inversion proprioception during walking, i.e., the Ankle Inversion Discrimination Apparatus—Walking (AIDAW). The aims of this study were: 1) to assess the test-retest reliability of the AIDAW in individuals with and without CAI; 2) to assess the discriminant validity of AIDAW for individuals with and without CAI; and 3) to assess the convergent validity of AIDAW, by examining the relationship between the AIDAW scores, ankle instability symptom CAIT scores, and functional balance YBT scores.Seventy participants volunteered to take part in this research. For both the reliability and validity studies, the G* Power software was used to determine the sample size. For the reliability study, the G* Power software was used to calculate the sample, with power = 0.7, significance level = 0.05, intraclass correlation assuming the null hypothesis = 0.5, intraclass correlation assuming the alternative hypothesis = 0.75. The software showed the minimum sample size was 29. Accordingly, we recruited 30 participants (15 CAI participants and 15 non-CAI participants). The sample size for the validity study was also calculated by G* Power software . The minimal sample size was determined as 38. Accordingly, another 40 participants (20 CAI participants and 20 non-CAI participants) were recruited. The inclusion criteria for the CAI group were: 1) a history of at least one ankle sprain with noticeable swelling and pain 12 months ago; 2) unable to perform daily activities for at least 1 day after the acute ankle sprain; 3) at least two ankle instability episodes within the past 6 months; 4) a score of less than 24 on the Chinese version of Cumberland Ankle Instability Tool (CAIT) . The excThis study was approved by the Committee for Ethics in Human Research at Shanghai University of Sport . All the participants provided signed informed consent before the data was collected.A piece of new equipment, the Ankle inversion Discrimination Apparatus during Walking AIDAW, , was purThe Y Balance Test Kit™ was used to test the dynamic balance ability of the lower limbs . The CAIFor test-retest reliability study, participants performed two sessions of the AIDAW proprioception tests with an interval between them of 7 days. For the discriminant and convergent validity study, participants completed the AIDAW proprioception test and the YBT in random order. The testing foot for the CAI group was the one with lower CAIT score, or the dominant foot for the non-CAI group.vice versa such that when the tested foot was left side, the walking direction was from right to left.For the AIDAW test, participants were required to walk 6 steps at their normal, comfortable speed for each trial. Before testing, participants were allowed to adjust their starting point to ensure the testing foot could step onto the testing platform in step 3. During the test, participants were instructed to stand with bare feet on one side of the walking platform (depending on which foot was going to be tested), with eyes looking forward . ParticiBefore data collection, there were 3 rounds of familiarization with the 4 ankle inversion positions, from 10 to 16° in order, and 12 trials in total, and participants were required to remember the feeling of each ankle inversion position. For the formal test, the 4 possible ankle inversion positions appeared randomly, and each inversion position appeared 10 times, for 40 trials in total. Participants were instructed to report their perceived ankle inversion position on each trial, without feedback on the correctness of their judgement. The actual presented positions and participants’ perceived positions were recorded on each trial for data analysis. Each participant would have 40 ankle inversion position stimuli and corresponding responses in total. The total test time was about 10 min.For the YBT, participants stood on the standing block with the tested leg, and used the other foot to push the slide block as far as possible in anterior, posteromedial, and posterolateral directions  Wilson . Before The CAIT questionnaire was completed during demographic data collection, and the order of AIDAW and YBT tests was random.p-value of <0.05 was set as statistical significance.All data analyses were performed with SPSS version 24 . A In order to calculate the AIDAW proprioceptive discrimination sensitivity score, the raw data of the 40 randomly assigned ankle inversion positions and their associated participant responses were entered into a 4 lculated . The ROClculated . AUC val90) was calculated with obtained ICC values using the following formula  and Bland-Altman plot were used. The ICC was calculated with a two-way fixed model, single measure type, and absolute agreement definition . ICC val formula . The SEMt-test was used to test the difference in AIDAW scores between the CAI and the non-CAI groups. The cutoff score for discriminating between CAI and non-CAI individuals was calculated from the ROC curve, and the optimal cutoff score was calculated by the maximum value of Youden’s index , 0.757 (95%CI = 0.425–0.911), and 0.761 (95%CI = 0.557–0.879), respectively. Bland-Altman plots of the whole group are shown in Regarding discriminant validity, the AIDAW scores in the CAI group were found to be significantly lower than in the non-CAI group . The optr = 0.51, p = 0.001) and the ankle instability symptoms CAIT score .For convergent validity, the difference analysis between the CAI group and healthy non-CAI group is shown in p = 0.055).Although the YBT scores were significantly correlated with the AIDAW scores, the YBT was not ideal for discriminating CAI from non-CAI participants   . These rConcerning the discriminant validity study, the AIDAW scores of the CAI group were significantly lower than for the non-CAI group. This result is consistent with what has been found in AIDAL testing . The AIDFor the convergent validity study, the correlation between the AIDAW score and CAIT score was moderate, also similar to that observed with the AIDAL . In compIn addition, we also found moderate correlation between the AIDAW scores and the final YBT scores. YBT performance reflects dynamic balance ability, which is fundamental for exercise and sport performance. To maintain balance, the brain has to integrate proprioception, vision and vestibular information . The fooThe AIDAW is the first tool developed for measuring ankle proprioception during walking based on AMEDA methods which could be used to identify CAI in clinical practice. In this study, we established the MDC values for CAI and non-CAI individuals, values which could be used to confirm a true and clinically important change. This study also found that ankle proprioception was related to dynamic balance ability and perceived instability, suggesting its importance in lower limb control. Therefore, the results provide essential information for use of the AIDAW in clinical management of CAI. However, in the current study only young participants were recruited, so the results here may not be generalized to other populations, such as older participants.In addition, compared to previous proprioceptive methods where participants were strapped on a machine and their vision and audition were blocked, the current AIDAW test was conducted under functional walking conditions, and thus the ecological validity of the test has been optimized. However, only six steps were performed, which is different from the number taken in normal walking activity.The novel AIDAW device is reliable and valid for evaluating ankle inversion proprioception during walking for individuals with and without CAI. Ankle inversion proprioception was significantly impaired in individuals with CAI, and the AIDAW can be used as a discrimination tool for separating CAI from non-CAI individuals. The AIDAW scores were significantly and positively associated with YBT and CAIT scores, suggesting that rehabilitation programs targeting ankle proprioception impairment may be beneficial in improving functional performance and symptoms in individuals with CAI.


Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The effects of HTLV-1 on health are not fully elucidated. Epidemiological studies have shown that the prevalence of HTLV-1 infection is high in patients with rheumatic diseases. The prevalence of comorbidities, such as Sjögren’s syndrome and rheumatoid arthritis (RA), is higher in patients with HAM/TSP than the in general population. Studies have shown the effects of HTLV-1-infection on the clinical course of RA. Major questions on the association between HTLV-1 infection and RA: (1) Is it possible that HTLV-1 infection causes RA? (2) Do patients with RA who are infected with HTLV-1 have different clinical features? (3) Are immunosuppressants associated with an increased prevalence of HAM/TSP or ATL in RA patients with HTLV-1 infection? Is ATL an immunosuppressive therapy-associated lymphoproliferative disorder? No large-scale studies have investigated the incidence of ATL in patients with RA. However, several studies have reported the development of ATL in patients with RA who have HTLV-1 infection. This review aimed to shed light on the association between HTLV-1 infection and RA and summarize the unmet medical needs of RA patients with HTLV-1 infection. Human Leukocyte Antigen–DR isotype genes and polymorphisms of multiple genes, also have been considered important for its etiology [Human T-cell leukemia virus type 1 (HTLV-1) is a human retrovirus that is a causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) ,3,4. HTLetiology ,19. Antietiology . Biologietiology . Howeveretiology ,22. For etiology ,24.The major research and clinical questions about HTLV-1 infection and rheumatic diseases reviewed in this article are as follows: (1) Is it possible that HTLV-1 infection is one of the causes of rheumatic diseases? (2) Do patients with rheumatic diseases display any different clinical features when they are HTLV-1 carriers? (3) Do immunosuppressants, including biologics and molecular-targeted DMARDs, increase the prevalence of HAM/TSP or ATL in RA patients with HTLV-1 infection? Should development of ATL be considered to be an immunosuppressive therapy-associated LPD? A search strategy was used to select articles on the association between HTLV-1 infection and RA, including case series, clinical research, cohort study, and meta-analysis, in PubMed. All studies were published from 1980 to 2022. The key words used to search the articles were as follows: HTLV-1, ATL, rheumatic manifestations, arthropathy, arthritis, RA, antirheumatic therapies, lymphoproliferative disorders, and HAM/TSP. This review aimed to provide novel information on the association between HTLV infection and RA and to summarize the unmet medical needs of RA patients with HTLV-1 infection in routine clinical practice.It remains unclear whether HTLV-1 infection etiologically contributes to the development of RA. Several epidemiological studies have shown that the prevalence of HTLV-1 infection is higher in patients with rheumatic diseases, such as RA, polymyositis, and Sjögren’s syndrome, than in healthy controls, such as blood donors ,26,27. ATax and HBZ genes [Tax-transgenic (Tg) mice but not in those of healthy Tg mice [Tax-Tg mice, TNF-alpha expression had not been observed in either arthropathic or healthy Tg mouse synovial tissues mRNA [Tax-Tg mice [HBZ-Tg mice [HTLV-1 primarily infects CD4+T-lymphocytes and is thought to alter their functions and lineages. It is possible that certain clones of HTLV-1-infected cells proliferate and alter both innate and acquired immunity of HTLV-1 carriers. Previous studies have suggested that the proinflammatory status of HTLV-1-infected cells is characterized by the presence of cytokines, such as TNF, IFN-γ, IL-6, IP-10, TGF, and GM-CSF ,37. BothBZ genes ,40. Tax,BZ genes ,42,43. E Tg mice . In the ues mRNA . Exocrin-Tg mice . Compare-Tg mice ,47. Thes-Tg mice ,47. Acco-Tg mice . IL-6 is-Tg mice . IL-6 in-Tg mice . HBZ act-Tg mice .t-bet gene with reduced expression of FoxP3 in the infected cells, resulting in their differentiation toward Th1 balance in HAM/TSP [HAM/TSP is a chronic inflammatory disease of the central nervous system that has high levels of HTLV-1 proviral load (PVL) and polyclonal expansion of HTLV-1-infected cells. In one study, peripheral blood mononuclear cells (PBMCs) isolated from patients with HAM/TSP showed autonomously produced inflammatory cytokines, such as IFN-γ, IL-6, and TNF-alpha . In furt HAM/TSP ,53,54. T HAM/TSP . CD4+CD2 HAM/TSP . Both HT HAM/TSP ,58,59,60In the inflammation sites of RA, HTLV-1-infected cells may have important roles in the exacerbation of inflammation. The levels of C-reactive protein (CRP), which is a marker of RA activity, have been shown to be higher in HTLV-1-positive patients with RA before receiving treatment with biologic DMARDs than the levels of CRP HTLV-1-negative patients . In addiMycobacterium tuberculosis (TB), Strongyloides stercoralis , EBV, cytomegalovirus, and Pneumocystis jirovejii [S. stercoralis has been observed in HTLV-1 carriers than in healthy subjects without HTLV-1 infection [HTLV-1 infection may alter the host immune responses against irovejii ,74. A hinfection . During nfection ,76. HoweS. stercoralis colitis after administration of a TNF inhibitor [Hashiba et al. reported a case of HTLV-1-positive RA patients who developed nhibitor . Recentlnhibitor . In thisnhibitor ,78. A menhibitor ,16. Atsunhibitor  suggestenhibitor . TherefoThe incidence of TB is reported to be higher in RA patients receiving anti-TNF therapy compared with the general population ,82. NatiIt is unknown if the association of rheumatic diseases and treatment against such diseases affects the natural clinical course of HTLV-1 infection, particularly in view of the development of HTLV-1-associated diseases, such as HAM/TSP and ATL.Patients with RA are considered to have a Th1- and Th17-dominant immune status. The immune status of HAM/TSP is also considered Th1 dominant. Therefore, whether or not RA affects the etiology and clinical condition of HAM/TSP is an important question. It is also important to know if the incidence of HAM/TSP in patients with RA