Adult T-cell leukemia-lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) infection that often occurs in HTLV-1-endemic areas, such as Japan, the Caribbean islands, Central and South America, Intertropical Africa, and the Middle East. In Japan, the nationwide estimation of the number of HTLV-1 carriers was at least 1.08 million in 2006–2007. Furthermore, in 2016, the nationwide annual incidence of newly infected with HTLV-1 was first estimated to be 3.8 per 100,000 person-years based on the age-specific seroconversion rates of blood donors in almost all areas of Japan. The incidence rate was three times higher in women than in men, and it was estimated that at least 4,000 new HTLV-1 infections occur yearly among adolescents and adults in Japan. As well known that HTLV-1 infection alone is not a sufficient condition for ATL to develop. To date, a variety of molecular abnormalities and host susceptibilities have been reported as candidate progression factors for the development of ATL in HTLV-1-carriers. In particular, quite recently in Japan, a variety of immunosuppressive conditions have been recognized as the most important host susceptibilities associated with the development of ATL from HTLV-1-carrier status. Furthermore, in 2013–2016 in Japan, a new nationwide epidemiological study of ATL was conducted targeting patients newly diagnosed with ATL in 2010–2011, from which the most current knowledge about the epidemiological characteristics of Japanese patients with ATL was updated as follows: (1) continuing regional unevenness of the distribution of people with HTLV-1, (2) further aging, with the mean age at diagnosis being 67.5 years, (3) declining M/F ratio, (4) increase of the lymphoma subtype, (5) sex differences in subtype distribution, (6) age differences in subtype distribution, and (7) comorbidity condition. In particular, 32.2% of ATL patients had comorbid malignancies other than ATL. However, the number of deaths due to ATL in Japan has been relatively stable, at around 1,000 patients annually, without significant decline from 1999 to 2017. Because the current epidemiological evidence about HTLV-1 and ATL is insufficient, further epidemiological studies are required. ATL was first discovered as a distinct clinical entity  in Japan in 1977 , followein vitro , and (4)in vitro .In the current World Health Organization classification of tumors of hematopoietic and lymphoid tissues , ATL is The diagnostic criteria for ATL and the classification of four clinical subtypes  were proposed for the first time by the Japanese Lymphoma Study Group in the early 1990s  based onThe majority of individuals with HTLV-1 infection and patients with ATL have been reported in Japan, the Caribbean islands, Central and South America, Central and South Africa, Aboriginal regions in Central Australia, parts of the Middle East and Melanesia, parts of Europe, and other small regions . MoreoveThe earlier version of this review summarized the literature published up to 2012 in Japan . This reSince the discovery of HTLV-1 in Japan in the early 1980s, the overall nationwide number of people with HTLV-1 infection has been estimated several times by multiplying the sex- and age-specific seroprevalence of HTLV-1 screening test results among first-time blood donors by age- and sex-specific demographic data across the whole of Japan in a specific year.The first estimation of the age-specific HTLV-1 prevalence in Japan was done in 1983 by using HTLV-1 screening test results from approximately 3,000 blood donors aged 40–64 years in selected areas in Japan  and idenThe most recent age-specific HTLV-1 prevalence and estimated number of HTLV-1-infected people in whole Japan were reported by Although knowledge about the pattern and trends of HTLV-1 seroprevalence is important for public health, information about the incidence of new HTLV infection is also important and essential for planning prevention efforts .p < 0.0001). The incidence was also higher in earlier birth cohorts, probably because of differences in lengths of the sexual transmission. After extrapolating the incidence rate to the Japanese general population, it was estimated that at least 4,000 new HTLV-1 infections will occur yearly among adolescents and adults in Japan.After the discovery of milk-bone transmission of HTLV-1 in Japan in the early 1980s, there were also several reports of heterosexual transmission of HTLV-1 in Japanese couples. In Japan, several government measures and actions have been implemented in place to prevent new HTLV-1 infection, including HTLV-1 antibody screening for pregnant women, the recommendation for mothers with positive results to refrain from breastfeeding, and HTLV-1 antibody screening of all donated blood since 1986. However, there have been no strategies to prevent new HTLV-1 transmission among adolescents and adults. After publication of Satake’s report, HTLV-1 infection is now recognized as a sexually transmitted infection (STI) in Japan. Other measures to control new HTLV-1 infection by treating HTLV-1 as a type of STI are expected in Japan. Quite recently, the Japanese Ministry of Health, Labour and Welfare has started to act to decrease HTLV-1 infection in Japan . FurtherMost published studies have estimated the incidence of ATL in a group by simply merging the number of cases of ATL with the number of people in a population, such as demographic statistics, blood donors positive for HTLV-1, or an existing group of HTLV-1 carriers. However, few prospective studies have investigated the incidence of ATL in asymptomatic people with HTLV-1 infection.Helicobacter pylori for gastric cancer, hepatitis B virus and hepatitis C virus for liver cancer, human papillomavirus for cervical cancer, Epstein–Barr virus for lymphoma, human herpes virus type 8 for Kaposi’s sarcoma, and HTLV-1 for ATL. According to the GLOBOCAN database of the IARC, the worldwide estimated number of newly diagnosed ATL cases in the years 2002, 2008, and 2012 were 3,340  has regularly reported the global burden of infection-associated cancers, such as re 3,340 , 2,100  in 2000 . This prFrom early 1980s to early 2000s in Japan, several epidemiological studies estimated that the annual incidence rate of ATL from HTLV-1 carriers among general population in HTLV-1 endemic areas, and reported that the rate was approximately from 60 to 130 per 100,000 HTLV-1 carriers with male predominant . FurtherIt has been well recognized that HTLV-1-infection alone is not sufficient to develop ATL from HTLV-1-carrier status. Regarding risk factors for the development of ATL from HTLV-1-carriers, so many risk factors have been documented in literature such as aging , family Recently, several clinical genetic/epigenetic studies of asymptomatic HTLV-1 carriers and patients with ATL have been conducted to identify the molecular entity responsible for the development and the prognosis of ATL , 2018. SStrongyloides stercoralis are a high-risk group for developing ATL because of the clonal proliferation of HTLV-1-infected lymphocytes and high proviral load  system in dually infected carriers, which may be a precipitating factor for ATL. Furthermore, S. stercoralis infection was related to higher numbers of ATL-related deaths.HTLV-1 carriers with abnormal immune systems are well-known to be at high risk of developing ATL. Several previous studies reported that HTLV-1 carriers co-infected with ral load . Satoh ePatients with rheumatoid arthritis (RA), Sjögren syndrome, and organ transplantation use strong immune suppressive therapies for long durations. Such strongly immunosuppressive states are also considered as high risk for the development of ATL from such patients with HTLV-1. There have been a non-negligible number of clinical reports of ATL developing among HTLV-1-positive patients in immunosuppressive status .Furthermore, recently, a small but not negligible number of cases of the development of ATL from HTLV-1-positive recipients undergoing strong immunosuppressive treatment after living-donor renal and liver transplantation have been reported from not only Japan but also other countries . One of Definite risk factors for the development of ATL from HTLV-1-carrier status have been well documented in many studies and I have also summarized regarding risk factors in the previous review article . As for The earlier version of this review summarized the literature published up to 2012 . At that(1)Sustained regional ubiquity: Of the 922 evaluable patients with ATL diagnosed in 2010–2011, 67.5% were still diagnosed in the Kyushu–Okinawa areas, where HTLV-1 infection is most prevalent, followed by 9.5% in Kinki and 6.4% in Kanto, which are the most populated areas of Japan and have the largest population inflow from other areas, including Kyushu–Okinawa.(2)Further aging: The mean age at diagnosis of ATL was 67.5 years in 2010–2011, which was older than the corresponding figures of 58.9 years in 1992–1993 and 56.9 years in 1984–1985 (984–1985 . The mai(3)Declining M/F ratio: The male/female ratio of numbers of ATL patients in the 1980s was 1.4; therefore, at that time, ATL was considered as a male-predominant disease. However, after the 1990s, the male-female ratio declined toward 1. Nevertheless, by considering the male/female ratio of Japan’s general population in 2010, which was 0.95 [was 0.95 , ATL is (4)Increase of lymphoma subtype: Among all registered patients, the proportion of lymphoma subtype increased from 21.0% in 1984–1985 to 21.4% in 1992–1993 and 25.7% in 2010–2011 (010–2011 .(5)Difference in subtype distribution by sex: There was a significant sex difference in the distribution of subtypes: the frequency of chronic type was higher in female compared with male patients; conversely, other types were more frequent in male than female patients. In particular, the lymphoma type was more frequent in male patients (60%).(6)Difference in subtype distribution by age: There was a significant age difference at diagnosis by subtype. Patients with chronic subtypes tended to be diagnosed at a younger age, whereas those with lymphoma subtypes were diagnosed at an older age.(7)Comorbid malignancies other than ATL: Of all the patients with ATL diagnosed in 2010–2011, 32.2% had one or more comorbid diseases, including (in descending frequency order) hematological malignancies, non-hematological malignancies, infectious diseases , neurologic diseases, autoimmune diseases, and other diseases (diseases . Among cFor HIV-infected patients, although the development of AIDS-defining malignancies has been well known, the development of non-AIDS-defining malignancies becomes another growing problem, recently . By consA possible reason for no significant association between HTLV-1 infection and any cancers other than ATL in the report of Schierhout et al. is that it might have been suffered to several limitations such that a half of evaluated data were all causes mortality for any cancers but not taken into account incidence rate of each cancer. Therefore, any cancers with a high cure rate are not reflected to the results. Furthermore, in their report, information on viral marker status such as HTLV-1 proviral load is lacked; therefore, it has been uncertain that the adverse health effect was definitely due to HTLV-1-infection.Unfortunately, information about the global burden of deaths from ATL is not available from any data source. Instead, information about deaths from ATL in Japan has been made available from “e-Stat”  at regulAlthough many studies of epidemiological evidence about ATL and HTLV-1 carriers from Japan have been published, reliable information about the annual incidence of ATL in the setting of longitudinal prospective studies has still been limited. Existing published data regarding predisposing factors and genetic abnormalities are still insufficient to explain the characteristics of ATL oncogenesis. Additional unknown factors may be involved in the development of ATL from asymptomatic HTLV-1 carrier status. Further well-designed epidemiological studies are needed to fully understand the relationship between HTLV-1 infection and ATL.In summary, in Japan, at least 1.1 million individuals are still infected with HTLV-1 , at leasMI designed and wrote the manuscript.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.


