Since the discovery of the human T-cell leukemia virus-1 (HTLV-1), cellular and animal models have provided invaluable contributions in the knowledge of viral infection, transmission and progression of HTLV-associated diseases. HTLV-1 is the causative agent of the aggressive adult T-cell leukemia/lymphoma and inflammatory diseases such as the HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Cell models contribute to defining the role of HTLV proteins, as well as the mechanisms of cell-to-cell transmission of the virus. Otherwise, selected and engineered animal models are currently applied to recapitulate in vivo the HTLV-1 associated pathogenesis and to verify the effectiveness of viral therapy and host immune response. Here we review the current cell models for studying virus–host interaction, cellular restriction factors and cell pathway deregulation mediated by HTLV products. We recapitulate the most effective animal models applied to investigate the pathogenesis of HTLV-1-associated diseases such as transgenic and humanized mice, rabbit and monkey models. Finally, we summarize the studies on STLV and BLV, two closely related HTLV-1 viruses in animals. The most recent anticancer and HAM/TSP therapies are also discussed in view of the most reliable experimental models that may accelerate the translation from the experimental findings to effective therapies in infected patients. Human T-cell leukemia virus type 1 (HTLV-1), isolated in the early 1980s from T cell lines derived from patients with cutaneous T-cell lymphoma and human adult T-cell leukemia, was the first human retrovirus to be discovered . Since tHTLV-1 persistent infection is likely associated with the ability of the virus to evade the host’s immune response. Immune evasion might correlate with high proviral load and thus to disease outcome. HTLV-1 infection occurs exclusively through cell-to-cell contact, and the number of infected cells in vivo significantly impacts on viral spreading . After pThe proviral genome is integrated into the host genome and contains, in addition to structural proteins Gag, Pol and Env, a unique pX region coding for several regulatory and nonstructural proteins Tax, Rex, p12, p13, p30 and HTLV-1 basic zipper protein (HBZ), which is encoded by the antisense viral transcript. Among them, Tax and HBZ are thought to play key roles in HTLV-1 infection and oncogenesis. Transgenic mice expressing Tax or HBZ develop neoplastic diseases, indicating that they function as oncogenes ,17,18,19Although intensive studies in recent years have contributed to shedding light on the mechanisms of viral replication and host response, several aspects of HTLV-1 pathogenesis remain poorly understood, including the intimate molecular mechanism(s) of tumorigenesis, the progression of HTLV-1 leukemia toward the aggressive form, and the possibility of enhancing the host response to avoid or at least delay disease onset.Here we will discuss the complex landscape of host–viral interactions in the context of HTLV-1 infection, pointing out key experimental systems  suitable for the study of HTLV-1 viral transmission, disease pathogenesis, and treatment and their recent contribution to the advancement of knowledge of HTLV infection.Initially, most of the data related to HTLV-1 infection, mode of transmission, pathogenesis as well as the description of the cellular pathways targeted by viral proteins were derived from studies of viral factors over-expressed in cells that are not targets of infection or from viral particles produced in cells transduced with the HTLV-1 proviral genome. Subsequently, the ability of HTLV-1 to infect different types of cells in vitro and transmit the infection via cell-to-cell contact has made it possible to generate HTLV-1-producing cell lines  by coculturing leukemic cells from ATL patients with human cord blood lymphocytes isolated from healthy subjects. HTLV-1-infected T cell lines have also been established from ATL patients, such as ATL-2 cells, MT-1, ATL-T, TLOm1, ED, ATL35-T, and ATL-55T. Commonly used cell lines in HTLV studies are listed in Studies conducted in primary T lymphocytes isolated from the peripheral blood of HTLV-1-infected individuals have added important elements to the knowledge of the mechanism of HTLV-1 pathogenesis, as these cells showed increased spontaneous IL-2-dependent proliferation when cultured in vitro. However, over time, proliferation often becomes independent of (IL-2), and these cell cultures generally represent outgrowths of clones that do not predominate in the leukemic patients, but rather are selected for growth in culture. Moreover, primary ATL cells express CD3, CD4 and CD25 but not CD7 in most cases, and 10–15% express both CD4 and CD8. Generally, signal transduction and gene expression alterations observed in ATL cells were also found in HTLV-1-infected and virus-expressing T cells, although viral genes, except for antisense transcripts, such as HBZ, are not expressed in ATL cells. Another important limitation in the study of HTLV-1 is the absence of a reliable system to measure de novo infection, due to the fact that HTLV-1 cell-free virus preparations are largely not infectious . Here weAs mentioned above, HTLV-1 transmission occurs through contact with infected cells present in body fluids such as in maternal milk, semen, or blood.In vitro studies conducted by coculturing a target cell with HTLV-1-infected T cell line confirmed that cell-free virus is poorly infectious and a virus-induced specialized cell-to-cell contact, based on specific interactions between cellular and viral proteins, is needed for an efficient virus transmission . Two typThe first mode of HTLV-1 transmission in cell models was reported by Iagakura et al. (2003), who observed the formation of virological synapse (VS) between fresh peripheral blood mononuclear cells (PBMC) isolated from HTLV-1-infected patients. Confocal microscopy studies have revealed that HTLV-1 Gag, Env proteins and genomic viral RNAs were unpolarized in isolated T cells but accumulated at cell–cell junction and transferred to uninfected T cells within 2 hours after contact. The formation of VS and the transport of viral proteins towards the VS and into the target cell requires the integrity of the microtubule cytoskeleton. The polarization of the microtubule organizing centers (MTOC) of the infected cell towards the cell–cell contact may be promoted by both the viral transactivator Tax-1 and the cell adhesion molecule ICAM-1, which is also induced by Tax-1 . ConsistElectron tomography studies on CD4+ T cells isolated from HTLV-1-infected individuals and MS-9 chronically HTLV-1-infected cell line have revealed the ultrastructure of the HTLV-1 VS and the requirement for cell contact and the HTLV-1-Env protein for HTLV-1 ,35,36. VRecent studies by coculturing HCT-5 cells with salivary gland epithelial cells have suggested that VB might facilitate the initial phase of transmission of HTLV-1 virions to non-immune cells, such as salivary gland epithelial cells ,40. Cellular conduits induced in the infected cells by the accessory protein p8 represent another route for HTLV-1 transmission. In addition to its role in increasing adhesion molecules association, such as Lymphocyte Function Associated Antigen 1/Intracellular Adhesion Molecules 1 (LFA- 1/ICAM-1) interactions, the combined use of live imaging and electron transmission microscopy (TEM) demonstrated that overexpression of p8 in MT-2 chronically infected cells increases the number and length of these conduits, as well as the number of contacts between infected and uninfected cells . A recenIn vivo HTLV-1 preferentially infects CD4+ T cells co-expressing CCR4 receptor and induces functional changes in the infected cells, mainly driven by oncoprotein Tax-1 . BesidesIn fact, monocytes obtained from ATL patients differentiate poorly into monocyte-derived dendritic cells (MDDCs) in vitro, have a reduced ability to present antigen, and have altered capacities to stimulate proliferation of allogeneic T lymphocytes .In vivo, HTLV-1 spreads through two different mechanisms: neo-infection of target cells or clonal proliferation of Tax-1-immortalized cells . The actThe interaction between HTLV-1 and the host immune response plays an important role in the outcome of HTLV-1-induced diseases . The firComparatively less information is available for the effect of other RFs on HTLV-1. TRIM5α was originally discovered to be an important determinant of the resistance of monkey cells to HIV-1 infection. Indeed, rhesus monkey TRIM5α (rhTRIM5α), but not human TRIM5α, potently limits HIV-1 infection in Old World monkeys by targeting the viral capsid, thus preventing the uncoating of the viral pre-integration complex. Unlike other RFs, the activity of TRIM5α is not antagonized by an accessory viral protein, since HIV-1 had evolved its capsid to avoid recognition by human TRIM5α, although it is still susceptible to the rhesus monkey version. Rhesus TRIM5α restricts a broad range of retroviruses including HIV-1, HIV-2, N-tropic murine leukemia virus (N-MLV), and equine infectious anemia virus (EIAV).Interestingly, genome sequence analysis of peripheral blood mononuclear cells (PBMC) from both HAM/TSP patients and AC identified specific TRIM5α polymorphisms associated with proviral load (PLV), indicating a possible role of TRIM5α in HTLV-1 replication . The samIn relation to SAMHD1, recent data obtained from 22 HAM/TSP patients and 61 AC, again demonstrated that the rs6029941 (A/G) polymorphism in host dNTPase SAMHD1 is associated with increased HTLV-1 PLV in HTLV-1-infected individuals, suggesting that the polymorphism could be a factor contributing to the development of the symptoms of the disease .Besides the classical RF, cellular miR-28-3p was found to suppress viral replication and gene expression in transiently transfected cells with an HTLV-1 molecular clone, by targeting a sequence localized within the viral gag/pol genomic viral mRNA. Indeed, cells expressing a high level of miR-28-3p were found to be resistant to HTLV-1 infection, suggesting a possible antiviral function of this cellular-derived miRNA. Consistent with this hypothesis, a single nucleotide polymorphism within the miR-28-3p target site in the Japanese ATK-1 viral genome strain renders this viral strain relatively resistant to the presence of miR-28, highlighting the role