who have been an HTLV-1 carrier or who acquired HTLV-1 infection in adulthood is higher than that in asymptomatic HTLV-1 carriers. Indeed, the prevalence of comorbidities of Sjögren’s syndrome and RA in patients with HAM/TSP was recently reported to be as high as 3.7% and 2.7%, respectively . This prWe must also consider the effect of the DMARDs that patients with RA are receiving. Indeed, worsening symptoms of both HAM/TSP and HTLV-1-associated uveitis were reported in an HTLV-1-positive patient with RA who received treatment with a soluble IL-6 receptor inhibitor (sIL-6Ri) . ConversPatients with RA reportedly have higher incidence rates of malignant lymphoma, although the surface phenotype of lymphoma found in RA is B-cell predominant . In addiIt is unclear whether DMARD treatment on RA must be recognized as an important risk factor for the development of ATL in HTLV-1-positive RA patients. MTX is well known as the anchor drug for RA and has immunosuppressive effects caused by inhibition of nucleic acid synthesis. Another DMARD, tacrolimus, specifically suppresses T-cell function by inhibiting calcineurin. Biologics and targeted synthetic DMARDs mainly inhibit the function of proinflammatory cytokines and immune response cells. It is unknown if there are any direct effects of these medicines on HTLV-1-infected cells in vivo; however, it is clear that these medicines cause various levels of immune suppression against anti-viral immunity. Several reports have suggested that antirheumatic therapies increased the incidence of viral reactivation, such as EBV, hepatitis B virus, and varicella-zoster virus. Since ATL could be considered to be one of the virus-associated LPDs, suppression of anti-HTLV-1 immunity may be involved in the progression of ATL . Indeed,In daily clinical practice of RA, several cases of ATL have been reported in patients with rheumatic diseases during treatment with DMARDs, including MTX ,106,107.Considering the rarity of HTLV-1 positivity in RA, it may be challenging to answer the following clinical questions: Is HTLV-1 an etiologic factor of RA? Do the clinical characteristics of RA patients with HTLV-1 infection differ from those who did not? Is there evidence showing that immunosuppressants are associated with an increased incidence of ATL in RA patients with HTLV-1 infection ? The pre


Background: There has been continuous debate among scholars regarding fertility transition in Africa. Two conclusions emerge: slow pace of decline because of weak facilitating social programs and high demand for large families amidst weak family planning programs. Accelerated fertility decline is expected to occur if there is both substantial decline in desired fertility and increased level of preference implementation. Despite these conclusions, there are also emergent exceptions in Africa, even among the Eastern African countries. Our motivation for the study of this region therefore lies in this context. First, the East African countries share some similarities in policy framework. Secondly, Rwanda and Kenya appear as exceptional in the drive towards accelerating further fertility decline. Fertility change therefore in any one country may have implications in the neighbouring country due to the commonalities especially in language, cultural traits, diffusion and spread new models of behaviour.Methods: With the utilization of DHS data, we analyse trends overtime in two specific features that scholars have indicated to slow or increase fertility decline. Using Bongaarts supply-demand framework, we first deduce trends in fertility preferences among women of reproductive age (15-49 years) and second, the extent to which women have been able to implement their fertility preferences during the course of fertility decline and subsequently decomposing these trends.Results: We found that with the rising aggregate of the degree of fertility preference implementation index, continuous declining trends in demand for births and subsequent increases in the contribution made by either or both the wanted fertility and the degree of fertility preference implementation index across categories that fertility transition is certainly on course in all countries albeit at different levels, thanks to the family planning.Conclusions: Family planning programs must therefore be accompanied by rigorous, consistent sensitization and public education. Over the last decade, there has been considerable debate among scholars on fertility transition in Africa. Two conclusions emerge: slow pace of decline  because Despite these conclusions, there are also emergent exceptions, such as Rwanda, Ethiopia, Malawi . In a seKenyan fertility began to rapidly decline in the 1980s, followed by a stall (1998–2003) and then another phase of decline (2003–2014) recently . TanzaniThe main reason for focus on these countries is that any fertility change in one country may have implications in the neighboring country, because neighbouring regions share common dynamics, including language and cultural traits that permit shared flow of ideas to eventually spread new models of behavior. We analyze trends in two specific features that scholars have indicated will slow or increase in fertility decline. First, trends in fertility preferences among women and secondly, the extent to which women have been able to implement their fertility preferences during the course of fertility decline. Because of heterogeneity in fertility change within countries, we focus on trends by sub-national regions and social class stratification, since understanding the differences in fertility according to socioeconomic status and how these differences evolved over the fertility transition period is of great importance in understanding fertility decline. Conclusions about fertility change have been based on mostly cross-national comparisons based on national data but there has been less attention to sub national differentiation. It has been observed that enormous heterogeneity exists in Kenya, where the wealthy and higher educated people have fertility desires close to replacement level, regardless of religion, while poor, uneducated