The most important drivers of HTLV-1-mediated transformation and proliferation are the tax and hbz viral genes. Tax, transcribed from the plus-sense or genome strand, is essential for de novo infection and cellular immortalization. Hbz, transcribed from the minus-strand, supports proliferation and survival of infected cells in both its protein and mRNA forms. Abrogating the function or expression of tax and/or hbz by genome editing and mutagenic double-strand break repair may disable HTLV-1-infected cell growth/survival and prevent immune modulatory effects and ultimately HTLV-1-associated disease. In addition, the HTLV-1 viral genome is highly conserved with remarkable sequence homogeneity, both within the same host and even among different HTLV isolates. This offers more focused guide RNA targeting. In addition, there are several well-established animal models for studying HTLV-1 infection in vivo as well as cell immortalization in vitro. Therefore, studies with HTLV-1 may provide a better basis to assess and advance in vivo genome editing against retroviral infections.CRISPR editing of retroviral proviruses has been limited to HIV-1. We propose human T-cell leukemia virus type 1 (HTLV-1) as an excellent model to advance CRISPR/Cas9 genome editing technologies against actively expressing and latent retroviral proviruses. HTLV-1 is a tumorigenic human retrovirus responsible for the development of both leukemia/lymphoma (ATL) and a neurological disease (HAM/TSP). The virus immortalizes and persists in CD4 Despitelization , while Hlization . Based olization . Severallization , and thegag, pro, pol, and env . The LTRs are exact duplicates which consist of a U3, R, and U5 region. These regions facilitate viral integration into the host genome and contain promoter elements, polyadenylation signal sequences, and other regulatory sequences necessary for proper viral transcription.HTLV-1 is a complex deltaretrovirus that contains the common retroviral structural and enzymatic genes;  and env Figure 1HTLV-1 infects an estimated 5–10 million individuals and is found in areas of endemic infection worldwide . Regions+ T cells, CD8+ T cells, B cells, dendritic cells, monocytes, and macrophages , an aggressive and fatal disease of CD4 T cells , and HTL T cells . The inc T cells . Disease T cells , and is  T cells . A recen T cells . Overallelopment . Therapi+ T cells. Tax also causes deregulation of the cell cycle by silencing cellular checkpoints that guard against DNA structural damage and abnormal chromosomal segregation, thus leading to the accumulation of mutations in HTLV-1 infected cells  and various cellular signaling pathways such as the CREB, NF-κB, and AP-1 pathways . Aberraned cells . Many ofed cells . Hbz alsed cells . Somewhaed cells . This acin vivo in approximately 103 to 106 clones of T cells that survive for the lifetime of the infected host  is transcribed in intense, intermittent bursts triggered by cellular stress and modulated by hypoxia and glycolysis /Cas9 genome editing is a relatively new technology that utilizes a guide RNA (gRNA) to target a site-specific DNA double strand break (DSB) by the Cas9 endonuclease. In human cells, DSBs are largely repaired by the error prone non-homologous end-joining pathway, which typically introduces insertions and deletions at the repair junction. Error-prone DSB repair can alter the reading frame of genes, disrupt DNA regulatory motifs, or disrupt the structures of encoded RNA elements. CRISPR is an innovative and powerful genome editing technology that has the potential for development as an HTLV-1 disease therapeutic strategy. In a 2013 report, zinc finger nucleases (ZFNs) that specifically recognized the HTLV-1 LTRs were utilized to disrupt LTR promoter function and inhibit the proliferation of HTLV-1-positive cell lines  would have the potential to effectively treat newly HTLV-1-infected individuals, asymptomatic viral carriers, and ATL and HAM/TSP patients. Also, given the nature of over-lapping reading frames between Hbz and the 3’LTR, and Tax and the 3’LTR, one can carefully design gRNAs that disrupt two viral elements at once.To date CRISPR editing of retroviral proviruses has been largely limited to HIV-1 . In contbz genes . Abrogatin vitro and in vivo models exist to study HTLV-1 immortalization, persistence, and tumorigenesis. Importantly, in vivo animal models could provide a system to eventually test delivery of CRISPR/Cas9 gene editing components in an animal model with known, effective, and measurable gRNA-viral targets. The different types of in vitro and in vivo HTLV-1 models are described below:Several in vitro using co-culture immortalization assays . The initiation of immortalization/transformation is apparent within 5–6 weeks following co-culture as detected by expansion of cells from the peripheral blood lymphocyte mixed cell population. This technique has been extremely useful for examining the requirement of specific gene products on HTLV-1-mediated immortalization in the absence of a functional immune system  mice inoculated subcutaneously with HTLV-1-infected cell lines  will develop tumors . The train vivo . The dec+ T-cells, and 3) development of lymphoproliferative disease. Importantly, disease in these mice can be induced using an infectious molecular HTLV-1 clone. Humanized mice can also be generated by intra-bone marrow injection of human CD133+ hematopoietic stem cells into NOG mice (Humanized immune system (HIS) mice model T-cell tropism and lymphoproliferative disease after HTLV-1 infection. The HIS mice are created by injecting human umbilical-cord stem cells into the livers of immunodeficient neonatal NSG mice, resulting in the development of human lymphocytes that appear phenotypically normal but cannot mount an adaptive immune response . HIS micNOG mice . Intrapein vivo study of the functional properties of HTLV-1 proteins  as well as cell immortalization in vitro. Studies with HTLV-1 may provide a better basis to assess and advance in vivo genome editing against retroviral infections.The progression from HTLV-1 infection to disease development  can take up to several decades. Thus, the integrated HTLV-1 proviral genome is clinically latent for long periods of time. The current lack of effective therapies for both ATL and HAM/TSP indicates a need for innovative clinical approaches. HTLV-1 Tax and Hbz are major drivers of transformation, proliferation, and immunological inducing effects. The viral LTRs are also integrally involved in these processes by driving viral transcription and directing integration into the host genome. Targeting any of these viral genes or elements with gene editing would unquestionably alter HTLV-1-infected cell growth/survival and prevent immune modulatory effects and ultimately HTLV-1-associated disease. HTLV-1 is an excellent model to advance genome editing technologies against actively expressing and latent retroviral proviruses. To date CRISPR editing of retroviral proviruses has been limited to HIV-1. In contrast, HTLV-1 offers more focused gRNA targeting because the viral genome is highly conserved with remarkable sequence homogeneity, both within the same host and even among different HTLV isolates. In addition, there are well-established animal models for studying HTLV-1 infection All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This research was supported by NIH R21AI142794 (PG and KY). Additional support was provided by The Ohio State University Comprehensive Cancer Center (AP).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.


Type-I transmembrane proteins represent a large group of 1,412 proteins in humans with a multitude of functions in cells and tissues. They are characterized by an extracellular, or luminal, N-terminus followed by a single transmembrane helix and a cytosolic C-terminus. The domain composition and structures of the extracellular and intercellular segments differ substantially amongst its members. Most of the type-I transmembrane proteins have roles in cell signaling processes, as ligands or receptors, and in cellular adhesion. The extracellular segment often determines specificity and can control signaling and adhesion. Here we focus on recent structural understanding on how the extracellular segments of several diverse type-I transmembrane proteins engage in interactions and can undergo conformational changes for their function. Interactions at the extracellular side by proteins on the same cell or between cells are enhanced by the transmembrane setting. Extracellular conformational domain rearrangement and structural changes within domains alter the properties of the proteins and are used to regulate signaling events. The combination of structural properties and interactions can support the formation of larger-order assemblies on the membrane surface that are important for cellular adhesion and intercellular signaling. In addition, transmembrane and membrane-associated proteins control cell-cell adhesion processes to form tissues and organs. Cell signaling and cell adhesion are dependent on protein-protein interactions at the extracellular side, protein conformations, and conformational changes play important roles in regulating these processes.Proteins at the cell surface play an important role in the formation and function of tissues. Transmembrane proteins can receive and transmit signals from the cell outside to the inside and www.uniprot.org)  fluid surfaces in which molecules can be inserted anisotropically or adsorbed reversibly . This deformability of membranes has been suggested to affect signaling processes. By alteration of surface to volume ratios in convex and concave protrusions, membranes may be able to control effective ligand to receptor concentrations  is broader than that of non-membrane bound proteins  is critical to proper developmental time course in haematopoiesis, melanogenesis, and spermatogenesis. The study of activation of KIT by SCF illustrates well-requirements for signaling in a crowded 2D environment, since these receptors undergo ligand induced oligomerization which exploits cooperativity derived from colocalization to the membrane Whitty,  Figure . Crystalcis dimerization of MAG represents a second example in which a very weak solution-state interaction with a Kd of 380 μM is still relevant in a functional setting . The dimerization of the MAG extracellular segment was first observed in the packing of crystals for structure determination and subsequently verified in solution experiments  Bazan,  composedc   and early to late endosomes (pH 6.3–5.5). Upon acidification, the β-propeller of Sortilin undergoes a conformational change and its top surface, negatively charged at neutral pH, becomes more neutral  domains and a SorCS membrane proximal (SoMP) domain for the three SorCS subfamily members, an epidermal growth factor-like (EGF), 11 low-density lipoprotein receptor type A repeats (LA) and six fibronectin type III (FN3) domains for the fifth family member SorLA. One of the roles of these additional domains is to modulate binding to the VPS10 platform. For example, SorCS2 has been shown to exist in at least two different conformations (Januliene et al., Ligand-induced type-I transmembrane receptor dimerization is the canonical mechanism to trigger cell signaling. Signaling in the absence of ligand is often actively prevented. This inhibition is required because receptors can encounter each other independent of ligand as a function of the local receptor concentration on the cell surface and this chance encounter may induce signaling (Atanasova and Whitty, Regulation of signaling by the conformational-change model in HER family members represents a well-described example of how an auto-inhibited conformation of a receptor prevents activation. In the auto-inhibited “compact” conformation of the HER extracellular segment, dimerization arms in domains II and IV interact and prevent high-affinity binding of the ligand (Cho and Leahy, PlexinA's receptor signaling is induced by Semaphorin ligands and controls nervous system development and plasticity. The PlexinA extracellular segment has a large unusual ring-like conformation that seems to be required to bring the PlexinA cytosolic segments of two receptors into close proximity in a Semaphorin-induced receptor dimer to trigger signaling (Janssen et al., The propensity of cell surface receptors to interact pre-ligand binding may serve a second role. It permits the local concentration of receptors and prepares them to respond rapidly by rearranging into a signaling competent form once ligand is bound . This meIt is interesting to note that whilst also homomeric class I cytokine receptors are believed to be dimers which become activated by a ligand-induced receptor conformational change (Atanasova and Whitty, Here we have discussed several extracellular molecular mechanisms employed by type-I transmembrane proteins in cell signaling and adhesion processes see also . The strSeveral outstanding questions that center on the mechanistic principles underlying the function of type-I transmembrane proteins may be addressed in the near future by structural studies; How are the structures and interactions affected by their physiologic transmembrane setting? What is the influence of protein distribution in space and time on the organization and function of type-I transmembrane proteins? In what way are the extracellular segment and cytosolic portion of type-I transmembrane proteins coupled to organize adhesion and signaling? Currently, there is no detailed structural data that shows the direct coupling of the extracellular part of the protein with its cytosolic part, although several attempts have been made toward this endeavor (Ge et al., All authors contributed to the conception and writing of the manuscript.NL was employed by the company Morphic Therapeutic. The remaining 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.