of miRNA in viral transmission .Another cellular factor exerting a potent antiviral activity against HTLV-1 is the MHC class II transactivator, also designated as CIITA, the master regulator of MHC-II genes transcription. First described in our laboratory, CIITA potently suppressed HTLV-1 replication by targeting the viral transactivator Tax-1 ,65. InteThe HTLV-1-transformed cell lines , ATL-derived cells  or transiently transfected cells  are the common cell models used to investigate the role of Tax and HBZ in cell signaling deregulation ,28. NF-κComparative studies with the less pathogenic HTLV-2 homolog, which expresses a Tax-2 and antisense protein APH-2, have highlighted the exclusive properties of Tax-1 and HBZ that may account for the divergences in HTLV types linked pathobiology ,76,77. THBZ presents an N-terminal transactivation domain that lacks in APH-2 and inhibits Tax-1 activity more efficiently than APH-2 ,82,83. TIn vitro cell models have demonstrated the direct interaction of Tax-1 with several factors of the NF-κB pathway, including NEMO/IKKγ, IKKα, TAB2, TRAF6, and the cross-talk factors IKKε and TBK1 ,88,89. ITax expression is silenced in the majority of ATL due to genetic alterations in the tax gene or DNA hypermethylation of the 5’-LTR . NeverthFinally, Tax-mediated NF-κB activation may have an impact on alternative splicing events, enhancing physical and functional interaction between p65 and the DDX17 splicing factor. By constitutive activation of NF-κB pathway, Tax may promote DDX17-dependent splicing regulation enhancing DDX17/p65 recruitment in intragenic region, thus altering splicing target specificity .Animal models, including mice, rats, rabbits, squirrel monkeys, baboons, macaques, and even fruit flies, although not the natural hosts of HTLV infection, may help in elucidating some aspects of HTLV infection, persistence, host immune response, and diseases-associated developments ,106,107.Although immunocompetent murine cells are not productively infected with HTLV-1, xenograft and transgenic mice are widely used for the study of HTLV-1 infection and related diseases. Starting from the late 1990s, when the C3H/HeJ and BALB/c strains were used to establish persistent infection injecting HTLV-1-producing MT-2 cell intraperitoneally in neonatal mice, several HTLV immunocompromised mouse models have been further developed ,161,162.SCID xenograft mouse models can reproduce some features of HTLV-1 disease, such as multiple organ engraftments with ATL cells, expression of parathyroid hormone-related protein (PTHrP), a mediator of hypercalcemia in ATL patients, and increased levels of IL2 Rα and β-2 microglobulin ,165,166.NOD/ SCID mice have recently been used to evaluate a new therapeutic agent for ATL. In this study, NOD/SCID mice were injected with S1T cells, an HTLV-1-infected CD4 + T cell line derived from an ATL patient, and treated with dorsomorphin, an inhibitor of the bone morphogenetic protein (BMP) and AMP-activated protein kinase (AMPK) pathway. The administration of dorsomorphin to NOD/SCID mice proved to be efficient in reducing tumor growth . In anotAntitumor effects of autologous Tax-specific cytotoxic T cell (CTS) have also been tested in NOG mice bearing human primary ATL cells. Tax-CTL treatment led to Tax-specific CTL infiltration in the tumor site, recognition and blocking of the proliferation of autologous ATL cells and prolongation of mouse survival , althoug−/− γc −/− (Rag1) and Bone Marrow Liver Thymic (BLT) humanized mice (hu-mice) were engrafted with human CD34+ hematopoietic stem cells and were able to reconstitute human macrophages, dendritic cells, T cells, and B cells [Humanized mouse models derived from mouse xenotransplanted with human cells or engineered to express human genes may be used to study human-specific function in physiological and pathological conditions, most of them related to the human immune system . In HTLV B cells . Both mo−/− c-kit+ engrafted with human tissues and NSG mice implanted with human fetal liver, thymus tissue and stem cells (BLT mice), the authors demonstrated that these humanized mice were highly susceptible to HTLV-1 infection with the rapid polyclonal proliferation of CD4+ CD25+ T cells, similarly to the events in the healthy carrier stage of HTLV infection, although they did not reproduce the monoclonal origin of ATL, as happens in humans [Recently, humanized mice that cannot mount an adaptive immune response were obtained by injecting human umbilical-cord stem cells into the livers of immunodeficient NSG mice, and these were applied in the study of T cell tropism and lymphoproliferation of HTLV . In thesn humans . As propIn addition to the numerous studies aimed at dissecting the molecular function of the Tax and HBZ viral protein in in vitro cellular model, as summarized in Transgenic mice have been generated mostly to analyze the oncogenic potential of Tax and HBZ viral protein. Indeed, Tax expression in transgenic mice is sufficient to induce tumors, confirming the in vivo oncogenic potential of Tax ,120. An To restrict Tax expression to the thymus, Tax transgenic mice have been generated using lymphocyte-specific protein–tyrosine kinase (lck) promoters. These Lck-Tax mouse models develop lymphoma and leukemia after a long latency period of almost 18 months and present most of the characteristics of acute ATL patients ,123. TaxAs for Tax, the in vivo role of HBZ has been studied in HBZ transgenic (HBZ-Tg) mice. HBZ is the only regulatory/accessory gene encoded by HTLV-1 to be expressed in all ATL patients and necessary for the proliferation of ATL cells . Mice exPBMC-humanized NSG mice and HBZ-transgenic (Tg) mice, which develop systemic inflammation, were recently used to validate the efficacy of administration of pentosan polysulfate (PPS), a semisynthetic glycosaminoglycan, to counteract HTLV-1 infection and pathological sequelae. PPS blocked HTLV-1 infection in huPBMC NSG and suppressed the development of dermatitis and lung damage in HBZ-Tg mice, supporting the therapeutic use of PPS in the treatment of HTLV-1-induced inflammatory diseases . Tax-transgenic (Tax-Tg) and HBZ Tg mouse models have contributed to identifying functional ATL stem cells (ATLSC) and determining that c-kit, a common surface marker of ATLSCs, is a key regulator of ATL disease initiation and progression . UnexpecRat models have been useful in the study of HAM/TPS pathology. HTLV-infected Wistar-King-Apekman (WKA) rat strain develops spastic paraparesis and clinical symptoms similar to the humans with HAM/TPS . Rats haThe role of host factors in supporting viral infection has also been investigated in rat models. Human CRM1 (hCRM1) protein, a member of the importin β family, acts as a cofactor of Rex-dependent viral mRNA transport. Transgenic CRMI1 rats intraperitoneally inoculated with HTLV-1-infected cells exhibited a much higher HTLV-1 viral production than wild type rats, and presented more extensive invasion of the thymus by HTLV-1, supporting the in vitro evidence of the key role of CRM1 in HTLV-1 infection . Rat modRabbits are well established and reproducible models to study HTLV-1 transmission, immune responses, and viral determinants required for HTLV infection. Rabbits can be infected with HTLV, but do not develop HTLV-associated diseases; nevertheless, they produce a persistent infection and represent a useful animal model for studying the early steps of infection ,149,186.Recently the NZW rabbit model was also used to study epigenetic regulation of HTLV-1 gene expression in vivo, demonstrating that the CCCTC binding site present in the overlapping p12 and HBZ sequences of the HTLV-1 genome is dispensable for persistent infection . ParticuNon-human primates are susceptible to HTLV-1 infection and develop HTLV-1-associated diseases, including leukemia. Squirrel monkeys, cynomolgus monkeys, rhesus macaques, and pig-tailed macaques have been used to study HTLV immune response, viral persistence, and ATL-like disease. In squirrel monkey Saimiri sciureus injected with HTLV-1-immortalized PBMCs, the spleen and lymph nodes were shown to serve as major reservoirs for HTLV-1 . p12, p3A Tax and HBZ transgenic Drosophila melanogaster fly model was recently proposed as a suitable model for studying HTLV-I transformation, persistence, and epigenetic modification. The in vivo fly model demonstrated that Tax activates the chromatin polycomb repressive complex 2 (PRC2), which acts on the regulation of the expression of genes involved in cell survival, proliferation, or apoptosis. In this model, HBZ does not induce transformation or NF-κB activation, but its expression abolishes Tax-mediated PRC2 activation in flies expressing both Tax and HBZ ,188.Bovine leukemia virus (BLV) is a retrovirus closely related to HTLV-1 that causes B-cell lymphoma in ~5% of infected animals and has been proposed as a model for investigating the transmission, latency and pathogenesis of both BLV and HTLV ,152. In STLV-1 naturally infects non-human primates such as the Japanese rhesus macaque, Mandrillus sphinx, and Papio anubis and, like HTLV-1, causes ATL adult T-cell leukemia and lymphoma. . CompareIn Papio papio baboons naturally infected with STLV-1, it was observed that the combined treatment with valproate, an inhibitor of histone deacetylases, and azidothymidine, an inhibitor of reverse transcriptase, caused a strong decrease in the proviral load and an increase in the STLV-1 specific cytotoxic T-cell population . Due to Despite recent advances in ATL treatment, including multiagent chemotherapy, allogeneic hematopoietic stem cell transplantation, anti-CCR4 monoclonal antibody, and antiviral therapy, the ATL prognosis remains poor. Cell and animal models, although they suffere limitations with respect to replicating HTLV-1 human infection and related diseases, have been and still remain extremely useful models for identifying new key host and viral factors required for HTVL replication ad pathogenesis. It is expected that these models will be improved following the recent advancement in cell-based technologies. Genome editing by CRISPR/Cas9 system targeting HTLV integrated genomes, single-cell analyses, immunogenic peptide design, RNA-based therapy, and improvement in drug delivery are all expected to contribute to the future development of novel and more effective therapies for HTLV-1 related diseases.