people, particularly those in Muslim communities, have virtually uncontrolled fertility (This study was guided byAccording tou is unwanted fertility (which can simply be expressed as Fo – Fw), andWhere Fn is total natural fertility and Ip is the degree of fertility preference implementation index. Ip ranges from 0 to 1. With full preference implementation, Ip = 1 (which implies that Fu = 0 and F = Fw) and Ip is 0 with no preference implementation .n is not the same as in total fecundity used in the Bongaarts proximate determinants but is taken to mean fertility level achieved in absence of contraception The above formulation shows that a change in wanted or natural fertility to the observed fertility decline depends on the average level of implementation index. Similarly, the fertility effect from a given change in the index of implementation depends on the average between natural and wanted fertility  collected between 1988 and 2015 for Kenya, Rwanda, Uganda and Tanzania from women of reproductive age (15 to 49 years). DHS data is highly comparable across countries and has been shown to be of high quality. DHS data often computes first the total fertility rates based on data for the three years preceding the survey for age group 15–49 expressed per woman and second the total wanted fertility rates for the three years preceding the survey for age group 15–49 expressed per woman. Total wanted fertility rate is calculated in the same way as the total fertility rate, but only including wanted births. However, an alternative method is also to utilize the formula for the proportion of women age 40–49 in union who want no more births. According to Bongaarts, Fw is the proportion of women who want more children and Wm (40–49) is the proportion of women in union aged from 40 to 49 who want no more births. Bongaarts, however, was cognizant of the fact that indeed there are limitations especially with the information on wanted fertility. The key limitations highlighted here are rationalization, involuntary and voluntary fertility limitation.Where Fp between 1998 and 2005 perhaps coinciding with the period of fertility decline. Contraceptive use also stagnated or stalled. Rwanda has experienced a rapid rise in Ip since 2005, which is consistent with the beginning of strength of family planning programs in the country. Uganda on the other hand is rising at a slower pace than its counterparts.This section presents the effect of both wanted fertility and preference implementation on fertility decline in the four countries based on the decomposition procedure. The analysis is restricted to the periods between 2000 and 2014. Positive values of wanted fertility, natural fertility and implementation index indicate contribution to decline while the negative values indicate tendency to increase fertility.Except for Uganda, in all the other countries, the ability to implement fertility preference contributed to nearly twofold decline in fertility. The results complement the assertion byp and contribution of wanted fertility and preference implementation to fertility change by region, presents variations in fertility preference implementation index and the contribution of wanted fertility and preference implementation to fertility decline at sub-national levels for the four countries. In Kenya, most regions have Ip above 0.85 except for North Eastern region with negative low value. Regions in Rwanda have Ip ranging 0.75 in Western region to 0.96 in Northern region. Tanzania has very wide variation in Ip, with the lowest (0.44) in Pemba south to the highest (1.00) in Kilimanjaro region. However, some regions experience unique results . Such results might arise due to measurement errors in wanted fertility rate or issues in the measurement of contraceptive use that is utilized in the estimation of natural fertility. However, the low fertility of Mtwara region did not arise from high contraceptive use but from long post-partum amenorrhea. Thus, it may be the case of the effects of other proximate determinants may also influence the estimation of Ip. The largest declines in fertility within the decade occurred in Regions of Rwanda (North (2.7-fold), West (2-fold) and East (1.9-fold)). This was followed by Kagera (1.8-fold) and Mbeya (1.7-fold) regions of Tanzania. The next highest decline occurred in South region of Rwanda, Northern of Uganda and Eastern regions Kenya followed by Mtwara region of Tanzania, Nyanza and Rift valley regions of Kenya. Most regions of Tanzania had minimal or no decline at all over the period assessed. The Nairobi region of Kenya, Morogoro and Pemba North regions of Tanzania equally registered no significant change in fertility . What isp was the greatest contribution to fertility decline for most of the regions. The contribution of Ip was highest in South and Kigali regions in Rwanda, Pwani and Lindi regions of Tanzania, where Ip contributed to over a twofold decline in fertility. Notwithstanding this, the Ip contributed least to fertility decline mostly in regions of Tanzania. These regions include Manyara, Rukwa and Iringa. Bongaarts’ formulation equation rule is that the index of implementation of fertility can only fall between zero and one. However, this has not been the case in some segments of the populations within this study. It calls for a reflection on some regions that registered erratic indices. These regions actually posted indices values either below zero or above unity. The regions were Pemba South of Tanzania North Eastern region of Kenya at −0.09-fold, while the indices beyond unity values were the Pwani, Mtwara, Lindi and Dar es Salaam in Tanzania, which therefore calls for revisiting the formula to include the effect of other related proximate determinants of fertility decline, acting as a normalization of sort.In Kenya, Rwanda and Uganda, the ability to implement fertility desires Ip increased with increase in level of education. Ip was also higher in urban areas compared to rural areas in all regions. In Kenya and Rwanda, fertility decline was higher in rural areas and among women with lower or no education. Tanzania posted mixed and unique results. In