Charcot–Marie–Tooth (CMT) disease is a hereditary neuropathy mainly caused by gene mutation of peripheral myelin proteins including myelin protein zero . Large myelin protein zero (L-MPZ) is an isoform of P0 that contains an extended polypeptide synthesized by translational readthrough at the C-terminus in tetrapods, including humans. The physiological role of L-MPZ and consequences of an altered L-MPZ/P0 ratio in peripheral myelin are not known. To clarify this, we used genome editing to generate a mouse line (L-MPZ mice) that produced L-MPZ instead of P0. Motor tests and electrophysiological, immunohistological, and electron microscopy analyses show that homozygous L-MPZ mice exhibit CMT-like phenotypes including thin and/or loose myelin, increased small-caliber axons, and disorganized axo–glial interactions. Heterozygous mice show a milder phenotype. These results highlight the importance of an appropriate L-MPZ/P0 ratio and show that aberrant readthrough of a myelin protein causes neuropathy. Otani et al. show that upregulation of large myelin protein zero (L-MPZ), an isoform of myelin protein zero (P0) which contains an extended polypeptide synthesized by translational readthrough, can cause neuropathy, using mice that produce L-MPZ instead of P0. This study suggests the importance of keeping L-MPZ low for the proper functioning of myelin. To perform this function, myelin-forming cells are involved in the formation and maintenance of functional axonal domains including the node of Ranvier, paranode, juxtaparanode, and internode. In the peripheral nervous system (PNS), myelin is produced by Schwann cells. To form or maintain myelin, unique cytoplasmic channels are present in PNS myelinated fibers, including Schmidt–Lanterman incisures (SLI) in myelin and Cajal bands in the outermost regions of myelinated Schwann cells1. Disorganization of these cytoplasmic channels is frequently seen in knockout mouse lines expressing neuropathy-like phenotypes5.Myelin acts as an electrical insulator to promote saltatory conduction of action potentials6. Adhesion activity is regulated by protein kinase C (PKC)-dependent phosphorylation in the cytoplasmic region of P07. The MPZ gene is one of the genes responsible for Charcot–Marie–Tooth (CMT) disease, which is also known as hereditary motor and sensory neuropathy (HMSN). Traditionally, CMT disease caused by MPZ mutations is categorized into three types based on electrophysiological characteristics, specifically: demyelinating, axonal, and intermediate8 (https://www.ncbi.nlm.nih.gov/books/NBK1358/). Many P0 mutations cause various types of HMSN, including CMT1B9. Additionally, heterozygote and homozygote P0 knockout mice exhibit CMT1B (demyelinating), or the more severe, Dejerine–Sottas syndrome-like phenotype, respectively12. Therefore, P0 is an essential molecule for myelin formation and maintenance in the PNS.Myelin protein zero  is a major peripheral myelin protein that acts as a homophilic adhesion molecule, and is crucial for compact myelin formation in the PNS13. Moreover, this L-MPZ-specific domain contains a putative PKC phosphorylation site with high antigenicity. L-MPZ is expressed during myelinogenesis, is localized in PNS myelin, and accumulates at cell–cell adhesion sites in transfected cells, suggesting that L-MPZ is involved in the formation and/or maintenance of myelin in the PNS. Because the sequences around the canonical stop codon and L-MPZ-specific domain of MPZ genes  are highly conserved from frog to human in the vertebrate phylogeny, and L-MPZ is present within the PNS of these species14, the appearance of L-MPZ may be important for myelin during the evolution of tetrapod limbs.Large myelin protein zero (L-MPZ) is a recently identified translational readthrough isoform of P0, which has an additional 63-amino acid sequence at the C-terminus of P015, yeasts16, and Drosophila17. To our knowledge, L-MPZ is the first reported common mammalian protein synthesized by the stop codon readthrough mechanism in the physiological state13. Following identification of L-MPZ, two additional, widely expressed mammalian readthrough isoforms were reported, namely extended isoforms of vascular endothelial growth factor A (VEGF-Ax)18 and aquaporin 4 (AQP4ex)19. Thus, translational readthrough may be a common physiological mechanism for translational regulation in mammals including humans20. Interestingly, VEGF-Ax has anti-angiogenic or weaker angiogenic activity compared with canonical VEGF21, while AQP4ex displays a crucial role for the anchoring of canonical AQP4 at the astrocyte end-feet and potential regulation of water permeability22. Together, these findings suggest that quantitative changes of translational readthrough isoforms affect the function of canonical molecules. However, because both P0 and L-MPZ are produced from the same P0 mRNA and are both localized in myelin, it is difficult to determine the functional significance of L-MPZ itself, and the influence of the L-MPZ/P0 ratio on myelin in vivo is still uncertain.Translational readthrough is a key mechanism in translational regulation, and is thought to expand the coding potential of restricted genomes in virusesMpz gene. As a result, these mice synthesize L-MPZ but not P0. Increased L-MPZ levels in heterozygous (Het) and homozygous (Hom) L-MPZ mice cause severe morphological and functional changes of myelin and axons that are similar to CMT pathologies.Accordingly, in this study, we sought to clarify the functional significance of changes in amount of the readthrough isoform, L-MPZ, in the PNS. Hence, we used a genome editing system to generate mice (L-MPZ mice) with mutation of the canonical stop codon in the To distinguish the function of L-MPZ from P0, we used the clustered regularly interspaced short palindromic repeats–CRISPR associated protein 9 (CRISPR–Cas9) genome editing system to generate mice that synthesize only L-MPZ, and not P0, by replacement of the canonical stop codon (TAG) with an alanine codon (GCG) . Angles formed between bilateral hindlimbs  was more prominent in Hom than Het mice. These results indicate that neurogenic muscular atrophy has developed in L-MPZ mice -positive fluorescence signal Fig. . Immunos1. Their function is not fully understood, but as connexin 32-containing gap junctions are localized in SLIs, they may be involved in the transport of small molecules between Schwann cell bodies and the innermost myelin layer23. Thus, we investigated SLI structure in L-MPZ mice using E-cadherin as a marker of SLI containing adherens junctions24. Although both L-MPZ Hom and Het mice displayed decrease of E-cadherin-positive funnel-like staining, this staining pattern was virtually absent in Hom mice  in L-MPZ Hom mice. Taken together, both cytoplasmic channels of myelin internodes are disorganized in L-MPZ mice.Cajal bands that run longitudinally on the surface of myelin internodes can be clearly distinguished from non-channel areas of the myelin surface by antibodies against periaxin or dystrophin-related protein 2 (DRP2)ody Fig. . In WT mted Fig. . In normƒ-ratio in L-MPZ mice  and total fiber diameter (DF). In large-caliber axons, the G-ratio was particularly large , respectively. In semithin sections, compact myelin sheaths were thinner in L-MPZ mice compared with WT mice Fig. , arrows,ice Fig. . The numice Fig. . The G-rrge Fig.  in L-MPZrge Fig. . These rrge Fig. .Fig. 3In25. Periodicity was larger in L-MPZ Hom mice  Fig. , arrows.ice Fig. .Fig. 4Ul28. These paranodal junctions act as a diffusion barrier of juxtaparanodal proteins including voltage-gated K+ channels such as Kv1.231. Triple staining of WT teased fibers using antibodies against neurofascin, Caspr, and Kv1.2 showed a pair of Caspr-positive paranodal regions flanked by Kv1.2-positive juxtaparanodes. In contrast, various abnormal staining patterns were detected in L-MPZ Hom and Het mice  family proteins and dystroglycan are involved in correct sodium channel clustering at the nodal axon and normal NCV35. Sodium channel clusters were present but nodal lengths became longer in L-MPZ mice compared with WT mice  and an axonal complex of contactin and contactin-associated protein 1 (Caspr/paranodin) are involved in paranodal axoglial junctionsice Fig. , includiice Fig. , suggest36. To examine the structural abnormalities causing molecular changes, we performed serial block-face scanning electron microscopy (SBF-SEM) and transmission electron micrograph (TEM) examinations using longitudinal sciatic nerve sections from L-MPZ mice. In SBF-SEM analyses, serial images of a single myelinated axon in sciatic nerves of L-MPZ Hom mice  stress contributes to the pathogenesis of neuropathy including CMTice Fig. . Furtherice Fig. . In addiWe also investigated glial fibrillary acidic protein (GFAP) as a non-myelinated Schwann cell marker and MBP as a myelin marker. L-MPZ Hom mice showed an increase of GFAP Fig.  and decr39. Therefore, the number of macrophage in sciatic nerves of L-MPZ mice were examined by immunostaining with an antibody against ionized calcium binding adapter molecule 1 (Iba1), a major macrophage marker. We detected many strongly Iba1-positive macrophages in sciatic nerves of L-MPZ Hom and Het mice , short internodal distances, increase of myelinated axons with smaller diameters, and disorganization of paranodal and juxtaparanodal structures were observed in L-MPZ mice. Increased Schwann cells containing single axons without myelin and extensive infiltration of macrophages with phagocytosis suggests that the abnormal myelin morphology likely caused demyelination. These changes were mainly observed in Hom mice, with Het mice showing milder changes. All of these abnormalities can be responsible for the motor symptoms. Two independent L-MPZ mouse lines generated by the CRISPR–Cas9 system exhibited the same neuropathy-like phenotype indicating that these pathologies were caused by replacement of P0 with L-MPZ and not by off-target genome editing. Thus, to the best of our knowledge, this is the first report showing that disturbance of physiological translational readthrough causes a disease in mammals.12. As shown here, although P0 was completely deficient (Hom) or clearly reduced (Het), the symptoms of motor disturbance in both L-MPZ Hom and Het mice were milder compared with P0-null mice. In P0-null mice, separation of extracellular IPL was observed10. However, in L-MPZ mice, IPLs in the remaining myelin looked normal and comparable to WT. Consequently, functional loss of P0 in extracellular IPL of L-MPZ mice appears to be compensated for by L-MPZ. Meanwhile, in L-MPZ mice, many intracellular MDLs were wide-open in myelin, and myelin outfoldings or tomacula (which are not observed in P0-null mice12) were frequently observed  knockout mice2, periaxin-null mice41, or O-GlcNAc transferase-conditional knockout mice5 exhibit tomacula and a loose myelin structure, as well as similar symptoms as L-MPZ mice. Especially in the periaxin-related latter two mouse lines, loss of Cajal band structure has been shown to slow conduction velocity and impair Schwann cell elongation during nerve growth, which causes shorter internodes41. Thus, abnormalities in Cajal bands may cause CMT-like neuropathy in mice. Our study shows that increase of L-MPZ in myelin causes loss of Cajal band structure with an increase in number of myelinated fibers with shorter internodal lengths in L-MPZ mice  has shown that mutant P0 is retained in the ER, and that the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) pathway is activated to increase the phosphorylated α subunit of eukaryotic initiation factor 2 (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in Schwann cells44. As shown in Fig. In many neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, polyglutamine disease, and amyotrophic lateral sclerosis, involvement of ER stress has been suggested31. Thus, in L-MPZ mice, discontinuous paranodal formation and loss of paranodal axo–glial junctions in some myelinated fibers may cause electrophysiological abnormalities. Furthermore, in the extracellular matrix surrounding nodes, which acts on stabilization of nodal proteins by axonal cytoskeletal scaffolds46, Schwann cell microvilli were disorganized and relative diffusion of sodium channels was observed  is required for formation and/or maintenance of myelin morphology and proper function.L-MPZ Het mice only produce P0 from a normal allele of the 50. Because an increase of L-MPZ/P0 ratio induces CMT-like neuropathy in mice, and L-MPZ is also produced by the same mechanism in humans, we should consider possible side effects when readthrough agents are used for treatment of diseases. Together with the present study, future studies that focus on changes of peripheral nerve in the absence of L-MPZ may ascertain the functional significance of the physiological readthrough mechanism in the PNS. Thus, L-MPZ mice will become a unique model of neuropathy caused by alteration of translational readthrough.In summary, we have shown that changes in amount of L-MPZ influences the formation and maintenance of myelin. Therefore, analyses of gene mutations that influence L-MPZ production and its function may be required for CMT patients of unknown etiology. In addition, many readthrough agents have recently been developed for treatment of severe hereditary diseases caused by nonsense mutations such as Duchenne muscular dystrophyChemicals were of the highest purity and obtained from Fujifilm Wako Pure Chemical Co., , Sigma–Aldrich Japan/Merck , Nacalai Tesque , and Kanto Chemical .51. L-MPZ founder mice were provided from the Laboratory Animal Resource Center, University of Tsukuba. For backcrossing, C57BL/6J mice were purchased from Charles River Laboratories Japan .L-MPZ mice were generated by replacement of the canonical stop codon (TAG) with an alanine codon (GCG) using the CRISPR–Cas9 genome editing system52. Allele-specific PCR of mouse tail genomic DNA was performed using Go Taq Green Master Mix  with the following S set primers: P0 STOP Allele Forward (P0_Stop_Allele_Fw), 5′-GGAGTCTCGCAAGGATAAGAGAT-3′ and MPZ mutant genome reverse (MPZ_Mut_Geno_Rv), 5′-TGGTTCTACTGGGGGACTTGACTG-3′; and A set primers: P0_MutA_Allele_Fw, 5′-GGAGTCTCGCAAGGATAAGAGAG-3′ and MPZ_Mut_Geno_Rv. The S primer set generated a 167 bp PCR product from WT and Het genomic DNA, while the A primer set generated a 167 bp PCR product from Het and Hom genomic DNA  in 0.32 M sucrose containing 5 mM Tris-HCl (pH 7.5), 2 mM ethylene glycol tetraacetic acid, 0.75 μM aprotinin, 1 μM leupeptin, 1 μM pepstatin A, and 0.4 mM phenylmethylsulfonyl fluoride (homogenization buffer), and if necessary, PhosSTOP  was added to prevent dephosphorylation. To remove chromosomal DNA, cell debris and fibers, homogenates were centrifuged at 800×g for 10 min, and supernatants were collected and stored as whole homogenate fractions at −80 °C. Protein concentration was determined using a bicinchoninic acid assay . Samples were examined by SDS–PAGE or Phos-tag SDS–PAGE and immunoblot analysis with various antibodies.Sciatic nerve homogenates were prepared from 10-week-old L-MPZ WT, Het, and Hom mice. Mice were anesthetized by intraperitoneal injection of anesthetic combination comprising 0.3 mg kg54 with minor modifications. Briefly, each sample was separated on either 7.5% or 12% SDS–PAGE gels and transferred to polyvinylidene fluoride membranes . Membranes were incubated for 1 h with blocking buffer containing 0.3% skimmed milk or 2% donkey serum in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 (T-TBS) at room temperature. Membranes were then incubated for 1 h with primary antibodies diluted in blocking buffer and washed three times in T-TBS prior to incubation for 1 h with secondary antibodies in T-TBS. After washing three times in T-TBS, immunoreactivities were detected using the ECL system . Both chemiluminescence and prestained size marker images were captured at the same field of view using LuminoGraphI . Size markers were aligned with positive bands on the same blot using software . For quantification, band intensities were measured using ImageJ (https://imagej.net/Welcome). To distinguish phosphorylated proteins, Phos-tag (50 μM)-containing 7.5% SDS–PAGE was performed, followed by western blotting according to the manufacturer’s instructions .Western blotting was performed as described previously−1 medetomidine, 4.0 mg kg−1 midazolam, and 5.0 mg kg−1 butorphanol) were sacrificed by cervical dislocation. Sciatic nerves were dissected and snap-frozen in liquid nitrogen. Frozen tissue was then ground into a powder using BioMasher II. Total RNA was extracted using Trizol . Quantification of mRNA of P0, MBP, or the housekeeping gene, hypoxanthine phosphoribosyl transferase (HPRT), was performed using a fluorescence-based real-time detection method. First strand cDNA was synthesized using ReverTra Ace qPCR kit . Real-time PCR was then performed using the CFX Connect Real-Time PCR Detection System  with the primer sets: P0_primer_Fw, 5′-CTTCAAGATGGACGCGACAC-3′ and P0_primer_Rv, 5′-AGAGTGTCTCAGCCTCCACAG-3′; MBP_primer_Fw, 5′-AACATTGTGACACCTCGAACA-3′ and MBP_primer_Rv, 5′-TGTCTCTTCCTCCCCAGCTA-3′; HPRT_primer_Fw, 5′-CCCTCTGGTAGATTGTCGCTTA-3′ and HPRT_primer_Rv, 5′-AGATGCTGTTACTGATAGGAAATCGA-3′. Relative copy number of P0 or MBP transcripts per HPRT transcripts was determined using calibration standards for each of the tested molecules.Anesthetized mice using anesthetic combination  and 4–5 min (later period)  is checked by the tail suspension testMotor function was also examined using a MK-630B rotarod . Mice were placed on a rubber-coated rod and two test trials were performed with an inter-trial interval of 10 min. After test trials, the rotarod test was performed. Rotation speed of the rod was 20 rpm with each 5 min trial. The latency was measured when the mouse stopped walking on the rod, either by falling off or by clinging to the rod, and mean latency from three independent trials was calculated for each mouse.59 with slight modifications. In brief, conduction speeds in motor nerve fibers innervating the plantar muscle was examined under anesthesia by intraperitoneal injection of anesthetic combination . Body temperature was monitored at the thigh and maintained around 30 °C using a heating pad. As the anode and cathode, two stainless steel subdermal needle electrodes  were placed in the plantar muscle for detection of CMAPs. Proximal and distal stimulation electrodes were inserted in the ankle and sciatic notch, respectively. Electrical stimulation (0.1 ms in duration) was applied every second using a constant voltage stimulator . To detect the amplified waveforms, a digital data recorder  was used. For recording and analyzing, WAVE SHOT 2000 software (Keyence) was used. The trigger level was set to 0.1 V for recording. Data were recorded for 10 ms  from 500 μs before the trigger. Distance between the distal and proximal electrodes and the difference in onset latency obtained by stimulation of each electrode were used for calculation of conduction velocity. Waveform duration was measured between the onset and endpoint of each CMAP.Motor NCVs were measured as previously described59. Sciatic nerves obtained from anesthetized 10-week-old mice were rapidly dissected and immediately fixed in ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) (pH 7.4) for 30 min. After fixation, nerves were cryoprotected with 10% sucrose in 0.1 M PB, and then later with 30% sucrose overnight at 4 °C. Next, nerves were frozen in OCT compound . Sections (10 μm) were cut using a cryostat , and placed on FRONTIER slide glass . For teased fiber preparation, sciatic nerves were transferred into 0.1 M PB (pH 7.4) after fixation. The nerves were then gently teased apart, spread on FRONTIER slide glass, and air-dried. If necessary, samples were incubated with −20 °C methanol for 5 min, and rinsed in phosphate buffered saline (PBS) before pre-incubation with 0.1 M PB (pH 7.4) containing 0.3% Triton X-100 and 10% normal goat serum (PBTGS) for 1 h. Afterwards, samples were incubated overnight at 4 °C with primary antibodies diluted to appropriate concentrations in PBTGS. Samples were thoroughly rinsed in PBS, followed by application of fluorescently labeled secondary antibodies (1:2000 in PBTGS) for 1 h at room temperature. Finally, labeled samples were rinsed with 0.01 M PBS every 5 min, three times. Samples were