Although uric acid (UA) is regarded as a risk factor for cardiovascular disease, whether UA is an independent risk factor contributing to coronary artery calcification in chronic kidney disease (CKD) is not well known. We evaluated whether UA level is associated with coronary artery calcium (CAC) score in a predialysis CKD cohort.A total of 1,350 subjects who underwent coronary computed tomography as part of the KoreaN Cohort Study for Outcome in Patients With Chronic Kidney Disease were analysed. We conducted a logistic regression analysis to evaluate the association between UA and the presence of CAC.P = 0.003). However, UA showed no association with CAC > 0 in multivariate analysis. Further analysis showed that UA showed a positive association with CAC > 0 only in estimated glomerual filtration rate (eGFR) > 60 ml/min/1.73 m2  but not in eGFR 30–59 ml/min/1.73 m2  or < 30 ml/min/1.73 m2 .CAC was detected in 705 (52.2 %) patients, and the level of UA was significantly higher in CAC > 0 patients. UA showed a positive relationship with CAC > 0 in age- and sex-adjusted logistic regression analysis (Odds ratio (OR) 1.11, 95 % confidence interval (CI) 1.04–1.19, UA level was significantly associated with CAC in early CKD, but not in advanced CKD.The online version contains supplementary material available at 10.1186/s12882-021-02463-2. Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD), accounting for more than half of the deaths . PatientCoronary artery calcification is significantly associated with the occurrence of major cardiovascular events. As measured by multi-detector computed tomography, the degree of calcification is one of the most widely accepted tools for detecting coronary atherosclerosis and estimating coronary risk assessment with high sensitivity and specificity . This toUric acid (UA), the end product of purine catabolism, has been in the spotlight for decades as a risk factor for cardiovascular disease. Many epidemiologic studies have revealed that hyperuricemia is associated with an increased risk of coronary heart disease , heart fThis was a cross-sectional study designed to assess the association between serum UA and CAC in patients with CKD. The study population consisted of participants in the KoreaN Cohort Study for Outcome in Patients With Chronic Kidney Disease (KNOW-CKD), a nationwide prospective cohort study, including CKD stage 1 to 5 nondialysis patients, that was designed to clarify the natural course and complication profiles of CKD. Details of the KNOW-CKD study’s rationale and design have been described previously  and the Baseline demographics and laboratory data were retrieved from the electronic data management system  with assistance from the Division of Data Management at Seoul National University Medical Research Collaborating Center. Socio-demographic characteristics, smoking history, medication, and comorbidities were collected at the time of enrollment. Anthropometric indexes including height, weight, waist circumference, hip circumference, and resting blood pressure (BP) were measured in the clinic. Laboratory data were analysed at the hospital laboratory of each participating centre. Serum samples were collected and sent to the central laboratory  to measure creatinine levels. The estimated glomerular filtration rate (eGFR) was calculated through the CKD-EPI equation .2 or more is in the coronary artery position, it is determined as positive. In each lesion, 1 point for 130 to 199 HU, 2 for 200 to 299 HU Points, 3 points for 300 ~ 399 HU, 4 points for 400 or more, were measured as the sum of these values.CAC score was evaluated at each research institution by an electron-beam computed tomography (CT) scanner or a multi-detector CT. Specifically, SOMATOM Definition Flash , SOMATOM Force , Revolution CT , Discovery CT750 HD , Brilliance iCT 256 slice , Brilliance 64 CT scanner , IQon Spectral CT  were used. The quantitative CAC score was assessed using the Agatston score ; If a leP-value < 0.05 was considered statistically significant. The adjusted odds ratios of CAC > 0 associated with risk factors were estimated using logistic regression analysis. Only covariates that were significant (p < 0.05) were retained in the multivariable analysis. The independent association between UA and CAC was also evaluated using restricted cubic splines. To visualise the association of UA and CAC, selected knots automatically, cubic splines were drawn by the “gam,” “mgcv” packages in R. Statistical analysis was performed using R software (version 3.2.2).Continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as numbers and proportions. The comparisons were made using the independent t-test or analysis of variance for continuous variables and the chi-squared test for categorical variables. A This study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the institutional review board at each participating hospital, including Seoul National University Hospital (1104-089-359), Seoul National University Bundang Hospital (B-1106/129-008), Yonsei University Severance Hospital (4-2011-0163), Kangbuk Samsung Medical Center (2011-01-076), Seoul St. Mary’s Hospital (KC11OIMI0441), Gil Hospital (GIRBA2553), Eulji General Hospital (201105-01), Chonnam National University Hospital (CNUH-2011-092), and Pusan Paik Hospital (11–091). All participants provided written informed consent.2, and the serum UA level was 7.0 ± 1.9 mg/dL. Of these, 645 (47.8 %) belonged to CACS 0 and 705 (52.2 %) belonged to CACS > 0. Table The mean age was 53.5 ± 11.9 years, and 758 (56.2 %) were men. Seventy (5.2 %) had a history of coronary artery disease, 499 (37.0 %) had diabetes mellitus, and 1286 (95.3 %) had hypertension. The mean eGFR was 56.7 ± 32.4 ml/min/1.73 mWe further divided as CAC 0, > 0–100, > 100–400, > 400. The participants with high CAC score were older and had higher proportion of male, coronary artery disease, diabetes mellitus and hypertension. The level of eGFR, calcium, LDL cholesterol, and HDL cholesterol were lower, whereas uric acid, phosphate, PTH, and 24 h urine protein were higher according to higher CAC level (Supplementary Table P = 0.003) was significantly associated with having CAC > 0. However, on multivariate logistic regression analysis, adjusted for age, sex, coronary artery disease, diabetes mellitus, use of lipid-lowering agents, mean BP, waist-hip ratio, eGFR, calcium, phosphate, LDL-cholesterol, HDL-cholesterol, PTH, and 24-hour urine protein, UA showed no association with CAC > 0 . History of coronary artery disease, history of diabetes mellitus, use of lipid-lowering agents, mean BP, and phosphate levels were positively associated with CAC in multivariate analysis.Table 2. UA level showed a positive association with CAC > 0  only in estimated GFR > 60 ml/min/1.73 m2  or 30 ml/min/1.73 m2 . Restricted cubic splines of UA on the CAC > 0, stratified by eGFR, showed that CAC increased steadily with higher UA levels only in estimated GFR > 60 ml/min/1.73 m2 (Fig. We further divided the participants by eGFR ≥ 60, 30–59, and < 30 ml/min/1.73 mIn the present study, we showed that over half of the CKD population had CAC. The patients with CAC > 0 had higher UA levels than those with CAC 0, and UA showed a positive relationship with CAC > 0 in age- and sex-adjusted logistic regression analysis. However, UA showed an association with CAC > 0 in multivariate-adjusted logistic regression analysis only in the early stage of CKD, and this association disappeared in advanced CKD.UA, formerly considered a significant antioxidant in humans, has been regarded as a risk factor for cardiovascular disease in the past two decades. Previous studies have shown that elevated UA levels could predict the severity and morphology of coronary atherosclerosis  and elevHowever, UA failed to show an association with CAC in patients with CKD, not only in this study population but also in another CKD cohort . The autUnlike UA, phosphate showed a positive association with CAC throughout the whole stage of CKD in this study. Phosphate is an integral component of hydroxyapatite, the calcium mineral seen in bone and vascular calcification. It is also a part of the signalling cascade that triggers vascular calcification . CalcifiThis study has some limitations. This is a cross-sectional study; therefore, we could not identify a longitudinal relationship between UA and CAC. Moreover, this study could not confirm that UA is associated with cardiovascular events in patients with CKD. CAC is a complex process; therefore, there could be confounders that we could not consider. However, this study is the first, to the best of our knowledge, to elucidate that UA showed a different relationship with CAC according to CKD stages.The UA level was significantly associated with CAC in early CKD but not in advanced CKD. Longitudinal studies are required to confirm the association between UA and CAC and the effect of UA lowering therapy for CAC in patients with CKD.Additional file 1: Supplementary table 1. Baseline characteristics of participants according to CAC 0, > 0-100, > 100-400, > 400.