general, the degree of fertility preference implementation index was higher among women of urban resident as well as among women with higher education. In Rwanda, both changes in wanted fertility and ability to implement fertility desires contributed to fertility decline across all the sub groups. However, in Kenya it was the ability to implement fertility desires that made major contribution to fertility decline across most of the subgroups. Uganda had almost similar results to Rwanda except that the contribution was lower. Similarly, Tanzania had similar results to Kenya, though with lower contribution values.p). The same is true for Uganda; however, Tanzania posted mixed results by different groups.In this study, we focused on the sub-national regions (provinces) of East African countries, analyzing trends in fertility preferences among women and the extent to which these women have been able to implement these fertility preferences during the course of declining fertility in these countries. The main reason was that fertility change in one country may have implications in the neighboring country because neighboring regions share common language and cultural traits that permit shared flow of ideas and eventual spread of new models of behavior. In Kenya and Rwanda, the most important contributor to fertility decline by region, place of residence and socio-economic groups is the ability to implement fertility desires . Access to the dataset requires registration and is granted to those that wish to use the data for legitimate research purposes. A guide for how to apply for dataset access is available at:https://dhsprogram.com/data/Access-Instructions.cfm. The DHS datasets used in the present study are listed inThe datasets analyzed during the current study are available in the MEASURE DHS repository  decline in wanted fertility, 2) increase in preference implementation and 3) change in natural fertility. Results show that the rise in the implementation index is the main driver of fertility decline. Although the study does not directly assess the impact of family planning programs, the authors note that these programs aim to help women in implementing their reproductive preferences and disseminate information about the benefits of contraception and smaller families.The total fertility rate is represented by three different abbreviations: TFR, F, and Fo. This is confusing and should be fixed. The first paragraph of the results section makes reference to Figure 2. This should be Table 2. The text is clearly written and the analysis is technically competent. Two minor edits:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Demography, Fertility, Family planningI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


We demonstrate a partially degenerated intermodal four-wave mixing (FWM) process realized in a few-mode nonlinear optical fiber, leading to the effective generation of visible red and blue light from 532 nm sub-nanosecond pulses. We present a self-seeded FWM configuration with a signal beam obtained in the additional section of the same type of fiber that ensures perfect matching between the seed and the Stokes beams. Over 40% of the wavelength conversion efficiency in the FWM process was obtained using a fiber length shorter than 1 m. H. Stolen [Light parametric conversion through a third-order nonlinear four-wave mixing (FWM) in optical fibers is a commonly utilized technique for frequency conversion. This process is described by χ. Stolen . The FWM. Stolen ,3 and mu. Stolen ,5,6,7,8,. Stolen ,10,11. A. Stolen  is the u. Stolen  and the . Stolen . Such co. Stolen . The pha. Stolen ,16, bire. Stolen ,18, or b. Stolen . One of . Stolen . In our In our work, we analyze the Stokes and anti-Stokes beams’ power and FWM process efficiency versus the power of a pump beam in both cases, with and without using an additional signal beam.01 mode at 1292 nm, for LP11 at 1290 nm, and the LP02 and LP21 modes at 1306 nm. As presented in 02/LP01 modes at 616.8 nm/467.7 nm or LP12/LP01 modes at 642.2 nm/454.1 nm, respectively.The IM-FWM was realized in a graded-index optical fiber with a core diameter of 27 μm and a maximum refractive index difference of 0.008. Such a fiber supports 42 mode groups at a wavelength of 532 nm, which may be treated as a multimode fiber. The effective refractive index and group refractive index values were calculated and are plotted in The detailed experimental setup is shown in Even though the power of the Stokes and anti-Stokes beams should increase along with the fiber length, the recorded beams’ powers are limited, predominantly by the Raman effect and due to temporal walk-off (insignificantly at such short propagation distance and sub-nanosecond pulse duration) . As the 01-like modal field profile in the linear propagation regime, both in the near- and far-field. Then, for the high-power pump beam propagation, the fiber position was adjusted again to maxim-ize the power of the Stokes/anti-Stokes beams at the output, however, continuously monitoring the modal field profile to keep the propagation of the pump beam pre-dominantly in the fundamental mode.The FUT was kept straight to avoid external strain and stress-induced birefrin-gence. Thus, we can assume that the polarization of light is preserved during propaga-tion. The fiber was pumped at 532 nm using a free-space beam coupling and mi-cro-translation stage supported by a piezoelectric drive to precisely adjust the position of the fiber. For the purpose of mainly fundamental mode excitation for the pump beam, the position of the FUT was carefully adjusted to get a clean LP01 mode. To fulfil this condition, we verified the quality of a Gaussian profile of a beam with a beam profiler (BP209-VIS) and CCD camera that visualizes the light intensity distribution. To ensure high coupling efficiency, we used a 3-Axis NanoMax Flexure Stage to generate a clean LP01-like green-color modal field both in the near and in the far-field. Using a microscope objective of NA = 0.25, a beam was focused to a spot of a waist diameter 2w0 = 2.8 μm and precisely coupled to a fiber of 0.8 m length. The