mounted on cover glasses using Vectashield with DAPI  and examined by BZ-X700 microscopy (Keyence) or FV-1000 confocal microscopy .Immunostaining methods were modified as described previously13. Rabbit polyclonal antibodies against Kv1.2 , GFAP , phospho-(Ser) PKC substrate , DRP2 , GRP78 (BiP) , Iba1 , and Mannose receptor (CD206) , chicken polyclonal antibody against Neurofascin , mouse monoclonal antibodies against Caspr , sodium channel (Pan) , beta dystroglycan DAG43/DAG31 , neurofilament , moesin , KDEL , actin, , GAPDH , and CD68 , rat monoclonal antibodies against MBP , laminin alpha-2 , and E-cadherin , and goat polyclonal antibody against P0  were purchased. Secondary antibodies including Alexa 488-conjugated and 594-conjugated species-specific antibodies , and aminomethyl coumarin-conjugated anti-chicken IgY antibody  were used for immunohistochemistry. For western blotting, horseradish peroxidase-conjugated anti-mouse, anti-rabbit, anti-rat, and anti-goat IgG antibodies  were used.Rabbit polyclonal anti-L-MPZ antibody  was produced as previously described60. Briefly, samples were treated with 2% osmium tetroxide (Nisshin EM Co.) and 1.5% potassium ferrocyanide in PBS for 1 h at 4 °C, followed by 1% thiocarbohydrazide for 20 min at room temperature, 2% aqueous osmium tetroxide for 30 min at room temperature, 2% uranyl acetate solution overnight at 4 °C, and lead aspartate solution for 30 min at 65 °C. Lead aspartate solution was produced by adding lead nitrate  into 0.03 M l-aspartic acid to a final concentration of 0.67% (pH 5.0–5.5). Samples were then dehydrated in a graded ethanol series, treated with dehydrated acetone, and embedded in Quetol 812 epoxy resin containing Ketjen black powder61. The resin was incubated at 70 °C for three nights to ensure polymerization. SBF-SEM observation of the myelin membrane was performed using Sigma or Merlin scanning electron microscopes  equipped with a 3View in-chamber ultramicrotome system . Serial image sequences were 6144 × 6144 pixels wide (4.7–6 nm per pixel) and 200–1000 slices in 80 nm steps. Sequential images were processed using FIJI (https://fiji.sc/), and analyses were performed using Amira .Eight-week-old mice were deeply anesthetized by intraperitoneal injection of anesthetic combination. Four WT and four L-MPZ Hom mice were perfused with 4% PFA and 2.5% glutaraldehyde in 0.1 M PB (pH 7.4), and incubated in the same fixative overnight. For TEM, samples were post-fixed in 2% osmium tetroxide  in PBS, dehydrated in a graded ethanol series, treated with dehydrated acetone, and embedded in Quetol 812 epoxy resin (Nisshin EM Co.). For observation by SBF-SEM, sample preparation, observation, and analyses of acquired data were performed as described previously with slight modificationsπ. Periodicity measurement was performed with FIJI by obtaining mean values of periodicity, and medians of outer, middle, and inner parts (three measurements in each part) in individual myelinated fibers. Macrophages in SBF-SEM images were identified as cells with relatively short but complex processes with a ruffled surface and abundant vacuoles and primary and secondary lysosomes. The number of macrophages was divided by the observation volume to obtain a macrophage density (per 106 μm3).For the percentage of axons without compact myelin that were individually ensheathed by Schwann cells, axons were counted in single EM images. Remak bundles were excluded from analyses. Diameters of axons and myelinated fibers were obtained by dividing the perimeter with The width of each ER profile was measured as the largest distance of the membranes which was perpendicular to the longer axis of the profile. The mean ER width of each cell was pooled from three wild-type and three homozygous mice.Muscle tissues were harvested from 4-month-old L-MPZ WT, Het, and Hom mice, and flash-frozen using dry ice–isopentane. Sections (10 µm) were cut by cryostat and placed on FRONTIER slide glass. For histological staining, slides were incubated with Mayer’s hematoxylin solution  for 15 min, followed by a 10 min wash. Slides were then incubated in 1% eosin solution  for 1 min. Next, slides were washed with tap water for 10 min. Afterwards, they were dehydrated successively with 70, 80, and 90% ethanol (EtOH) for 1 min, three times with 100% EtOH for 1 min, and three times with xylene for 1 min. Samples were mounted with MGK-S (Matsunami Glass) and covered by cover slips, and observed using a BZ-X700 microscope (Keyence).U-test, Student’s t-test, or one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test or two-way ANOVA followed by Bonferroni multiple comparison tests were performed. p-values < 0.05 were regarded as statistically significant. Details of each statistical test are indicated in figure legends. Non-numeric data are shown as representative results from more than three independent experiments.Statistical analyses were performed using PRISM 5 . Data were expressed as mean ± standard error (SE) or standard deviation (SD). For comparisons, Mann–Whitney Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary ItemsSupplementary Data 1Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Reporting SummaryPeer Review File


Hyperuricemia, which has been considered as a cause of gout and nephrolithiasis has recently been suggested to be associated with hypertension, coronary heart disease, heart failure, atrial fibrillation, insulin resistance, and nonalcoholic fatty liver disease. Several clinical and experimental studies have supported uric acid (UA) as an independent risk factor for predicting disease development along with the traditional risk factors. The mechanism by which UA causes cardiometabolic disease has not been fully elucidated to date; however, it has been explained by several hypotheses such as oxidative stress, reduced nitric oxide bioavailability, inflammation, endothelial dysfunction, and so on. Although evidence of the preventive and therapeutic effects of UA lowering therapy on cardiometabolic diseases is still insufficient, it is expected to be considered as a new treatment strategy for such diseases through additional, carefully designed, large-scale clinical studies. Uric acid (UA) is the end product of purine metabolism in humans, it is formed from xanthine by xanthine oxidase enzyme through several steps and excreted into the urine. UA is endogenously synthesized mainly in the liver, intestines, muscle, and vascular endothelium , 2. ExogIn many mammals, UA is converted to highly soluble allantoin and maintained at very low levels  .Pathologically, increased serum UA levels lead to crystal (monosodium urate [MSU]) precipitation in the joints, soft tissue, kidneys, and other organs, which in turn causes various diseases , 9. It hThe role of UA in CVD or cardiometabolic disease is still controversial because it is perceived that UA plays a protective role in oxidative stress. Otherwise speaking, UA acts as an active oxygen scavenger in the human body and has an antioxidant effect that prevents cardiovascular diseases (CVD), such as atherosclerosis , 18. In However, several clinical and epidemiological studies have presented the relationship between UA and various disorders including CVD, metabolic syndrome, and kidney disease, thereby overwhelming the beneficial effects of UA –25 Fig.. FurtherSeveral studies have demonstrated that hyperuricemia is associated with hypertension development –28. A reSLC2A9 and GLUT9, associated with the regulation of UA levels in the human body, are known to be associated with hypertension development [In experimental studies, it was suggested that UA inhibits nitric oxide release from the endothelial cells, activates the renin-angiotensin system, and increases oxidative stress, which damages the endothelial cells and causes vasoconstriction, leading to hypertension development –37. In aelopment –41.CVD is the leading cause of death worldwide, with ischemic heart disease accounting for nearly 50% of all CVD mortalities, according to the World Health Organization (WHO) estimates . Since tOn the contrary, some studies including ours, have argued that serum UA did not meaningfully improve the prediction of CHD in the general population and was not associated with all-cause and cardiovascular mortalities , 58. FurHyperuricemia is frequently found in heart failure patients, and UA levels are elevated in more than half of the hospitalized chronic heart failure patients , 62. HypRecent studies have shown that elevated UA levels are associated with increased risk of atrial fibrillation. A European cohort study involving approximately 6000 patients showed that high baseline UA levels increased the risk of atrial fibrillation . AdditioMetabolic syndrome (MetS), once called ‘Syndrome X’ by Raven in the year 1988, is a group of risk factors that increase CVD . From thNon-alcoholic fatty liver disease (NAFLD) refers to the condition of fat infiltration in the hepatic parenchyma without alcohol abuse, which can potentially lead to liver cirrhosis or liver cancer, and is known to be associated with coexisting conditions such as obesity, type 2 diabetes mellitus, and hyperlipidemia . RecentlAs gout prevalence has increased over the past decades, there has been a growing interest in UA, the causative agent of gout. Although there are some controversies due to the ambivalent nature of UA in the human body, several clinical and experimental studies have shown that UA is associated with CVDs and MetS. Moreover, a number of studies have revealed that UA is an independent risk factor for these diseases, suggesting that UA may be a potential therapeutic target for cardiometabolic disease patients, especially those with hyperuricemia, as UA plays a central role. But, there has been some controversy about the causal relationship between hyperuricemia and cardiometabolic disease; uncertainties exist regarding the mechanism of UA-induced cardiometabolic disease. Therefore, it is necessary to elucidate the causal relationship between UA and cardiometabolic disease by further well-controlled, large-scale studies as well as reveal the potential therapeutic and preventive effects of UA lowering therapy in cardiometabolic disease.