Metopimazine (MPZ) is a peripherally restricted, dopamine D2 receptor antagonist used for four decades to treat acute nausea and vomiting. MPZ is currently under clinical investigation for the treatment of gastroparesis (GP). MPZ undergoes high first‐pass metabolism that produces metopimazine acid (MPZA), the major circulating metabolite in humans. Despite a long history of use, the enzymes involved in the metabolism of MPZ have not been identified. Here we report a series of studies designed to identify potential MPZ metabolites in vitro, determine their clinical relevance in humans, and elucidate the enzymes responsible for their formation. The findings demonstrated that the formation of MPZA was primarily catalyzed by human liver microsomal amidase. Additionally, human liver cytosolic aldehyde oxidase (AO) catalyzes the formation of MPZA, in vitro, although to a much lesser extent. Neither cytochrome P450 enzymes nor flavin‐monooxygenases (FMO) were involved in the formation MPZA, although two minor oxidative pathways were catalyzed by CYP3A4 and CYP2D6 in vitro. Analysis of plasma samples from subjects dosed 60 mg of MPZ verified that these oxidative pathways are very minor and that CYP enzyme involvement was negligible compared to microsomal amidase/hydrolase in overall MPZ metabolism in humans. The metabolism by liver amidase, an enzyme family not well defined in small molecule drug metabolism, with minimal metabolism by CYPs, differentiates this drug from current D2 antagonists used or in development for the treatment of GP. Metopimazine acid is the predominant circulating metabolite, present at many fold the plasma concentrations of the parent, and yet the metabolic route to that metabolite has not been defined. Through a series of studies, we identified potential MPZ metabolites in vitro, determined their clinical relevance in humans, and elucidated the enzymes responsible for their formation. ABT1‐aminobenzotriazoleAOaldehyde oxidaseAUCarea under the concentration‐time curveBNPPbis‐p‐nitrophenyl phosphateCYPcytochrome P450FAAHfatty acid amide hydrolaseFMOflavin monooxygenaseGPgastroparesisHKMhuman kidney microsomesHLChuman liver cytosolHLMhuman liver microsomesKPipotassium phosphate bufferLC–MSliquid chromatography‐mass spectrometryMPZmetopimazineMPZAmetopimazine acidMPZHmetopimazine hydroxylated metaboliteMPZSmetopimazine sulfoxideNADPHPAR, peak area ratioUGTuridine 5’‐diphospho‐glucuronosyltransferase1Metopimazine (MPZ) is an approved drug in France under the brand name Vogalene®  that has been used for many years for the short‐term treatment of nausea and vomiting. A new salt formulation of MPZ is in clinical development in the US for the treatment of gastroparesis, a chronic disorder of the stomach characterized by delayed gastric emptying without evidence of mechanical obstruction.D2/D3 selective, dopamine receptor antagonist that is peripherally restricted.While much is known about the pharmacology of MPZ, the metabolism of this drug is not fully elucidated. MPZ is deaminated to form metopimazine acid (MPZA), the major circulating metabolite, present at much higher plasma concentrations than the parent. At clinical doses of 20 and 50 mg of MPZ, MPZA accounted for 80% or more of the circulating drug‐related material in plasma following a single dose taken either preprandially or postprandially.In this paper, we describe the multidisciplinary approach designed to determine the main human metabolites and elucidate the enzymes involved in MPZ metabolism. This entailed a thorough in vitro characterization of metopimazine metabolites derived from cellular and in vitro incubations as well as exploring the clinical relevance by looking for these metabolites in samples of healthy volunteers who were administered a 60 mg single dose of metopimazine. The limited role of cytochrome P450s in the metabolism of metopimazine has been more thoroughly defined. Most importantly, using a series of recombinant enzymes, inhibitor phenotyping and a variety of conditions, we identified the enzymes involved in the first pass metabolism of MPZ to form MPZA.2The following experiments were performed in an exploratory manner.2.1MPZ, MPZA, and metopimazine sulfate (MPZS) were supplied by Pacific Pharmaceutical Services. Bupropion, dextromethorphan, diclofenac, midazolam, paclitaxel, phenacetin, testosterone, itraconazole, montelukast, quinidine, sulfaphenazole, benzydamine, bis(4‐nitrophenyl) phosphate (BNPP), 7‐ethoxycoumarin (7‐EC), menadione, methimazole, phthalazine, and β‐nicotinamide adenine dinucleotide phosphate  were purchased from Sigma‐Aldrich. Chlorzoxazone and 1‐aminobenzotriazole (ABT) were purchased from Tokyo Chemical Industry. Coumarin was purchased from Acros Organics. (S)‐Mephenytoin, (+)‐benzylnirvanol, and tranylcypromine were purchased from Toronto Research Chemicals. Furafylline was purchased from Cayman Chemical. Pooled human liver microsomes  and human kidney microsomes  were purchased from BioIVT. Pooled human liver S9 (50‐donor mixed gender) and human intestine S9 (10‐donor mixed gender) fractions and pooled human liver cytosol  were purchased from Xenotech. Supersomes™ CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, FMO1, FMO3, and FMO5, were purchased from Corning Inc.. CTL P450 (Supersomes™ containing only P450 reductase and cytochrome b5) was also purchased from Corning, Inc., and used to normalize the protein content in the CYP reactions. Upon receipt, all test systems were stored at ≤−70°C until use.2.22.2.1®, Millipore) to 100 mM. The 100 mM potassium phosphate buffer was prepared by adding the monobasic potassium phosphate solution (100 mM) to dibasic potassium phosphate solution (100 mM) until the pH value adjusted to 7.4, 8.4, or 9.4. The resulting 100 mM potassium phosphate buffer (KPi) was stored refrigerated (4°C). The NADPH solution  was prepared fresh in KPi buffer prior to use and used in the reactions at a final concentration of 1 mM.Monobasic potassium phosphate (1 M) and dibasic potassium phosphate (1 M) stock solutions were diluted with water (Milli‐Q2.2.2Stock solutions of MPZ at 20 mM (Lot No. AP237759063‐076‐01) were prepared in DMSO and stored at −20°C until use. Prior to incubations, 1 or 10 µM of MPZ  was prepared. Primary stock solutions (Stock A) of positive control probe (7‐EC) were prepared in ethanol and stored at −20°C until use. The concentration of Stock A was 100 mM. On the day of the experiment, subsequent working solution of the positive control was prepared (from Stock A) at 100× the incubation concentration using appropriate buffer (Stock B).Primary stock solutions (Stock A) of positive control probe  were prepared in DMSO and stored at −20°C until use. The concentration of Stock A was 100 mM (for benzydamine) and 1 mM , respectively. On the day of the experiment, subsequent working solution of the positive control was prepared (from Stock A) in KPi buffer (Stock B).2.2.3The clinical study was conducted at Biotrial Rennes, in accordance with the Declaration of Helsinki. The study (NG100‐101) was a double‐blind, placebo‐ controlled, ascending single‐ and multiple‐dose study of the safety, tolerability, and pharmacokinetics of metopimazine administered orally to healthy adult subjects. The study protocol, informed consent form, and appropriate related documents were approved by the institutional review board, and all subjects provided written informed consent prior to participation in the study.In this study, sequential ascending single doses were administered to 4 cohorts of 8 participants . In the morning, after at least 10 h of fasting overnight, eight healthy adult subjects , were orally administered metopimazine (or a matching placebo). Cohort 1–4 received a matching placebo or ascending doses of Vogalene (metopimazine) capsules at the doses of 15, 30, 45, or 60 mg, respectively. Blood samples were collected at the following time points: predose and 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 3, 4, 6, 8, 12, 18, and 24 h postdose; plasma samples were obtained by centrifugation of the blood samples. A comprehensive safety and pharmacokinetic analysis was performed for each cohort for the SAD and MAD parts of the study and will be presented elsewhere. A fit‐for‐purpose discovery method was used to analyze 4 subjects from the highest single dosed cohort for the presence of metabolites.Plasma concentrations of metopimazine, metopimazine acid and metopimazine sulfoxide were quantified using LC–MS/MS methods.2.2.4g. After centrifugation, supernatant was removed and stored frozen in a −70°C freezer until analyzed by LC–MS/MS.The pharmacokinetics of metopimazine (parent) and metopimazine acid (metabolite) were evaluated in male and female Sprague Dawley rats following a single oral dose of 45 mg/kg metopimazine (free base). Three animals per sex in each dose group were sampled at each timepoint after administration . The rats (rattus norvegicus) were obtained from Envigo RMS, Inc. and were 2 months old. Animals were weighed prior to each dose administration for the purpose of dose calculation. Whole venous blood samples of approximately 0.5 ml were collected from a peripheral vein of animals for determination of exposure to metopimazine and metopimazine acid. Three animals per sex were sampled at each timepoint. Samples were collected in tubes containing K2EDTA with protease inhibitor cocktail  and placed on ice until processed for plasma by centrifugation under refrigeration for at least 10 min at 1912 x 2.2.5g. After centrifugation, the supernatant was removed and stored frozen in a −70°C freezer until analyzed by LC–MS/MS.The pharmacokinetics of metopimazine (parent) and metopimazine acid (metabolite) were evaluated in three male, beagle dogs following a single oral dose of 10 mg/kg metopimazine (free base). The dogs were obtained from Marshall BioResources. Animals were weighed prior to each dose administration for the purpose of dose calculation. Metopimazine was administered in size 13 gelatin capsules. Whole venous blood samples of approximately 1.0 ml were collected from a peripheral vein of all animals for determination of metopimazine and metopimazine acid. Samples were collected at the following timepoints: 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h after test article administration. All animals were sampled at each timepoint. Samples were collected in tubes containing K2EDTA and placed on ice until processed for plasma by centrifugation under refrigeration for at least 10 min at 1912 x 2.2.62 followed by the addition of NADPH (1 mM) to initiate the reaction. The reaction was incubated for 0 and 60 min. The total percentage of organic solvent in the incubation mixture was ≤0.1%. The reactions were terminated by the addition of sample mixture of acetonitrile and ethanol . The positive control substrate, 7‐EC, was incubated in triplicate in an identical manner as MPZ for S9 fractions.An initial experiment was conducted with S9 fractions from liver and intestine to assess the role of liver and intestinal enzymes in the metabolism of MPZ. The reaction mixture contained 2.5 µM MPZ, pooled human liver or intestine S9 fraction (1 mg/ml S9 protein), 100 mM KPi buffer (pH 7.4). The reaction mixture was pre‐incubated for 3–5 min at 37°C in a humidified incubator filled with 5% CO2.2.7To conduct the metabolic incubation in liver microsomes in the linear range, protein concentration and the reaction time were optimized.Optimization of protein concentration: For protein concentration optimization, MPZ at 1 µM was incubated with varying concentration of HLM proteins , 100 mM KPi buffer (pH 7.4). After pre‐incubation for 3–5 min at 37°C, the reaction was initiated by the addition of NADPH (1 mM) for 60 min in triplicates.Optimization of Incubation Time: For incubation time optimization, MPZ at 1 µM was incubated with pooled HLM  and 100 mM KPi buffer (pH 7.4). After pre‐incubation for 3–5 min at 37°C, the reaction was initiated by the addition NADPH (1 mM) incubated for 0, 10, 20, 40, 60, and 90 min to determine the optimal incubation time (linear range). All metabolic reactions in this study were terminated by the addition of chilled organic solvent  containing internal standards, unless otherwise indicated.2.2.8To determine the role of microsomal CYP enzymes in the metabolism of MPZ, MPZ was incubated with pooled HLM at 1 and 10 µM in triplicate and the disappearance of MPZ and the formation of known metabolites  was assessed.Initially, two sets of incubations were conducted with HLM , 1 and 10 μM MPZ, 100 mM KPi buffer (pH 7.4) where one set was in the presence and the other set was in the absence of 