fiber was kept straight to avoid additional strain and prevent higher-order modes’ excitation, keeping predominantly the fundamental LP01 mode. In order to prevent damage to the end of the optical fiber, the average power of a pump beam was limited to 10 mW (a peak pulse power ~20 kW). Depending on the measurement, the output beam was routed between the power meter/optical spectrum analyzer (OSA) and a prism (or set of two prisms) combined with a CCD camera. Such a switchable configuration was utilized for data acquisition concerning the output power, spectral characteristics and modal profiles of the pump, Stokes and anti-Stokes beams.Among other possibilities, intermodal phase-matching in FUT can be achieved when the 532 nm pump propagates in the fundamental LPIn the second part of our work, we investigated the seeded FWM process using an additional low power signal beam of a wavelength matched to the Stokes beam that was co-coupled and co-propagated with an intense pump beam. Such an approach minimizes the impact of stimulated Raman scattering on the optical output field. With an additional seed beam, the pump beam is more efficiently converted to the Stokes and anti-Stokes frequencies, as the signal beam is gained from the beginning of the fi-ber, compared to a case with spontaneous generation Stokes/anti-Stokes generation from vacuum noise. As a result, the FWM conversion efficiency significantly increases.2 method [Despite the advantages, the approach with the additional seed beam also has its drawbacks. One is the necessity to use a laser source with a wavelength adjusted to the Stokes or the anti-Stokes waves, which is not a significant problem in the infrared (IR) pumping regime. In a visible frequency range, of which the most common and easily obtainable wavelength is 532 nm (the second harmonic of Nd:YAG laser), the wavelengths of the Stokes and anti-Stokes beams are both in the visible red and blue light range. Thus, matching with the wavelength of a signal beam requires an appropriate, preferably tunable, light source of coherent light. Moreover, a proper modal field distribution that corresponds to the appropriate order mode in the optical fiber should also be ensured. The proper evaluation of the HOM modes number for the generated Stokes/anti-Stokes beams can be realized, using, for example, the S2 method ,22. The 2 method ,24. Ther2 method  concerni2 method ,27, wherThe experimental setup for the self-seeded IM-FWM process is presented in The red-wavelength Stokes beam generated in one section of FUT was utilized as a signal beam and coupled with an intense pump beam (532 nm) to the second segment of the same type of fiber. Because both beams are pulsed, it is also crucial to match both pulses in the time domain. For that purpose, a pump beam coupled to the second segment of the FUT was routed through a delay line that compensates for the additional optical path taken by a signal beam .In the first part of the experiment, we characterized the optical field  at the fiber’s output versus the power of a power pump. The generation of Stokes and anti-Stokes waves of wavelengths of 617 nm and 470 nm, respectively, is observed for the average pump power of about 5.0 mW. When the pump is not appropriately aligned, the FWM efficiency is significantly lower, and other spectral components or a mix of higher-order modal profiles can be visible for the Stokes/anti-Stokes waves. For the minimization of the optical power of a pump beam that results in the efficient generation of FWM spectral components, the position of the fiber was precisely re-adjusted with the use of piezoelectric actuators: first, to keep a clean Gaussian-like green spot of a pump Beam, visible in the far-field, as well as to maximize the power of the Stokes and anti-Stokes spectral components.The spectra of the optical output field as a function of the pump’s power are presented in 01 mode while the FWM generated waves propagate in one of the HOM, which confirms an inter-modal character of the observed nonlinear process.Almost 20% conversion efficiency was obtained in a single pump beam configuration. It was calculated as a percentage ratio of the Stokes and anti-Stokes waves powers at the output  to the pump beam power coupled to the optical fiber. However, based on the FWM efficiency versus input power characteristic presented in The performed self-seeded IM-FWM process consists of two parts. As presented in 12 that corresponds precisely to the mode number of a Stokes beam, generat-ed spontaneously in the second stage of FUT. The output field of a signal beam that propagates without an intense pump beam (inset of 12) has not been achieved. Notwithstanding, the wavelength of a signal beam corresponds precisely to the Stokes beam and can be effectively gained along with the propagation in the second fiber. We think improvements in the signal beam coupling that would lead to the selective exci-tation of only the LP12 mode would enable even greater efficiency of the self-seeded IM-FWM process.As presented in the inset of The presented approach is universal and provides good compatibility between the Stokes and the signal beams in the self-seeded IM-FWM process. It can also be applied to other types of optical fibers without introducing additional modifications to the experimental system. Due to the same modal profiles of the pump and signal beams, the efficiency of a seeded IM-FWM process would be significantly increased. Experimental results of the self-seeded IM-FWM are presented in FMF fibers have more freedom to design the fiber parameters than single-mode ones. The presented self-seeded IM-FWM configuration and the appropriate nonlinear optical fiber can be potentially applied in the construction of nonlinear optical frequency converters. Proper system design and utilization of the all-fiber technology could also contribute to more compact dimensions of the final device and increase the system’s stability. By managing the refractive index profile, we can directly affect the values of propagation constants for a given guided mode and meet the phase-matching condition for the desired wavelengths.