Leishmania strains. The in vitro antileishmanial activity against the intracellular forms was six and three-fold higher compared to extracellular forms of L. amazonensis and L. infantum, respectively. UA also showed to be a potent antileishmanial drug against both VL and CL manifestations of the disease in experimental models. UA parenterally administered at 5 mg/kg for seven days significantly reduced the parasite burden in liver and spleen not only in murine acute infection but also in a chronic-infection model against L. infantum. In addition, UA ointment (0.2%) topically administered for four weeks diminished (50%) lesion size progression in a chronic infection model of CL caused by L. amazonensis, which was much greater than the effect of UA formulated as an O/W emulsion. UA played a key role in the immunological response modulating the Th1 response. The exposure of Leishmania-infected macrophages to UA led to a significant different production in the cytokine levels depending on the Leishmania strain causing the infection. In conclusion, UA can be a promising therapy against both CL and VL.Leishmaniasis affects around 12 million people worldwide and is estimated to cause the ninth-largest disease burden. There are three main forms of the disease, visceral (VL), cutaneous (CL), and mucocutaneous (MCL), leading to more than one million new cases every year and several thousand deaths. Current treatments based on chemically synthesized molecules are far from ideal. In this study, we have tested the in vitro and in vivo efficacy of ursolic acid (UA), a multifunctional triterpenoid with well-known antitumoral, antioxidant, and antimicrobial effects on different  Leishmania spp. are known to transmit the disease to humans. According to the species and the differences in virulence, the infections may vary from simple cutaneous lesions to mucocutaneous ulcers or visceral diseases [Leishmaniasis affects around 12 million people worldwide and is estimated to cause the ninth-largest disease burden [diseases ,4.The chemotherapy of leishmaniasis has been based on the use of antimonial compounds, such as sodium stibogluconate and meglumine antimoniate, which are far from being the ideal treatments as they display a variable efficacy against VL and CL, cause severe side effects and are parenterally administered . CurrentCurrently, major research efforts are focused on discovering new natural compounds with antileishmanial properties . Many acTriterpenoids are polycyclic compounds derived from the linear hydrocarbon squalene and comprise one of the most promising groups of phytochemicals due to its multifunctionality in the treatment of a broad range of diseases ,19,20,21Trypanosoma cruzi [Plasmodium falciparum [Leishmania spp [Ursolic Acid (UA) is a ubiquitous triterpenoid in the plant kingdom . UA is ama cruzi , Plasmodlciparum , and Leiania spp .Mycobacterium tuberculosis, UA has shown the ability to inhibit the expression of certain inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and TGF-β [Toxoplasma gondii, leading to an increased production of NO, ROS, IL-10, IL-12, granulocyte macrophage colony stimulating factor (GM-CSF) and interferon-β, while reducing the expression of IL-1β, IL-6, TNF-α and TGF-β in infected immune cells [Regarding the immunological effect of triterpenes, several studies have shown that they possess anti-inflammatory activity, mainly caused either by inhibiting enzymes involved in the production of eicosanoids, such as cyclooxygenases, or inhibiting the release of cytokines . This annd TGF-β ,33. UA ane cells .L. amazonensis and L. braziliensis. This activity has been attributed to the fraction of Baccharis uncinellase containing oleanolic and ursolic acids [L. infantum [Petiveria alliaceae showed similar activity against L. amazonensis associated with programmed cell death of parasites and an increase in NO [Several plant extracts from Brazil have shown in vitro activity against ic acids . The samic acids . In addiinfantum . UA extrse in NO .Leishmania species in order to select an adequate dose for in vivo efficacy studies. Moreover, topical formulations containing UA were developed, and its efficacy was tested in a chronic model of CL. The activity of parenterally administered UA was assessed on both an acute and chronic model of VL.The aim of this study was to evaluate the in vitro activity of UA on promastigotes and intracellular amastigotes of different 2 = 0.9999).UA is a triterpenoid with negligible aqueous solubility. For this reason, it was compounded in semisolid formulations to facilitate its solubility and its topical administration. UA was readily dispersed using a mixture of glycerin: propylene glycol  that avoided drug agglomeration after mixing with the preformed emulsion or Orabase in the case of the cream or ointment, respectively. Both formulations exhibited a pH of 5.5, which is suitable for skin administration. UA was chemically stable over one month at 25 ± 2 °C. Consistency, color, and pH were kept unchanged over this period of time. UA concentration was determined using a linear regression calibration curve between 0.78 to 200μg/mL . These results suggest a good UA bioavailability from the site of injection to the target organs. No evident signs of toxicity, like loss in weight or hair loss, were observed at the end of the experiment in any of the animals, which suggests that UA was well-tolerated by the infected mice at the administered dose.Supported by the in vitro leishmanicidal efficacy in both intracellular and extracellular forms, UA was also evaluated in vivo in a murine model of acute infection by UA was also evaluated in vivo in a chronic infection model of VL developed in hamsters. Similarly, UA was administered by the intraperitoneal route at 5 mg/kg daily for seven consecutive days. The results are summarized in p < 0.05) 42% and 50.3% reduction in the lesion size compared to the untreated group at weeks 10 and 15. However, parasites were not completely eradicated after UA topical administration as inflammation increased at week 15.Prior to topical treatment, disease progression was similar in all animals, and no statistically significant differences were observed after 35 days post-infection . Daily tIn a subsequent study, a higher dose of UA (0.5%) was tested . Daily tL. infantum and L. amazonensis. On the splenocytes, out of the 17 studied cytokines, the following ones showed a significant increase in the presence of UA: GM-CSF, IFN-γ, IL-4, IL-6, IL-9, IL-10, and decrease of RANTES  naïve splenocytes from mouse spleen and (ii) infected macrophages with f RANTES . NeverthLeishmania-infected macrophages to UA led to a significant different production in the cytokines levels depending on the Leishmania strain causing the infection. Except for the production of TNF-α that was exacerbated in both cases, several differences were observed in the levels of: GM-CSF, IFN-γ, IL-1b, IL-2, IL-6, IL-10, and MCP-1.It is also key to investigate the production of cytokines on infected macrophages and not only just on lymphocytes. The exposure of L. infantum-infected macrophages, the production of GM-CSF, IL-6, and IL-10 was augmented compared to the untreated control while the levels of IFN-γ, MCP-1, and IL-1b were diminished significantly (p < 0.05) when L. amazonensis infected macrophages were exposed to UA  and amastigotes  [Terminalia arjuna displaying an IC50 of 3.51 μg/mL against L. donovani promastigotes [Pseudelephantopus spicatus (peruvian plant), which exhibited a high in vitro activity  against L. amazonensis [Tan et al. extracted UA from  7.0 nM) . Moulishstigotes . Odonne zonensis .Miconialang sdorffii exhibited a very low antileishmanial activity against L. amazonensis promastigotes (IC50 de 350 μM) [50 of 20 and > 25 μM against L. amazonensis and L. infantum promastigotes, respectively [On the contrary, UA extracted from  350 μM) . Chemicaectively .This variable efficacy is one of the major problems of natural compounds. Extraction and purification processes should also be standardized in order to obtain a compound with reproducible characteristics, making its further use in clinical studies easier. For this reason, chemical synthesized UA (purchased by Sigma-Aldrich) was used to perform the in vitro and in vivo assays.The higher leishmanicidal activity of UA against intracellular compared to extracellular forms has also been reported by other authors . This fa1 dependent granulomatous response after multiple weeks [Parenteral administration of UA at 5 mg/kg for seven consecutive days has shown to significantly reduce parasite growth in liver and spleen in both acute and chronic infection models of VL. In the acute-infection mouse model, although there is parasite growth in the liver and spleen, the infection is usually resolved by a Thle weeks . Howeverle weeks . It has le weeks . In thisResults obtained after UA topical administration against CL were also encouraging suggesting that: (i) higher doses than 0.5% topically administered can lead to an altered immune-reaction in the cutaneous infected lesion, (ii) UA ointment formulation has a better permeability across the skin than UA cream, and (iii) a successful therapy against CL could be achieved if prolonged topical treatments using UA ointment (0.2%) are applied. Despite the fact that creams combine properties, such as lubrication, occlusion, and hydration, that lead to higher patient compliance and better suitability for cutaneous lesions , oinLeishmania infection is attributed to the development of a Th1-type immunity characterized by the production of cytokines, such as IFN-γ, TNF-α, IL-12, and GM-CSF, which can lead to the activation of macrophages and production of nitric oxide (NO).The immune response associated with the protection against a L. infantum-infected hamsters was characterized by an increase in the IL-10 levels at all the doses tested of UA and a decrease in the IFN-γ at higher doses [Passero et al. demonstrated that the production of cytokines in an in vivo model of er doses . Our in er doses .Other studies have shown that the antileishmanial activity of UA depends on two factors: the parasite strain as well as the interaction with the immune system. The production of IFN-γ in infected macrophages seems to be more regulated by the presence of the parasites rather than the concentration of UA. This can explain the lack of efficacy of UA in the chronic models of VL .Leishmania [L. infantum along with higher levels of TNF-α. This can explain the higher activity of UA observed against amastigotes compared to promastigotes.Other studies have shown that GM-CSF has a beneficial output in the disease progression, especially in CL, because it triggers the activation of macrophages to kill ishmania ,48. GM-Cishmania . In our All chemicals used, unless otherwise stated, were obtained from Sigma-Aldrich  or Panreac S.A.  and used without further purification.UA cream consisted of an aqueous phase with tween 80 (6%) dispersed in water and an oil phase constituted by white wax (1%) and cetyl alcohol (15%) melted at 60⁰. The aqueous phase was heated at 60⁰ and then was added onto the oil phase at the same temperature. Continuous stirring was required until the cream reached the desired consistency at room temperature. Finally, UA (0.5%) dispersed in a mixture (1:1) of glycerin:propylene glycol (12%) was added.