1 mM ABT, a non‐specific CYP inhibitor for 60 min. This allowed the assessment of total CYP contribution for MPZ metabolism. Then, CYP‐specific chemical inhibitors were used to assess the contribution of individual major CYP‐isoforms where HLM were incubated with 1 or 10 μM MPZ in the presence and in the absence of each CYP specific inhibitor at 37°C for 60 min. The following CYP‐isoform specific chemical inhibitors were used; furafylline (10 µM), tranylcypromine (10 µM), montelucast (0.5 µM), sulfaphenazole (5 µM), (+)‐benzylnirvinol (5 µM), quinidine (1 µM) and itraconazole (2.5 µM), for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5. Parallel incubation with seven CYP specific probe substrates  were included in as positive controls. For negative control reaction, MPZ was incubated at 37°C in duplicate in a reaction mixture devoid of NADPH. All other procedures were the same as described previously.2.2.9Recombinant CYP isoforms (Supersomes™) were used to determine the contribution of CYP isoforms in the metabolism of MPZ. MPZ at 1.0 µM was incubated in triplicate with individual CYP Supersomes™ . The final P450 content in the incubation mixtures was 50 pmol/ml. Protein concentrations for all CYP isoforms were normalized to the recombinant CYP containing the highest protein concentration. The reaction mixture containing MPZ or CYP substrates, KPi buffer (pH 7.4), and CYP‐ enzyme was pre‐incubated at 37°C for 3–5 min. All other procedures were the same as described previously. The marker substrates  were incubated at 1.0 µM in parallel. The negative control for incubation reactions incubated in the absence of NADPH under identical conditions.2.2.10MPZ, at 1.0 and 10.0 µM, was incubated with HLM or HKM to determine the contribution of microsomal FMOs on the metabolism of MPZ.A reaction mixture containing KPi buffer (pH 8.4), HLM or HKM (0.5 mg/ml protein) and MPZ (1.0 and 10.0 µM) was pre‐incubated for 3–5 min at 37°C. The reaction was initiated by the addition of NADPH into the reaction mixture. The reaction was conducted for 0 and 60 min. A positive control  was incubated in duplicate for 0 and 20 min. Parallel incubation was conducted by adding 1 mM methimazole, a non‐specific FMO inhibitor, under identical conditions. Heat‐inactivated microsomes (50°C for at least 5 min) was incubated under identical conditions negative control. All other procedures were the same as described previously.2.2.11MPZ at 1.0 µM was incubated in triplicate with individual recombinant FMO . Protein concentrations for all rFMO isoforms were 0.05 mg/ml. A positive control  was incubated for 20 min. The reaction mixture containing MPZ or FMO substrate, KPi buffer (pH 8.4), and FMO Supersomes™ was pre‐incubated at 37°C for 3–5 min. All other procedures were the same as described previously. The negative control reactions were incubated absence of NADPH.2.2.12t = 0 min incubation. Then 10 µM MPZ was added into the remaining mixture and incubated at 37°C on an orbital shaker for 60 min. A parallel set of incubations was conducted with 10 µM MPZ in the presence at 2 and 16 µM, of BNPP (a known inhibitor of amidase). All reactions were in triplicate. The total percentage of organic solvent in the incubation mixture was ≤0.1%. The reactions were terminated by the addition of chilled acetonitrile: methanol, 90:10, (v/v). The samples were vortex‐mixed and stored at ≤−70°C and were subjected analyzed by LC–MS/MS.Prior to the reaction for amidase activity, the reaction mixture containing KPi buffer  and HLM was pre‐incubated at 50°C for 5 min to inactivate CYP and FMO activities in HLM. The mixture was placed on ice to cool down. An aliquot was taken out which was considered as 2.2.13The metabolism of MPZ was evaluated in the human liver cytosolic incubation. A metabolic incubation was performed with HLC (1 mg/ml protein) and 10 µM MPZ at 37°C for on an orbital shaker for 60 min. A parallel set of incubations were also conducted 10 µM MPZ in the presence of 100 µM menadione, a known inhibitor of human aldehyde oxidase. Another set of incubation was also conducted 1 µM phthalazine, a known substrate for aldehyde oxidase as positive control, under identical conditions.2.2.14® tubes. It was important to avoid hemolysis during blood collection. After collection, the blood samples were centrifuged within 60 min, at 3000 rotations per minute for 10 min at 5°C. The resulting plasma was separated into 2 equal aliquots of 1000 µl  and transferred to polypropylene tubes, which were labeled, frozen within 120 min of blood collection and stored at –70°C until required for bioanalysis. Immediately prior to bioanalysis, samples were thawed and a 200 µl aliquots were removed and analytes extracted using acetonitrile protein precipitation. The extracted samples were analyzed twice, once for quantification of MPZ and MPZA and again for MPZS quantification as described below.Venous blood samples (6 ml) for the determination of plasma concentrations of MPZ and metabolites (MPZA and MPZS) were drawn by direct venipuncture or via an intravenous catheter into K2EDTA Vacutainer2.2.15An MRM LC–MS/MS method was developed for MPZ and metopimazine acid (MPZA) in rat, dog, and human plasma. The method was run in reverse phase in positive ion mode. Using an LC–MS/MS system that consisted of a Shimadzu Nexera X2 LC‐30 UPLC system (Shimadzu Corporation), and an API 4000 triple quadrupole mass spectrometer (SCIEX) running Analyst software 1.4. LC separation conditions were achieved for MPZ and MPZA using a Supelco Discovery HS F5 50 × 4.0 mm 3 µm column (Sigma‐Aldrich). D6‐isotopically labeled internal standards were used for each analyte. The following m/z transitions were monitored: metopimazine 446.3 → 141.1 and metopimazine acid 447.2 → 142.1. A calibration curve was generated for the quantification of MPZ and MPZA.2.2.16An MRM LC–MS/MS method was developed to look for metopimazine sulfoxide (MPZS) in human plasma. The method was run in reverse phase in positive ion mode. The system was identical to what was described above except that a slightly longer column was used, a Supelco Discovery HS F5 100 × 4.0 mm (3 µm) column (Sigma‐Aldrich). A D6‐isotopically labeled internal standards was used for MPS and the m/z transition monitored was 462.3 → 98.1. A calibration curve was generated for the quantification of MPZS in human plasma.2.2.17LC separation conditions were developed for MPZ and its metabolites (MPZA and MPZS), 7‐hydroxycoumarin, each metabolite of CYP probe substrate, benzydamine N‐oxide, and phthalazine. The LC–MS/MS system consisted of a Shimadzu Nexera X2 LC‐30 UPLC system (Shimadzu Corporation), and an API 4000 triple quadrupole mass spectrometer (SCIEX) was used for analysis. All analytes were analyzed using electrospray ionization (ESI) in positive ion mode except for 7‐EC, which used negative ion mode. The LC–MS/MS system was controlled by Analyst software .For each analytical method, a calibration curve was generated for the quantification of MPZ and its two metabolites (MPZA and MPZS). Due to lack of the reference standard, the hydroxylated metopimazine (MPZH) was determined qualitatively based on peak area ratio (PAR) of presumed hydroxylated analyte to the IS. PAR was also used for all positive control compounds.2.2.18t = 0, 100%). The percent of parent remaining values were calculated according to the following equation:The degree of disappearance of each analyte was evaluated by comparing the PAR of analyte to IS or concentration in different sampling time points to the PAR of time zero,  presented in the data tables are based on “precision as displayed” numbers . GraphPad Prism (version 8) was used for figure generation.Noncompartmental Pharmacokinetics analysis was performed in Phoenix 64 Winnonlin version 8.1.2.2.19http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY,Key protein targets and ligands in this article are hyperlinked to corresponding entries in 33.1Upon incubation of MPZ with HLM, three primary metabolites were found on full scan LC–MS analysis. Based on peak area, the largest signal was found at m/z 447.14, which corresponded to MPZA, the major in vivo metabolite in humans. Two other isobaric peaks were found at m/z 462.15, consistent with MPZH and MPZS metabolites . To look for the presence of these metabolites in vivo, plasma was sampled over 24 h from 4 subjects from the highest SAD cohort. Following a single oral dose of 60 mg of metopimazine, the mean clinical exposure parameters are provided in Table 3.3Given that MPZA was the predominant circulating metabolite in humans, bioanalytical methods were developed to quantify both parent and the major human metabolite. To determine the pharmacokinetic properties of metopimazine and its acid metabolite in rats and dogs, single doses of metopimazine (free base) were administered orally to each species and plasma samples were taken over time and analyzed. A noncompartmental analysis of the bioanalytical data was performed and the pharmacokinetic parameters determined. Table Interestingly, the MPZA metabolite to parent ratio is far lower in dogs and particularly lower in rats than in humans, indicating MPZA is disproportionately produced in humans.3.4To determine the involvement of cytochrome P450 enzymes, MPZ was incubated in HLM at 1 and 10 μM in the presence and absence of 1‐ABT, a known non‐specific CYP inhibitor, and with and without NADPH cofactor addition to the incubation mixture. As shown Figure 3.5Since MPZA formation was not mediated by cytochrome P450 enzymes, other enzymes, FMO and hydroxylase, were investigated. To determine the contribution of microsomal FMOs on the metabolism of MPZ, incubations of MPZ were done in mixed, pooled HLM or HKM at 1 and 10 µM in triplicate in the presence of NADPH at pH 8.4 (a pH that should minimize CYP involvement) with and without an FMO inhibitor (methimazole). An HLM control was included in which HLM was pre‐incubated at 50°C for 5 min to inactivate CYP and FMO activities. Considerable formation of MPZA was observed with all conditions in HLM at pH 8.4 and was increased with increasing concentration of MPZ.Incubation of MPZ with HKM at pH 8.4 did not produce any metabolites Figure . TogetheIn order to track down what hydrolase activity was responsible for the metabolism of MPZ to MPZA, an HLM incubation was performed under previous conditions except at increasing pH values 7.4, 8.4 and 9.4. MPZA formation was actually faster the higher the pH, including very strong formation at pH 9.4, consistent with amidase activity Figure . In addiThe formation of MPZA was also observed in the incubation of 10 μM MPZ in human liver cytosol (HLC). HLC catalyzed the formation of some MPZA over 60 min of incubation, which was inhibited (48%) when 100 μM menadione, a known inhibitor of human AO, was included in the incubation Table . These fThese results clearly demonstrate a plausible MPZ metabolic scheme in which the major in vivo metabolite, MPZA, is catalyzed by a liver amidase with some potential contribution from aldehyde oxidase. The oxidative metabolites are catalyzed by CYP3A4 and CYP2D6 in vitro but are very minor pathways in vivo and have a negligible impact on human metabolism and clearance Figure .4Clinical pharmacokinetic studies with MPZ have demonstrated that MPZA is the major in vivo metabolite in humans and based on AUC exposure, it accounts for over 90% of circulating drug‐related material.In this study, MPZ was shown to undergo hydrolysis in human liver microsomes to form MPZA. This conversion of MPZ to MPZA is not inhibited by any individual CYP inhibitor, including ABT, a non‐specific inhibitor of CYPs. This activity is not dependent on NADPH and increases under conditions above physiologic pH. Amidase/hydrolases, such as fatty acid amide hydrolase (FAAH) are known to have optimal activity in the pH range of 8.5–10.There are over 100 human genes that encode hydrolases that include esterases, amidases, peptidases and other enzymes.Amidases, a class of hydrolase enzymes, are ubiquitous and their functions vary widely.While liver amidase conversion appears to be the primary biotransformation enzyme involved in MPZ metabolism, another enzyme that may contribute to the formation of MPZA is the cytosolic enzyme AO. We observed the conversion of MPZ to MPZA in HLC, though to a much lesser extent than HLM. This activity was moderately inhibited by menadione, a known inhibitor of AO, suggesting the possible contribution of AO to MPZ metabolism. AO is a metalloenzyme that contains molybdenum cofactor and typically shows hydroxylase activity.Although the hydrolysis of the amide bond in metopimazine