The skin epidermis is a stratified multilayered epithelium that provides a life-sustaining protective and defensive barrier for our body. The barrier machinery is established and maintained through a tightly regulated keratinocyte differentiation program. Under normal conditions, the basal layer keratinocytes undergo active proliferation and migration upward, differentiating into the suprabasal layer cells. Perturbation of the epidermal differentiation program often results in skin barrier defects and inflammatory skin disorders. The protein kinase mechanistic target of rapamycin (mTOR) is the central hub of cell growth, metabolism and nutrient signaling. Over the past several years, we and others using transgenic mouse models have unraveled that mTOR signaling is critical for epidermal differentiation and barrier formation. On the other hand, there is increasing evidence that disturbed activation of mTOR signaling is significantly implicated in the development of various skin diseases. In this review, we focus on the formation of skin barrier and discuss the current understanding on how mTOR signaling networks, including upstream inputs, kinases and downstream effectors, regulate epidermal differentiation and skin barrier formation. We hope this review will help us better understand the metabolic signaling in the epidermis, which may open new vistas for epidermal barrier defect-associated disease therapy.The skin epidermis, with its capacity for lifelong self-renewal and rapid repairing response upon injury, must maintain an active status in metabolism. Mechanistic target of rapamycin (mTOR) signaling is a central controller of cellular growth and metabolism that coordinates diverse physiological and pathological processes in a variety of tissues and organs. Recent evidence with genetic mouse models highlights an essential role of the mTOR signaling network in epidermal morphogenesis and barrier formation. In this review, we focus on the recent advances in understanding how mTOR signaling networks, including upstream inputs, kinases and downstream effectors, regulate epidermal morphogenesis and skin barrier formation. Understanding the details of the metabolic signaling will be critical for the development of novel pharmacological approaches to promote skin barrier regeneration and to treat epidermal barrier defect-associated diseases. The skin is composed of three layers: the epidermis, dermis and the deeper subcutaneous tissue. The epidermis is a stratified squamous epithelium that serves as the primary interface between the body and the outside environment, providing a life-sustaining permeability barrier by preventing water and heat loss and protecting the body from various environmental insults. In response to danger signals or injury, the epidermis needs continuous self-renewal throughout life to maintain the barrier function . DisruptStudies with gene-targeted mutations in mice have unraveled important regulatory networks over the past decades, including signaling pathways and transcriptional factors that control epidermal morphogenesis and the barrier formation . Among tThe epidermis is developed from a single-layered epithelium, originated from the surface of the ectoderm at around embryonic day 9.5 (E9.5) in mice. Signals from dermal mesenchymal cells together with intracellular master regulators, such as p63, direct the epithelial cells towards differentiation and stratification to yield the spinous, granular and cornified layers. A fully functional epidermis is nearly completed by E17.5 .In postnatal life, the epidermis continuously rejuvenates itself to replenish the shed corneocytes, dying or damaged cells upon injury. To achieve this, the proliferating keratinocyte stem/progenitor cells in the basal layer lose their mitotic activity, migrate upwards and differentiate into spinous layers. After detaching from the basement membrane (BM), the cells start the terminal differentiation program, known as cornification or keratinization. Dynamic cell morphological changes and sequential expression of specific markers, including keratins and barrier proteins, are characterized by the terminal differentiation program. Ultimately, the flattened squames form the outmost cornified layer, which provides the major protective barrier function of the skin  Figure . The homThe mTOR protein is atypical serine/threonine kinase and was firstly identified through genetic screening as a nutrient sensor, regulating cell growth and proliferation ,13. SubsBy interacting with various regulatory proteins, mTOR forms two complexes with distinct structure and function, referred to as mTOR complex 1 (mTORC1) and mTORC2 . mTORC1 mTORC1 regulates cell growth and metabolism by responding to multiple upstream inputs such as growth factors, nutrients, energy and oxygen stress ,16 Figu. In respIn contrast to mTORC1, the specific molecular mechanism by which mTORC2 is regulated is less understood. However, it is known that in response to growth factor stimuli, mTORC2 is primarily controlled by the PI3K pathway. PIP3 can directly bind the unique mTORC2 component mSIN1 to alleviate mSIN1-modulated auto-inhibition ,23. InsuUpon activation, mTORC1 enhances primarily anabolic metabolism, including protein, nucleotide and lipid biosynthesis and glucose metabolism and decreases catabolic metabolism including suppression autophagy by phosphorylating several related effectors . RibosomWhile the cellular processes controlled by mTORC2 signaling are less understood than mTORC1, accumulating evidence shows that mTORC2 contributes to modulating cell migration, metabolic processes, cell survival and proliferation . When mTAlthough the general role and function of mTOR signaling at the cellular level is relatively well characterized, the physiological function of mTOR signaling in specific tissues and organs is still not well defined. Given the fundamental role of mTOR signaling in cell growth and metabolism, constitutive knockout of the core component often results in embryonic lethality ,44. CondIGF-1 and insulin represent the major growth factors that can promote mTOR activity and regulate tissue growth and development. They mediate their effects by binding to IGF receptors  or insulin receptor (IR), respectively. Studies with their deficient mice showed that IR is not required for epidermal