® , a plasticized hydrocarbon gel composed of gelatin, pectin, and sodium carboxymethyl cellulose.UA ointment was prepared by dispersing the UA (0.2 or 0.5%) in a mixture (2:1) of glycerin:propylene glycol (5%), which, in a second step, was mixed with Orabaseg for 10 min, and supernatant was collected and injected in the HPLC. HPLC was equipped with a Jasco PU-1580 pump, a Jasco AS-2050 Plus autosampler, and a Jasco UV-1575UV–visible detector . Integration of the peaks was performed with the program Borwin 1.5 for PC . The samples (100 µL) were chromatographed over a ThermoHypersil BDS C18 reverse-phase column   at a flow rate of 1 mL/min. Elution was carried out isocratically with a mobile phase that consisted of a methanol:water  mixture filtered through a 0.45 µm hydrophilic polypropylene filter membrane . UA was detected at a wavelength of 210 nm, and its retention time was 5 min.UA concentration was assessed by HPLC. Formulations (50 mg) were weighted and dissolved with methanol (100 mL). Samples were centrifuged at 3000× Leishmania were used: an autochthonous isolate of L. infantum (MCAN/ES/96/BCN150) kindly given by Prof. José M. Requena ; L. braziliensis 2903, L. amazonensis (MHOM/Br/79/Maria), and L. guyanensis 141/93 were kindly provided by Prof. Alfredo Toraño . Promastigotes were cultured in Schneider’s Insect Medium supplemented with 10% heat-inactivated Foetal Bovine Serum (FBS) and 1000 U/L of penicillin plus 100 mg/L of streptomycin in 25 mL culture flasks at 26 °C.The following species of 5 parasites/well) were cultured in 96-well plastic plates. Compounds (UA and miltefosine) were dissolved in dimethylsulfoxide (DMSO) and different concentrations of each  were carried out up to 200 µL final volume. Growth control and signal-to-noise were also included. The final solvent (DMSO) concentrations never exceeded 0.5% (v/v) warranting no effect on parasite proliferation or morphology. After 48 h at 26 °C, 20 μL of a 2.5 mM resazurin  solution was added to each well, and the plates were returned to the incubator for another 3 h. The Relative Fluorescence Units (RFU) (535–590 nm excitation–emission wavelength) was determined in a fluorometer . Growth inhibition (GI) was calculated by the following equation:The assay was performed as previously described . Briefly50 (concentration of the compound that produced a 50% reduction in parasites) using a multinomial probit analysis incorporated in SPSS software v21.0 .All tests were carried out in triplicate. Miltefosine was used as the reference drug and was evaluated under the same conditions. The efficacy of each compound was estimated by calculating the IC4 J774 macrophages and stationary promastigotes in a 1:10 ratio were seeded in each well of a microtiter plate, suspended in 200 μL of culture medium and incubated for 24 h at 33 °C in 5% CO2 chamber. After this first incubation, the temperature was increased up to 37 °C for another 24 h. Thereafter, cells were washed several times in culture medium by centrifugation at 1500 rpm for 5 min in order to remove free non-internalized promastigotes. Finally, the supernatant was replaced by 200 μL/well of culture medium containing 2-fold serial dilutions of the test compounds . Growth control and signal-to-noise were also included. All tests were carried out in a triplicate assay. Following incubation for 48 h at 37 °C, 5% CO2, the culture medium was replaced by 200 μL/well of the lysis solution (RPMI-1640 with 0.048% HEPES and 0.006% SDS) and incubated at room temperature for 20 min. Thereafter, the plates were centrifuged at 3500 rpm for 5 min, and the lysis solution was replaced by 200 μL/well of Schneider´s insect medium. The culture plates were then incubated at 26 °C for another 4 days to allow the transformation of viable amastigotes into promastigotes and proliferation. Afterward, 20 μL/well of 2.5 mM resazurin was added and incubated for another 3 h. Finally, fluorescence emission was measured, and IC50 was estimated as described above. All tests were carried out in triplicate. Miltefosine was used as the reference drug and was evaluated at the same conditions.The assay was carried out as previously described . Briefly4 cells/well) in 96-well flat-bottom microplates with 100 μL of RPMI 1640 medium. The cells were allowed to attach for 24 h at 37 °C, 5% CO2, and the medium was replaced by different concentrations of the compounds in 200 μL of medium and exposed for another 24 h. Growth controls and signal-to-noise were also included. Afterward, a volume of 20 μL of the 2.5 mM resazurin solution was added, and plates were returned to the incubator for another 3 h to evaluate cell viability. The reduction of resazurin was determined by fluorometry as in the promastigote assay. Each concentration was assayed three times. Cytotoxicity effect of compounds was defined as the 50% reduction of cell viability of treated culture cells with respect to untreated culture (CC50).The assay was carried out as previously described . J774 maMesocricetus auratus) of 60–70 g were obtained from Harlan Interfauna Ibérica S.A. . Animals were allocated in plastic cages in a 12 h dark–light cycle animal with controlled temperature (25 °C) and humidity (70%). Water and food were unrestricted throughout the study. All the animals were handled according to the European Union legislation Directive 2010/63/EU and Spanish law Real Decreto 53/2013 on the protection of animals used for scientific purposes with an ethical committee code CEXAN170415.Male BALB/c mice of 20–25 g body weight and male golden hamsters . Parasite burden in liver and spleen was estimated by the limit dilution assay described above [Chronic infection in Golden hamster was developed, as previously described by Dea-Ayuela et al. . Each haed above .7 stationary promastigotes of L. amazonensis in Schneider medium was injected subcutaneously in the footpad of the left hind paw at day 0. Right hind paw was used as a negative control (no infection). After 5 weeks post-infection, chronic CL was developed, and topical treatment with UA started. In a first experiment, UA ointment (0.2%) was administered once daily for 4 consecutive weeks. The other group received no treatment. Disease progression was monitored at weeks 0, 5, 10, and 15 using a Vernier caliper to measure footpad size. In a second experiment, hamsters were randomly split into three groups of six animals. One group was treated with UA ointment (0.5%), and the other group was tread with UA cream (0.5%). As in the first experiment, the third group received no treatment (control group).This experiment was carried out as described by Morais-Teixeira et al., with some modifications . HamsterLeishmania infection was carried out as described previously. After the incubation with 200 μL/well of culture medium containing UA at the IC50 concentration previously determined for each of the species (L. infantum and L. amazonensis) for 48 h the supernatants were collected and stored at −80 °C for cytokine quantification.In vitro 9 cells/L, were cultured in RPMI 1640 medium containing 10% fetal bovine serum and incubated at 37 °C for 24 h. After incubation for 24 h, the supernatants were collected and stored at −80 °C for cytokine quantification.Cell suspension of splenocytes was prepared from the spleen of non-infected BALB/c mice and then was rinsed with RPMI 1640 medium three times. Then, 5 mL of the cell suspension was added to 1 mL of ammonium chloride 0.8% to isolate the splenocytes, and the suspension was again washed three times with RPMI 1640 medium. The splenocytes, 2.5 × 10® array , a multiplexed sandwich ELISA-based quantitative array platform [® 4000B Scanner, UCM Facilities, Madrid, Spain).The analyses were carried out by using the Quantibodyplatform . Ninetee2SO4, and the optical densities (OD) were read at 450 nm.A sandwich ELISA kit was used to determine the concentrations of TNF-α according the manufacturer’s instructions. Briefly, standards and samples (50 µL) were diluted with 50 µL of commercial diluting solution in triplicate and incubated for 2 h at 37 °C. Plates were washed five times, and 100 µL of conjugated anti-TNF-α was added and incubated for 1 h at 37 °C. Finally, 100 µL of TMB was added as substrate and incubated for 30 min in dark at room temperature. The reaction was stopped with 3M H50) and cytotoxicity effect (CC50) of compounds were calculated from Probit analysis using SPSS v21.0 software  Finally, selective index (SI) was calculated defined as the ratio between CC50/IC50.The efficacy against the parasite .The analysis of cytokines in vitro was performed by Tukey‘s HSD test using the statistical program IBM®  and IBM® SPSS v21.0. Statistical significance was set at a p-value < 0.05.The group mean, standard deviation, standard error, and the differences were compared by the Mann–Whitney U nonparametric test, using the statistical programs of Microsoft Excel 2011Currently, plant-based therapies have shown great potential in the treatment of leishmaniasis. However, it is challenging to find a compound that possesses efficacy not only against cutaneous but also against visceral leishmaniasis. In this work, we have demonstrated that UA, a ubiquitous triterpenoid in nature, has a potent antileishmanial efficacy against both VL and CL, having a direct effect on the immunological response of the host. UA parenterally administered at 5 mg/kg significantly reduced parasite growth in liver and spleen not only in acute-infection but also in a chronic infection model. In addition, UA ointment (0.2%) topically administered could constitute a promising therapy against CL being able to penetrate across the skin and to diminish disease progression. In conclusion, formulating UA with suitable excipient can lead to promising treatments for cutaneous and visceral leishmaniasis.


Tetraspanins comprise a family of proteins embedded in the membrane through four transmembrane domains. One of the most distinctive features of tetraspanins is their ability to interact with other proteins in the membrane using their extracellular, transmembrane and cytoplasmic domains, allowing them to incorporate several proteins into clusters called tetraspanin-enriched microdomains. The spatial proximity of signaling proteins and their regulators enables a rapid functional cross-talk between these proteins, which is required for a rapid translation of extracellular signals into intracellular signaling cascades. In this article, we highlight a few examples that illustrate how tetraspanin-mediated interactions between cell surface proteins allow their functional cross-talk to regulate intracellular signaling. Different mechanisms have evolved how these signaling complexes propagate the signals into the interior of the recipient cell. Signaling by G-protein-coupled receptors (GPCRs) is mediated by conformational changes induced by ligand binding resulting in the dissociation of heterotrimeric G-proteins and activation of signaling cascades through the generation of second messengers . Signaliomplexes .Signaling events are frequently restricted to specific subcellular sites. This site-specific localization of signaling events can be mediated by physically linking the signaling molecules to cell adhesion receptors present at specific locations, such as sites of cell–matrix adhesion like focal adhesions (FA) , or site2 [Tetraspanins  proteins), a family of integral membrane proteins consisting of 33 members in humans , 9, have2 , 26, 27.2 . The assAngiogenesis describes the formation of new blood vessels from the pre-existing vasculature . It is iTetraspanin CD63 is highly expressed in endothelial cells  and inteSimilar to CD63, tetraspanin CD9 interacts with various integrins including a number of β1 and β3 integrins, but at the same time with a number of IgSF members . CD9 hasTetraspanin CD9 is also expressed on platelets in which it is the most abundant tetraspanin localized at the surface . Among oIn the absence of CD9, the binding of fibrinogen to stimulated platelets and the inclination to thrombus formation are significantly enhanced, suggesting that CD9 inhibits αIIbβ3 integrin-mediated signaling . SimilarTetraspanins CD9 and CD81 interact with members of a small subfamily of the IgSF of proteins, which is characterized by a conserved Glu-Trp-Ile (EWI) motif. Two members of this family, i.e., EWI motif-containing protein 2 (EWI-2) and EWI-F, have been identified as tetraspanin partners on the basis of their interaction with CD9 and CD81 –52. EWI-The association of CD9 and CD81 with EWI-2 has been characterized in more detail. The interaction of EWI-2 with CD9 and CD81 is most likely direct and mediated by the extracellular domains of the proteins with contribution of the transmembrane domain and Cys residues in the cytoplasmic domain of EWI-2 that are palmitoylated , 52, 53.The regulation of EWI protein interactions with other integral membrane proteins by tetraspanins can be mutual and even occur in a negative manner. As opposed to the recruitment of EWI-2 to α3β1 integrin by CD9 and CD81 to allow functional interaction between EWI-2 and the integrin, the recruitment of CD9 and CD81 to TGFβ receptors is prevented by EWI-2 to limit TGFβ signaling .TGFβ signaling plays a central role during epithelial-to-mesenchymal transition (EMT), a process that is