appears to be catalyzed to a greater degree by a microsomal liver amidase, cytosolic AO can catalyze this reaction in vitro. While AO is known to catalyze a wide variety of biotransformations that include both oxidase activity and reductase activity,We have demonstrated that the formation of MPZA from MPZ is primarily catalyzed by human liver amidase, with the potential for contribution from human liver cytosolic aldehyde oxidase, however to a much lesser extent. Clinical studies have demonstrated that conversion of MPZ to MPZA is by far the major metabolic process in humans, although this is not the case in the rat and the dog. Human liver cytochrome P450 enzymes are not involved in the formation of this major metabolite; however, they do appear to convert MPZ to 2 minor metabolites in vitro. Analysis of clinical samples from subjects receiving a 60 mg single dose of metopimazine indicates that MPZS is a very minor circulating metabolite and if MPZH is present it is only in transient trace amounts.MPZS is predominantly catalyzed by CYP3A4. The metabolic scheme in humans is shown in Figure Liver amidases are not commonly thought of as drug metabolizing enzymes for small molecules and there is a dearth of literature on predicting human clearance when amide hydrolysis is the principal metabolic route. As additional examples of amide hydrolysis appear, species differences may be better understood and potential in vitro to in vivo extrapolation (IVIVE) approaches may be developed. In the case of metopimazine, the fact that amide hydrolysis is the main step in human MPZ metabolism contrasts markedly with other drugs used or in development for the treatment of GP. This may make combination and concomitant therapies more feasible for the treatment of GP with less drug‐drug interaction possibilities.Dr. Busby has received consulting fees from Neurogastrx. Drs. De Colle and Wax have stock options in Neurogastrx.Busby, De Colle, Sadeque, Shen participated in research design. Cai, Yang, and Ramos conducted experiments. Cai, Venkatarangan, and Sadeque performed data analysis. Busby, Cai, De Colle, Sadeque, Shen, Wax and Yang wrote or contributed to the writing of the manuscript.This paper is the original and authentic work of the authors. All authors read and approved the final manuscript.


Greece has a mean age of first motherhood at 31.5 years, higher than the European average age of 29.4. Delaying conception, however, may be an important non-reversible cause of infertility. The aim of this study was to identify possible knowledge deficits regarding fertility in young adults.This was an online survey of young adults, regarding information on intention to parenthood and knowledge on issues affecting fertility. This study was conducted from February to December 2020, aiming for a representative sample of Greek men and women aged 18 and 26 years. The questionnaire was designed by a multidisciplinary group based on the Cardiff Fertility Knowledge Scale, which contained 22 multiple-choice or Likert-scale questions.We obtained responses from 1875 young adults, whose mean age was 22.1 years. About 91.8% of men and 94.0% of women declared an intention to have children, out of which 44.0% wanted to have two and 29.0% three children. About 52.0 and 50.8% men and women, respectively, aimed to start a family between 31 and 35 years. Residents of rural areas and those with a lower education level more likely aimed to have children before the age of 30. The most prevalent answers for age of ideal parenthood were between 26 and 30 years for a woman and 31–35 years for a man. Smoking, alcohol consumption and sexually transmitted infections were identified as factors affecting both female and male fertility. Half of men and women, respectively, overestimated general success rates of reproductive techniques.The knowledge of fertility, particularly with regards to assisted reproductive techniques’ success rates, may be overestimated as more young adults plan for having children after the age of 30. According to recent data, Greece is among the countries presenting the highest rates of first childbirth among women at age 40 and over, estimated at 5.3%, with the mean age of motherhood steadily increasing and is currently 31.5 years, while the European average age is 29.4 years  may offer a solution, they provide by no means a guarantee. Still, the media are overwhelmed by news of celebrities and social influencers who have their children during the fifth or- sometimes sixth- decade of life . This inPromoting reliable information and realistic expectations regarding the reproductive ability should empower young people to make true informed choices about their future fertility, thus deciding or not to delay childbearing. A recent report on fertility awareness among medical students in three European countries showed that Greek students were relatively realistic about the prospects of fertility decline with age. This, however, may reflect the level of information that is obtained in the obstetrics and gynaecology curriculum, rather than knowledge and attitudes applicable to the general population . The aimThis was an online survey of fertility awareness among young men and women aged 18–26 years in Greece.A multidisciplinary team, consisting of obstetricians and gynaecologists, fertility specialists, academic teachers and statisticians, discussed the aim of the study and the formulation of a questionnaire. A thorough literature search was performed to identify similar studies and questionnaires that could be used as a basis of the study. We based the questions on the Cardiff Fertility Knowledge Scale , 8, but The final questionnaire was anonymous and comprised of 22 questions. Out of them, five questions were on demographics , four questions regarding the intention to have children, four questions regarding factors that affect male and female fertility, along with the ideal age to father or bear a child and finally nine questions regarding ART, including egg donation, egg freezing and success rates of ART, in general, and in women over the age of 40 years.The questionnaire was piloted for brevity and clarity in a sample of 20 young adults, and final corrections were made accordingly. It was then transcribed in an electronic format via google forms platform for easier dissemination.Cronbach’s alpha coefficient for the questionnaire omitting the five questions regarding demographics- was 0.79.The study protocol received ethical approval from the scientific board of Alexandra General Hospital (Reference No. 693/02-09-2019). At the onset of the electronic form, we included information for the participant and an option to proceed to the questionnaire after checking an online consent form. The questionnaire was anonymous and did not ask for sensitive personal information.In order to achieve a representative sample of the relevant population, we calculated the minimum number of participants, so as to achieve a 95% confidence interval and a margin of error of 3%. We thus aimed for a minimum sample of 1,067  for analysis.Descriptive statistics were used to describe demographic variables and fertility knowledge. Categorical data were compared using chi-squared tests. T-tests were used to compare scores of knowledge depending on the gender. A Our sample consisted of 1,875 participants, of which 1,133 (60.4%) were women and 742 (39.6%) men. Out of them, 21 (1.1%) already had children, and their demographics are shown in About 91.8% of men and 94.0% of women revealed that they want to have children in the future, and out of them, 120 (6.4%) would want to have one, 821 (43.8%) two, 375 (20.0%) three and 126 (6.7%) more than three children. There was no statistical difference between men and women in their intention.Subsequently, when asked about the likely age they would have children, 52.0 and 50.8% of men and women, respectively, aimed to have children between 31 and 35 years. Those who wanted to have more than three children planned to have them before the age of 30. However, there were 224 participants (11.9%), of which 118 women, who wished to have three or more children after the age of 30.P = 0.014). Similarly, secondary school graduates were statistically significantly more likely to want to have children under the age of 30, in comparison with university students or graduates . There was no statistically significant difference in the number of children planned, depending on the place of residence or education level.When comparing participants according to place of residence, those residing in a rural area were more likely to aim to have children before the age of 30 than those residing in urban centres  and 31–35 years for a man (47.0%). Slightly more men than women agreed that the ideal age for a woman to have children is between 26 and 30 years of age . However, 41.2% of women (484) were seeing themselves likely to have children in this age margin, and 56.0% were more likely to have children after the age of 30  or previous abortions were considered to play a significant role in male and female fertility. It is, however, notable that prior use of condoms was among the least stated factors affecting either male or female fertility. Usage of condoms prevents STIs, and as such, we expected it to be identified as an important factor interfering with future fertility. It may, however, be that respondents did not fully appreciate the reasoning of the question. In future questionnaires, we could consider incorporating an explanatory sentence on this or adding further questioning on alternative ways to prevent STI transmission, such as frequent testing for highly prevalent STIs or having long-term, stable sexual relationships.According to similar studies from other parts of the world, fertility awareness tends to be low to moderate , 16 irreWith regards to egg freezing, about half of participants correctly stated that this should ideally take place prior to 30 years of age. However, only about one-third had a clear positive attitude towards this. With fertility declining with age and women favouring career over childbearing, social egg freezing is an appealing option for fertility preservation. More women are storing their oocytes to maintain the potential to have a baby in the future. However, they tend to proceed to egg freezing when they are already in their later 30s when the success rates of the method are limited . FurtherIn this study, the main barrier to social egg freezing was cost. A proportion of participants also mentioned that they would prefer natural methods of conception or that they were uncertain about results. This proportion was similar to the number of undecided participants regarding the recourse to reproductive fertility methods, in general. Lack of time had the least impact on their decision to future egg freezing.With regards to ART cycle success rates, about one-third of participants correctly appreciated this to be in the range of 30% . FurtherThe majority of participants were living in urban centres, which is typical of the current demographic distribution in the country  and, as Several countries have already introduced relevant educational programmes. For example, the British Fertility Society, in partnership with the Royal College of Obstetricians and Gynaecologists, other learned societies and non-profit institutions introduced the ‘Fertility Education Initiative’, aiming at providing accurate information about fertility at schools and promoting fertility awareness . On a sireproduktivlivsplan.se’ (Social media and internet websites can also influence and inform the public about infertility , as showplan.se’ . Howeverplan.se’ .In conclusion, the results of this project provide a snapshot of what are the current knowledge and intentions of young men and women in Greece regarding childbearing and fertility. Although there are intentions for childbearing before the age of 30, and despite adequate understanding of the limitations of ART on improving fertility, there are still a significant proportion of young adults who lack the necessary information about factors leading to declining fertility. Although social and financial reasons may be at the heart of postponed motherhood, improving fertility awareness through specific educational programmes can provide with the necessary knowledge to make informed decisions about deferring or not childbearing.With this in mind, we suggest the introduction of relevant educational programmes for the general public, including the incorporation of fertility awareness in the sexual and relations education curriculum, following the Australian and Swedish paradigm.