development, whereas mice deficient for IGF-1 or IGF-1R display severe epidermal hypoplasia ,88. In lDiGiovanni J et al. generated a mouse model with enforced expression of IGF-1 in epidermis. In contrast to IGF-1 deletion, these mice display a wrinkled and thickened epidermis. In addition, persistent overexpression of IGF-1 leads to epidermal hyperplasia, hyperkeratosis and cutaneous tumorigenesis, identifying the high concentration of IGF-1 resulting in the malignant behaviour of skin papillomas . With CRGlucose is the primary source of cellular energy and glucose deprivation can suppress mTORC1 signaling through the major energy sensor AMP-activated protein kinase (AMPK) . GlucoseSlc3a2), a heterodimeric amino acid transporter positively modulates mTOR activity in vitro [Slc3a2 deletion in mouse epidermis results in aberrant skin homeostasis, including delayed hair growth, wound healing and impaired keratinocyte proliferation [mTORC1 activation is also regulated by amino acid availability, especially leucine and arginine . Howeverin vitro . Boulterferation , indicatHyperactivated PI3K/AKT/mTOR molecular cascade has been extensively described in skin cancers and hyperplasia diseases ,93. RecePI3K-dependent kinase-1 (PDK1) is a serine/threonine, transmitting PI3K-dependent signaling to downstream kinases, such as AKT, in response to insulin/IGF-1 . During Mtor, Raptor and Rictor, respectively [Mtor or Raptor ablation exhibit severe defects in epidermal morphogenesis and skin barrier formation during embryogenesis, resulting in neonatal lethality. Basal cell growth and proliferation are likely affected upon on mTORC1 inactivation [We and others recently demonstrated that mTOR complexes, as the core elements of the signaling cascade, are essential for embryonic epidermal development and skin barrier formation ,62. We gectively . Mice witivation ,62. Additivation . Besidestivation .Rictor deletion can survive but exhibit epidermal hypoplasia and an abnormal differentiation process [In contrast, mice with epidermis-specific  process ,64. Mech process ,61,80. O process . IntriguThe activity of PI3K/AKT/mTOR signaling pathway can be induced by depleting their negative regulators genetically. PTEN inhibits mTOR activity by dephosphorylating PIP3 back to PIP2  Figure . In factThrough their downstream effectors, activated mTOR signaling regulates a wide range of biological processes . In thismTORC1 promotes protein synthesis mainly by activating S6K and inhibiting 4E-BP . Phenoty4E-BP-dependent translation regulates many physiological and pathological processes associated with metabolism. Two independent studies showed that 4E-BP1/2 double knockout mice are more sensitive to diet-induced obesity, indicating resistance to insulin when compared with wild-type mice ,74. ConsAtg7, encoding a key serine/threonine kinase required for autophagosome formation, has no obvious effects on epidermal differentiation and skin barrier function formation [Atg7 likely suppresses carcinogen-induced protumorigenic inflammatory microenvironment and tumorigenesis of the epithelium [Atg7 have impaired wound closure [Autophagy is characterized by formation of double-layer-membrane autophagosomes, which then translocate to lysosomes, where their cargo is degraded to prevent cell damage and provide energy for cells under conditions of starvation . mTORC1 ormation . Howeverithelium . Recentl closure . HoweverPKC, a family of protein kinase, can be phosphorylated by active mTORC2. Inactivation of mTORC2 results in reduced both PKC protein and phosphorylation levels . To dateTranscription factor forkhead-box protein O1 (FoxO1) is regulated by IGF-1 via AKT-dependent phosphorylation, thereby resulting in nuclear exclusion and proteasomal degradation of FoxO1 . p63 genActivated mTORC1 can promote the translation of transcriptional factors such as Hypoxia-inducible factor-1 alpha (HIF-1α). HIF-1α is a ubiquitously distributed transcription factor involved in multi cellular and physiological processes . It is wMYC is a key transcription factor positively regulated by mTOR. Recent studies underline its importance in skin homeostasis and several mouse models have been generated to explore its effects on epidermis . In miceThe epidermis is an active site of lipid metabolism. During keratinocyte differentiation, epidermal lipids are synthesized and extruded into extracellular space to form a lipid matrix, composing a protective epidermal barrier together with cornified keratinocytes in the cornified layer. Disturbed lipid metabolism and altered lipid composition often lead to skin barrier defects . EmerginActivated AKT triggers expression of a number of lipogenic genes . InteresEpidermal barrier function is established and maintained through a complex and highly coordinated stratification program. Studies over recent years have identified mTOR signaling networks, including IGF-1/IGF-1R, PI3K/AKT and mTOR complexes as critical mediators in epidermal barrier formation and homeostasis. These signaling cascades regulate protein and lipid synthesis, transcription, and cytoskeleton reorganization, which in turn impact keratinocyte proliferation, cell polarity, and the stratification program. Their genetic inhibition abrogates and impairs epidermal morphogenesis and skin barrier formation. Increased mTOR activity, on the other hand, may result in skin cancer and other hyperplasia skin diseases. As a result, it appears that a delicate balance of mTOR signaling activity is critical for skin health and their dysfunction or dysregulation might be implicated in the pathogenesis of a variety of skin diseases.In recent years, a wealth of knowledge has accumulated on the understanding of the nutrition signaling network in regulating cell growth and metabolism . New regLastly, beyond epidermal keratinocytes, it is equally important to determine the specific roles of the mTOR signaling network in the other skin cell types, including but not limited to immune cells, fibroblasts or adipocytes. Given the hyperactivation of mTOR signaling in many pathological conditions, such as cancer and psoriasis, their roles in pathogenesis of skin diseases are now well recognized. A better understanding of the complex network of molecular players in skin pathologies will ultimately be important for exploring strategies for medical therapy.