critical during development but when inadvertently reactivated in the adult organism contributes to tumor progression by facilitating invasion and metastasis . TGFβ siA recent study indicates that the tetraspanin partner proteins EWI-2 negatively regulates TGFβ signaling, and that EWI-2 regulates TGFβ signaling activity through CD9 and CD81 . In earlThe B cell receptor (BCR) mediates the antigen-specific activation of B-lymphocytes leading to the production of antibodies. The BCR consists of membrane-bound immunoglobulins (Ig) which bind the antigens, and of the non-covalently bound co-receptors CD79A and CD79B  which form a heterodimer that initiates intracellular signaling . The intIn the absence of antigens, the BCR is not silent but generates a low strength signal that is required for the survival of mature B cells . AntigenMore recent evidence provided a deeper insight into the role of CD81 in regulating BCR signaling through CD19. In resting B cells, IgM- and IgD-based BCRs as well as CD19 are compartimentalized in nanoclusters at the B cell surface. Disrupting the actin cytoskeleton results in increased diffusion of IgM and IgD nanoclusters triggering cytoskeleton-dependent BCR signaling. This observation is interpreted in a way that the BCR is kept immobile in a nanocluster and that its diffusibility is required for efficient BCR signaling. Of note, CD19 is organized in a separate nanocluster, and its diffusibility is not dependent on the actin cytoskeleton but on its association with CD81 . In the One of the most distinctive features of tetraspanins is their ability to associate with other proteins using all of their topological doamins, i.e., their extracellular domains, transmembrane domains and cytoplasmic domains. Together with their ability to undergo homophilic and heterophilic interactions in cis with other tetraspanins, this ability allows tetraspanins to assemble large protein complexes into microdomains. Not surprisingly, tetraspanin-enriched microdomains localized at the plasma membrane are frequently involved in intercellular communication, which requires a rapid assembly of signaling complexes in response to an extracellular signal at a specific subcellular site. Rapid signaling requires close spatial proximity of primary signaling factors, such as growth factor receptors or adhesion receptor-associated signaling molecules, with their regulators. Tetraspanins are ideally suited to assemble protein complexes in which signaling molecules and their regulators are spatially segregated to prevent activation in the absence of agonist but on the other hand are close enough to allow rapid cross-talk in the presence of agonists. Recent observations in other protein-enriched signaling units, i.e., integrin-based adhesion complexes, indicate that signal propagation occurs in the absence of changes in the composition of the adhesion complex, but is regulated by the relay of phosphotyrosine-dependent signals . As outl


We have little information about the definite role of the thioredoxin antioxidant complex system during viral infection, particularly during human T-cell lymphotropic virus type 1 (HTLV-1) infection and the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) state. Therefore, we conducted comprehensive next-generation sequencing (NGS) analysis to determine Trx system expression changes in three categories of subjects: sero-negative HTLV-1 individuals, asymptomatic HTLV-1 people and HAM/TSP patients. We found that Trx capacity is reduced in the HAM/TSP state compared to healthy individuals, which indicates increasing inflammation and reduction of apoptosis, which might contribute to the progression of inflammation in the spinal cord, which in turn may develop into the HAM/TSP state. Human T-cell lymphotropic virus type 1 (HTLV-1) is one of the most important retrovirus type C members, affecting 5–10 million people worldwide .HTLV-1 is the aetiological agent of serious disorders such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukaemia/lymphoma (ATLL) . AlthougThe most important hallmarks of HAM/TSP include chronic inflammation in the cerebro-spinal fluid (CSF); increased plasma levels of pro-inflammatory cytokines ; CD4+ T cells, which continuously and spontaneously produce IFN- ϒ, TNF-α, IL-6 and IL-1β; and increased levels of CXCL10, CXCL9 and CXCR3 in the CSF, as well as numerous indicators of chronic inflammation in the CSF . In relaThe Trx system is one of the most important antioxidant systems in the body that protects the cells from degradation due to oxidative stress and also inhibits apoptosis through AP-1, NF_κB and AMPK . AccordiThe aim of this study was to evaluate the expression changes of Trx system genes  and some of the most important genes responsible for T cell proliferation and apoptosis.P-value assessment and a P-value of less than 0.05 was considered significant. Negative LogFC values indicated downregulation and positive LogFC values indicated overexpression (www.hiv.lanl.gov/content/sequence/HEATMAP/heatmap.html)  and HAM/TSP patients. The recorded expression profiles for the Trx system, BCL2, TP53, caspase 3, caspase 8 and TNFR genes of 117 patients, including 29 healthy donors, 50 ACs and 38 HAM/ TSP patients, were evaluated. GEO2R software was used to determine differentially expressed genes (DEGs) and logarithm fold change. DEGs were calculated based on Benjamini–Hochberg false discovery rate (FDR)-adjusted pression . Moreoveap.html) .We found that Trx capacity dysregulation occurred in the HAM/TSP state. Based on the DEG results, it was found that the expression of Trx reductase and BCL2 was decreased in the HAM/TSP group compared to the AC and healthy donor groups. Further, the expression levels of Trx, scavenger ROS, TNFR (Fas) and caspase 3 in the healthy group decreased steadily compared with those in the AC and HAM/TSP patients. However, the expression levels of BCL2 and TP53, which are the most important of the genes responsible for cell survival, were increased in the AC and HAM/TSP groups compared with the healthy control group .In general, our observations revealed that the Trx system function decreases in HAM/TSP patients, whereas the expression of inducer genes responsible for cell proliferation increases. Based on the available documents, apoptosis, cell survival, tissue invasion and proliferation processes are the most important signalling pathways in HAM/TSP pathogenesis . Stimulaet al., HTLV-1 Tax can suppress apoptosis through the inhibition of TP53 [As mentioned above, free radicals, particularly reactive oxygen species, are produced during the inflammation process, which causes cell damage and cell death by triggering TNFR and caspases, but these agents are resolved by the thioredoxin redox system activities . Accordi of TP53 . Therefo of TP53 .et al. have suggested that it may be appropriate to predict the progression towards the HAM/TSP by assessing the altered expression of genes responsible for the antioxidant process [et al. showed that the expression level of Trx system genes in HAM/TSP patients was significantly decreased compared to that in ACs and normal individuals [Studies about the expression changes of the genes responsible for the antioxidant function during HTLV-1 infection are quite limited. However, according to previously published reports, antioxidant capacity is reduced in HTLV-1 infection . Shomali process . Corroboividuals . The TrxFinally, the molecular signalling networks based on KEGG pathway data were proposed in order to better understand the role of the Trx system during the HTLV-1 infection .According to preset results, the thioredoxin redox system inhibits the inflammatory process and cell damage with the support of anti-apoptotic biomarkers (e.g. BCL2) and through the inhibition of various inflammatory signalling pathways, including NF_κB, JNK/STAT, MAPKs, PI3K/AKT and mTOR. This efficiently reduces the inflammatory response and development into HAM/TSP. A recent clinical trial study confirmed the anti-inflammatory, immunomodulatory and anti-proliferative effects of the reducing agents when administered with IFN-α for the treatment of HAM/TSP. Therefore, the evaluation of cellular antioxidant capacity changes during HTLV-1 pathogenesis could be a new approach in the determination of the diagnostic biomarkers of HTLV-1 infection and development of a new generation of anti-HTLV-1 drugs.


Drosophila melanogaster [The Hedgehog signaling pathway was first discovered in 1980 during a large-scale genetic screening seeking to find mutations that affect larval body segment development in the fruit fly, International Journal of Molecular Sciences, entitled “Hedgehog Signaling in Organogenesis and the Tumor microenvironment”, thus includes four original articles and five reviews that provide new insights regarding the roles of Hedgehog signaling in organogenesis and the tumor microenvironment.This Special Issue of the Tarulli et al., report on “Discrete Hedgehog Factor Expression and Action in the Developing Phallus”, and they describe a potential developmental interaction involved in urethral closure that mimics bone differentiation and incorporates discrete Hedgehog activity within the developing phallus and phallic urethra .Takebe et al., examined Gli-CreERT2; tdTomato mice, and they demonstrate that the SHH-Gli1 signaling pathway is involved in intramembranous and endochondral ossification during the fracture healing process .Takabatake et al., describe “The Role of Sonic Hedgehog Signaling in the Tumor Microenvironment of Oral Squamous Cell Carcinoma”, and their findings revealed that (1) autocrine effects of SHH induce cancer invasion and (2) paracrine effects of SHH govern parenchyma–stromal interactions of oral squamous cell carcinoma .El Shahawy et al., propose that “Sonic Hedgehog Signaling Is Required for Cyp26 Expression during Embryonic Development”, and they explain that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning .Hosoya et al., provide an overview of recent advances related to the role of SHH signaling in tooth development, homeostasis, regeneration, and the regulatory mechanism of stem cell properties in the dental mesenchyme from experiments using tamoxifen administration in iGli1/Tomato mice .Jeng et al., extensively review the recent progress made in the field of “Sonic Hedgehog Signaling in Organogenesis, Tumors, and Tumor Microenvironments”, focusing on the combined use of SHH signaling inhibitors and chemotherapy/radiation therapy/immunotherapy targeting cancer stem cells .Hyuga et al., contribute a comprehensive overview of the “Hedgehog Signaling for Urogenital Organogenesis and Prostate Cancer: An Implication for the Epithelial-Mesenchyme Interaction (EMI)” and compare possible similarities and divergences in Hedgehog signaling functions and the interaction of this signaling with other local growth factors between organogenesis and tumorigenesis. They discuss two pertinent research aspects of Hedgehog signaling: (1) the potential signaling crosstalk between Hedgehog and androgen signaling and (2) the effect of Hedgehog signaling between the epithelia and the mesenchyme on the status of the basement membrane with extracellular matrix structures located on the epithelial–mesenchymal interface .Bechtold et al., offer a thorough review of the recent progress made in studies on the roles of Indian Hedgehog signaling in temporomandibular joint (TMJ) formation, and they discuss important findings regarding the involvement of Hedgehog signaling in TMJ development during embryonic and early postnatal stages as well as in the establishment and postnatal maintenance of TMJs, plus the possible involvement of Hedgehog pathways in osteoarthritic conditions .Haraguchi et al., provide a detailed discussion about “Recent Insights into Long Bone Development: Central Role of Hedgehog Signaling Pathway in Regulating Growth Plate”, and they review the multiple roles of the Hedgehog pathway in the regulation of growth plate formation and differentiation, as well as longitudinal bone development and skeletal disorders .The Editor hopes that these articles will help readers update their knowledge about the role of Hedgehog signaling in physiology and pathology. The efforts of the authors who contributed their excellent articles to this Special Issue are greatly appreciated.