The period fertility in China has declined to very low levels, and the completed cohort fertility rate (CFR) has also decreased significantly. However, the exact fertility rate remains controversial. While the tempo effect has played a significant role in China’s period fertility decline, child underreporting has to be taken into consideration in China’s fertility research.By using the census data from 1982 to 2010, and the 1% population sample survey data from 1995 to 2015, we systematically analyzed China’s fertility and its trends since the 1980s using period fertility measures, adjusted period fertility measures, cohort fertility measures, and indirect estimation methods.The results show that marriage postponement significantly affects the TFR decline. Even after eliminating the tempo and parity structure effect, the adjusted TFR has fallen below 1.5, and the first-order fertility rate dropped to 0.9 in 2015. The CFR for women aged 45–49 declined from 5.37 in 1982 to 1.62 in 2015 mainly because of a decrease in fourth and higher-order births from 1982 to 1990, a decrease in second and third births from 1990 to 2000, and a decrease in second births from 2000 to 2015. Indirect estimation methods yielded a TFR in the range of 1.5–1.6 for the period 2000–2010 and an average TFR of 1.49 for the period 2011–2020.The traditional norm of universal marriage and childbearing for Chinese women is changing. China’s fertility has been steadily declining, as measured by both period and cohort indicators. Following the historical change, fertility may continue to decline even after introducing the universal three-child policy in China in 2021. China’s fertility level has continuously declined over the past decades. The total fertility rate (TFR) decreased from 5.81 in 1970 to 2.75 in 1979. In the 1980s, the TFR fluctuated above the replacement level. Since the 1990s, the fertility rate has declined to below the replacement level . China’sIn China’s fertility decline over the past decades, marriage and childbearing postponement played a considerable role. Research in western countries shows that marriage postponement is a major driving factor of TFR decline –14. BecaSome studies have reported the effect of tempo and parity change on fertility. The parity progression ratio—a measure proposed by Feeney 1985) , Ma et a85 , Ma eChinese women’s completed cohort fertility rate (CFR) also exhibits a significantly decreasing trend. Census data show that the average number of children born to women aged 45–49 years decreased from 5.37 in 1982 to 1.84 in 2010 . The cohChild underreporting is an issue that has to be considered in China’s fertility research . StudiesThe decline in fertility and births in China has been a concern for the government. After a steep decline of annual births from 14.65 million from 2019 to 12.00 million 2020, the number of annual births further declined 10.62 million, which has stirred up hot debate in China. The pandemic of COVID-19 accounted for partly the decline in birth numbers, but the main driver was the fertility decline due to low fertility intention and postponement in childbearing . To counIn the wake of the universal three-child policy and pessimistic fertility prospect, we expect to systemically depict the fertility trend over the past four decades and provide some reference for future study. We investigate the effect of marriage and childbearing postponement on China’s fertility decline, depict the fertility change trend after eliminating the tempo effects and examine the historical course of China’s cohort fertility decline. We apply period fertility measures, adjusted period fertility measures, cohort fertility measures, and indirect estimation methods to data from censuses, and 1% population sample surveys and annual one in thousand population sample survey since 1982.This study analyzes fertility using the following four groups of measures and methods: (1) period fertility measures, including period total fertility rate (TFR), age-specific fertility rate (ASFR), mean age at childbearing (MAC), total marital fertility rate (TMFR), ratio of TFR to TMFR, standardized TFR, and decomposition of TFR variations; (2) adjusted period fertility measures, including period parity progression ratios (PPPRs) and period total parity progression fertility rates (PTPPFR), tempo-adjusted TFR, and tempo- and parity-adjusted TFR; (3) cohort fertility measures, including the proportion of women with at least N births, completed cohort fertility rate (CFR), cohort parity progression ratio, and decomposition of CFR changes; (4) indirect estimation methods, including variable-r method, P/F ratio method, reverse survival method, and projection simulation method. We didn’t list all the detailed formulas here due to space limit. Please refer to “In China, several government agencies provide demographic statistics. The National Health Commission (into which the former Population and Family Planning Commission was merged) is responsible for implementing and monitoring family planning policies and provides statistics of annual births. As births and birth rates are measures for assessing the performance of family planning work, the birth statistics provided by the family planning agency may be unreliable , 41. ChiChina’s fertility decline was accompanied by data quality issues, particularly large-scale underreporting of births , 45. StuChina Population and Employment Statistical Yearbook 2018. This study did not adjust the tabulated data. The 2020 population census has been completed. However, except for publicly announced birth number and TFR, we could not get further information from 2020 population census and so we didn’t include in-depth analysis on 2020 data in this paper.We used the tabulated data of 1982, 1990, 2000, and 2010 censuses and the 1% national population sample surveys in 1995, 2005, and 2015. The 0.95‰ case data contained in the 2000 census were also used. The mean ages at childbearing in 1986, 1996, and 2011 were computed using the age-specific fertility data tabulated in the Figure We calculated the singulate mean age at marriage (SMAM), as shown in Table Table The mean age at childbearing (MAC) is presented in Table Variations in MAC are caused by the interaction of parity structure and parity-specific childbearing age. Between 1982 and 1995, the TFR of third- and higher-order births decreased rapidly, as did the proportion of third- and higher-order births to total births, resulting in declining mean ages at childbearing. After that, the effect of delayed childbearing started to become apparent, resulting in rising mean ages at childbearing. This pattern of childbearing age variations has been extensively verified in Western countries .The TMFRs presented in Table Table As shown by Jiang et al. 2019) [9 [1], thFigure Table Note that, the period parity progression fertility rate for first births was 0.86 in 2015 Table , the \doTable Table The decline in cohort parity progression ratios led to the decline in CFR. The cohort of women aged 45–49 had 5.37 live births in 1982. This figure decreased to 2.36 in 2000, 1.84 in 2010, and 1.62 in 2015.Table Table Concerning the results of the variable-r method, the underreporting of children in the 2000 census was more serious relative to that in 1990 and may affect the results. In this article, we applied the variable-r method directly with the census data, and the adjusted data that increased the population aged 0–9 in 2000 by 10%. The TFR estimated by variable-r method for 1990–2000 using the census data was 1.58 and was 1.74 using the adjusted data. The TFR for 2000–2010 was 1.68 when using census data directly and 1.52 when using the adjusted data. The results are similar to those by Cai (2008) , Chen (2The TFR estimates by the P/F ratio method with data from a single census are 2.29, 1.43, and 1.50, for 1990, 2000, and 2010, respectively. The average TFRs for 1990–2000 and 2000–2010 estimated by applying the P/F ratio method to data from two censuses  are 1.35The TFR estimates for 1982, 1990, and 2000 using the reverse survival method are higher than the figures obtained in the censuses. The TFR estimate for 2000 is 1.54, much higher than the 1.22 obtained by the census data.Table One noteworthy thing is that, before 2018, the projected TFRs were higher than the officially published figures. But for the three consecutive years 2018, 2019 and 2020, the projected TFRs were lower than the officially published. The reason for this reversal needs to be further explored.China’s fertility and births have attracted much attention with the implementation of its strict birth control policy, which has been highly controversial . With thDelayed marriage has a significant effect on fertility decline. This effect is reflected in the change in the age-specific fertility pattern, the increase in the mean age at childbearing, the declining TFR/TMFR ratio, and the standardization and decomposition of TFR. Delayed marriage contributes 0.42 during 1990–2000 and 0.17 during 2000–2010 to the decline in TFR. Age at first marriage is certain to increase, and delayed marriage and childbearing will further contribute to the fertility decline. China adopted the universal three-child policy in 2021. However, the people’s response suggests that the fertility rate will not increase significantly. China has now turned to comprehensive pronatalist policies nationwide, but whether these policies will help increase fertility remains to be seen.Adjusted period fertility measures show that, after eliminating the tempo and parity structure effects, the adjusted TFR was below 1.5 in 2015, and the tempo- and parity-adjusted TFR for first births was 0.9 in 2015. Universal marriage and childbearing was the traditional norm for Chinese women, but this social pattern is changing. The proportion of childlessness is increasing, leading to a declining fertility rate for first births. Historical data in Europe show that the rising proportion of childless women played a significant role in further declining the already low fertility . In EastCohort fertility measures show a marked decline in CFR. During 1982–1990, the decline in CFR was mainly driven by the decline in fourth and higher-order births, during 1990–2000 mainly by the decline in second and third births, and during 2000–2015 mainly by the decline in second births. It is reasonable to predict that future fertility declines will be strongly driven by declines in first births or increases in the proportion of childlessness.The indirect estimation techniques revealed TFR in the range of 1.5–1.6 for 2000–2010 and an average TFR of 1.5 for 2011–2020. Despite the slightly higher fertility rates in 2016 and 2017 due to the universal two-child policy introduced in 2016, China’s TFR fell to below 1.5 in 2018 and 1.3 in 2020. 2021 witnessed a further decline in birth numbers and the TFR was roughly estimated at 1.07. China’s TFR is unlikely to recover markedly in the near future, and it may remain at a very low level for a long time.Scholarships reached a consensus that both the birth control policy and socioeconomic developments contributed to the fertility decline , 56, 59,The present study has certain limitations. Firstly, data quality is a concern in researching China’s fertility. We tried to adjust the TFR with an indirect estimation method and adjusted indicators. Nevertheless, the general description of this paper depends largely on the raw data from the census. Secondly, we computed many indicators but could not reach a generally accepted fertility level. The fertility level remains controversial. Thirdly, we did not analyze the data by residence or educational attainment, which may shed more light on the fertility trend in China with rapid urbanization and expansion of high education. Lastly, we did not include detailed data from the 2020 population census due to the data unavailability. Despite these limitations, the results presented here are still helpful in interpreting China’s fertility and population characteristics and may serve as a reference for future adjustment and improvement of China’s fertility policy.


Rspo2 in mice led to supernumerary lower molars, while teeth forming at the normal positions showed no significant anomalies. Because multiple Rspo genes are expressed in the orofacial region, it is possible that the relatively mild phenotype of Rspo2 mutants is due to functional compensation by other RSPO proteins. We found that inactivating Rspo3 in the craniofacial mesenchyme caused the loss of lower incisors, which did not progress beyond the bud stage. A simultaneous deletion of Rspo2 and Rspo3 caused severe disruption of craniofacial development from early stages, which was accompanied with impaired development of all teeth. Together, these results indicate that Rspo3 is an important regulator of mammalian dental and craniofacial development.Development of the teeth requires complex signaling interactions between the mesenchyme and the epithelium mediated by multiple pathways. For example, canonical WNT signaling is essential to many aspects of odontogenesis, and inhibiting this pathway blocks tooth development at an early stage. R-spondins (RSPOs) are secreted proteins, and they mostly augment WNT signaling. Although RSPOs have been shown to play important roles in the development of many organs, their role in tooth development is unclear. A previous study reported that mutating  Tooth development in mammals is guided by reciprocal and dynamic interactions between the oral ectoderm and the underlying neural crest-derived mesenchyme. These interactions are mediated by many secreted signaling molecules and transcription factors, which are expressed in specific patterns. Signaling molecules involved in the process include fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), wingless-type MMTV integration site family (WNT), and sonic hedgehog (SHH). The transcription factors involved include members of msh homeobox (MSX), distal-less homeobox (DLX), paired box (PAX), and LIM homeobox proteins (LHX) ,2,3.The first morphological sign of tooth development appears as localized thickening of the oral ectoderm  at the 11th day of mouse gestation (E11). The epithelium further thickens and invaginates into the mesenchyme to form a dental placode E12) and then a bud E13), with the mesenchyme cells condensing around the bud. Subsequently, a signaling center called an enamel knot is established in the epithelium, and it directs further morphogenesis into the cap stage (E14.5) and the bell stage (E16.5). Differentiation of ameloblasts and odontoblasts from the epithelium and the mesenchyme, respectively, occurs during the late bell stage (E18.5) . We. WeRspo3Rspo2 and Rspo3 during craniofacial development in mammals. A recent publication showed that Rspo2 and Rspo3 synergistically regulated craniofacial and dental development in fish [Rspo genes, which made it important to examine mouse mutants. While further investigation is necessary to determine the molecular and cellular changes in the mouse double mutants, our data establish that Rspo3, along with Rspo2, is a crucial regulator of oro-facial development from early stages.The current study also provides the first description of the phenotypic consequences from the simultaneous loss of  in fish . HoweverRspo3 is important for development of the lower incisors in mammals, regulating the bud-to-cap transition. Furthermore, Rspo2 and Rspo3 have overlapping yet crucial roles in early development of the face in mammals.


We argued that transcript level profiles of target genes established from in vitro SE induction as reference compared to virus-induced profiles can identify differential virus traits that link to harmful reprogramming. To validate this hypothesis, we selected a standard set of genes named ‘ReprogVirus’. This approach was recently applied and published. It resulted in identifying ‘CoV-MAC-TED’, a complex trait that is promising to support combating SARS-CoV-2-induced cell reprogramming in primary infected nose and mouth cells. In this perspective, we aim to explain the rationale of our scientific approach. We are highlighting relevant background knowledge on SE, emphasize the role of alternative oxidase in plant reprogramming and resilience as a learning tool for designing human virus-defense strategies and, present the list of selected genes. As an outlook, we announce wider data collection in a ‘ReprogVirus Platform’ to support anti-viral strategy design through common efforts.Reprogramming of primary virus-infected cells is the critical step that turns viral attacks harmful to humans by initiating super-spreading at cell, organism and population levels. To develop early anti-viral therapies and proactive administration, it is important to understand the very first steps of this process. Plant somatic embryogenesis (SE) is the earliest and most studied model for  Effective immunologic protection contributes to resilient behavior of higher organisms. It is essentially based on the diversity of innate and adaptive cell responses and cell memory tools –4. Immunin vitro as a model for a resilient response upon severe stress of highly variable origins  and glutathione-S-transferase transcripts relative to several other tested genes , which can cause nitration and subsequent inhibition of a broad range of cellular protein functioning and nitro-oxidative stress   .AOX at transcriptional level was shown to be important for positive effects on performance (AOX for predicting plant robustness from early reprogramming has been substantiated (Tobacco mosaic virus (TMV) infection. AOX transcript accumulation took place when cytochrome-c-oxidase (COX) was inhibited by TMV, or NO or KCN.Mitochondrial AOX was proposed as functional marker for plant cell reprogramming . It demoformance , 91. Recantiated . In planantiated . It invoantiated . Fu et aantiated  revealedAOX transcript levels is typical and coincides with critical phases of de novo induced morpho-physiologic events . This included carrot SE induction and seed germination  AOX is critical for mitochondrial ROS signal transduction towards mitochondria-nucleus retrograde communication  both under phosphorylating (presence of ADP) and non-phosphorylating (absence of ADP) conditions (AOX in transgenic plants resulted in high ethanol production associated with injuries (The extraordinary role of AOX for reprogramming involves four major aspects for cell and tissue determination: (a) ibration ; AOX wason of NO . Cvetkovon of NO  demonstra et al.  found tha et al.  showed tnication –110, (c)nication , 112, annication , 113–117nication . AOX actnication . In AOX-nditions . Lack ofinjuries . Thus, AViruses are known to ‘abuse’ host cell’s competence and structures for reprogramming. Any virus infection provokes struggling for commanding coordination of the host cell program and this starts in the initially infected cells. Therefore, it is challenging to early stop virus-induced harmful reprogramming and avoiding at the same time suppressing the host’s defense and survival strategy. As reviewed in Costa et al. Preprint , virusesBased on this knowledge and the characteristics of ‘reprogramming for survival’ during SE induction and supported by our validating results on the overall approach Preprint  we selecin vitro - screening of a diversity of virus types and variants. It should be applied under commonly accepted standard conditions in relevant human cells or tissues of primary importance for defined diseases. Currently, the profile ‘ReprogVirus’ was used by our team to trace corona virus-related reprogramming , such as deep embedding clustering and GANs  .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.FunCROP net members and wrote the manuscript. RB contributed to manuscript writing and prepared overall ms for submission. PQ supports this initiative through his competence in Artificial Intelligence methodologies. SRK helped BA-S in overall FunCROP group coordination. KJG helped in writing manuscript parts related to NO metabolism. All authors contributed to the article and approved the submitted version.BA-S initiated scientific approach and concepts in close collaboration with JHC and CN, coordinated their final development for the presented perspective through common discussions among all BA-S, GM, and RS acknowledge support for academic cooperation and researchers mobility by the India-Portugal Bilateral Cooperation Program (2013– 2015), funded by “Fundação para a Ciência e Tecnologia” (FCT), Portugal, and the Department of Science and Technology (DST), India. GM is grateful to UGC, India, for doctoral grant from BSR fellowship. JHC is grateful to CNPq for the Researcher fellowship (CNPq grant 309795/2017-6). KTL is grateful to CNPq for the Doctoral fellowship. SA is grateful to CAPES for the Doctoral fellowship. KJG, MO and BA-S acknowledge support by the India-Portugal Bilateral Cooperation Program ‘DST/INT/Portugal/P-03/2017’. MO Research is partially supported by National Funds through. FCT.Fundação para a Ciência e a Tecnologia, projects UIDB/04674/2020 (CIMA). BR and SS acknowledge stay support provided by DBT-TDNBC-DEAKIN – Research Network Across continents for learning and innovation (DTD-RNA) at The Energy and Resources Institute (TERI), India. BAS wants to thank Dr. Natascha Sommer for helpful discussions and comments on part of the manuscript during its development on the background of her experience as medical doctor in the group of Prof. Dr. Norbert Weissmann, Chair for ‘Molecular Mechanisms of Emphysema, Hypoxia and Lung Aging’ at the Universities of Giessen and Marburg Lung Center (UGMLC), Germany, and as investigator involved in mitochondrial redox biology also by help of transgenic AOX-mice. BA-S recognizes internal forum discussions at the University of Évora, Portugal, that helped stimulating the integration of research on viruses into our running plant research approach on cell reprogramming. CN acknowledges the international scientific network BIOALICYTED, which contributed to establish FunCROP contacts.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.