Obstructive sleep apnea and hypopnea syndrome (OSAHS) are characterized by repeated pauses in breathing during sleep, usually associated with sleep interruption and decreased oxyhemoglobin saturation. Cephalometric analysis has become an important method in diagnosis, reporting specific craniofacial characteristics such as posterior air pharyngeal space, tongue length and hyoid position, which may predispose some people to develop SAHOS. The purpose of this revision is to present several anatomic aspects by cephalometric analysis that may have a predisposition to the development of upper airway occlusion. Obstructive sleep apnea-hypopnea syndrome (OSAHS) are characterized by repeated episodes of obstruction of the upper airways during sleep, usually associated with sleep interruption and decreased oxyhemoglobin saturationDiagnosis of OSAHS is confirmed by polysomnography, where apnea in adults is considered a pause in breathing for 10 seconds or more, and hypopnea is considered a 50% reduction of the air flow for a period equal to or higher than 10 seconds, associated with over 3% decrease of oxyhemoglobin saturation and/or a minor awake. A person is classified as apneic when he/she presents 5 or more respiratory events (apneas and/or hypoapneas) per hour of sleepSurgical treatment has not shown to be effective, although it has improved along with the development of etiological diagnostic techniques, that is, the identification of the obstructive point of the airways. Several methods have been used with this objective, among which we have cephalometryCephalometric radiography is a technique used for the diagnosis of craniofacial deformities2, whereas it is 5.3 cm2 in healthy individuals. Length of the soft palate is 48mm in individuals with apnea, whereas it is around 35mm in healthy people. This marked increase of the soft palate leads to reduction of the nasopharynx and increased contact between the soft palate and tongue, leading to a collapse in this areaRivlin et al.Skeleton measures have a significant role in the dimensions of upper airways (UA). The main parameters of pharynx are the posterior air space, that is, the distance between the pharynx posterior wall and base of tongue or soft palate. This region may be divided among three different levels: upper posterior air space ; middle ; and inferior . Air space reduction is evident among OSAHS patients, especially at the uvula level and mandible planePosition of the hyoid bone is significantly important. In healthy patients, the hyoid bone is found at the C3-C4 cervical vertebrae, while in OSAHS patients it is usually at C4-C6 level2 and PaCO2). The hypoglossal motor system is stimulated by adrenergic, serotoninergic and cholinergic neurotransmitters via hypoglossal nucleus situated in the central nervous systemNearly 58% of apnea patients presented mandibular micrognathism and retrognathism in relation to the maxillaEndo et al.The most common cause of OSAHS in children is adenotonsillar hypertrophyAttention must be paid to the fact that children submitted to adenotonsillectomy as treatment for OSAHS may present snoring relapse during teenager years. This fact may be related to the consequences of craniofacial changes, probably due to early onset of VAS obstruction2O resulted in UA collapse in men, while in women it represented a patent UA. UA collapse occurred in women only under a pressure of -18 cmH2O.The collapse mechanism of UA in men and women is different and is related with local anatomical differencesNon-obese OSAHS patients tend to present the following anatomical craniofacial characteristics: caudal hyoid, increased soft palate dimensions and consequent anteroposterior reductions of the airways at the soft palate level, reduction of anteroposterior region of nasopharynx and oral pharynx. Obese OSAHS patients presented the above findings plus increased volume of tongue and anterior hyoid bone. Lower and anterior position of hyoid bone in obese patients seems to be related to increased fat deposition on the tongue, which increases its volume.OSAHS patients presenting maxilla and mandible within normal limits, that is, 96mm and 125mm, respectively, may reveal a retropositioned mandible as well as caudal hyoid boneCakirer et al.Another important feature that should be considered is that most snoring individuals with OSAHS are mouth breathers during sleep. As mouth opens 1.5cm, there is a 1cm-dorsal displacement of the mandible angle, resulting in a 1cm-reduction between the pharyngeal posterior wall and the tongue dorsum. This new tongue posture causes static and dynamic stimuli over the palatine veli, uvula, tonsillar pillars, palatine tonsils and epiglottis, leading to hypotony and hypertrophy of these structures. With muscular loosening and action of inhaling pressure during sleep, there is pharyngeal collapse and, consequently, OSAHSThe cephalometric analysis is valuable for the etiology of OSAHS and should be considered among routine exams. Cephalometry is a diagnostic procedure to collect information on skeleton abnormalities and soft tissues of patients with OSAHS


Hedyotis diffusa Willd, known to suppress GCa without identified target. CYP19A1  was correlated to GCa prognosis. Relatedly, Ar silencers, which halt the expression of Ar exhibited anti‐GCa effects in experimental models, are currently being investigated.Gastric cancer (GCa) is a malignancy with few effective treatments. Ursolic acid (UA), a bioactive triterpenoid enriched in Hedyotis diffusa Willd ethanol extract, UA, or 5‐fluracil were applied onto AGS, SC‐M1, MKN45 GCa cells for cancer inhibition tests. Immunoblot for measuring gene expressions upon drug treatments, or gene knockdown/overexpression. Treatments were also applied in a MKN45 implantation tumor model. A web‐based GCa cohort for Ar expression association with prognosis was performed.The docking simulation score of UA was compared with Ar inhibitors, e.g., letrozole, exemestane, in Ar protein crystallization. Hedyotis diffusa Willd, enrich with UA, exhibited cytotoxic activity against GCa cells. Molecular docking simulations with the 3D Ar structure revealed an excellent fitting score for UA. UA increase cytotoxic, and suppressed colony, in addition to its Ar silencing capacity. Moreover, UA synergistically facilitated 5‐FU, (a standard GCa treatment) regimen in vitro. Consistent with those results, adding estradiol did not reverse the cancer‐suppressing effects of UA, which confirmed UA acts as an Ar silencer. Furthermore, UA exhibited tumor‐suppressing index (TSI) score of 90% over a 6‐week treatment term when used for single dosing in xenograft tumor model. In the clinical setting, Ar expression was found to be higher in GCa tumors than normal parental tissue from the TCGA (The Cancer Genome Atlas) cohort, while high Ar expression associated with poor prognosis. Together, the results indicate UA could be used to treat GCa by silencing Ar expression in GCa. Hedyotis diffusa Willd ethanol extract could be an functional food supplements.The ethanol extracts of  Ursolic acid (UA) has been discovered to inhibit gastric cancer without identified target. This article defined UA as is aromatase silencer, of which turn off aromatase expression rather than suppressing enzymatic activity. Interestingly, the well‐known anticancer compound ursolic acid (UA) was found to have a high fitting score when used in such a simulation. Its outstanding fitting score trajectory indicated that it has mechanism of action similar to that of exemestane in suppressing GCa cells. The current report characterized pharmacological mode of action for the well‐known anticancer natural compound UA that can suppress GCa cell growth.In order to target Ar, we would like to find out novel compound to evaluate its possibility to suppress GCa cells. Small molecule compounds from natural products are important resources for new drug discovery and development. In the current study, we accessed 2073 compounds from the TDEC  was obtained from the RCSB Protein Data Bank (https://www.rcsb.org/).x = 86.57, y = 54.12, and z = 45.85. All the parameters for the docking were set to default except that the maximum number of energy evaluation increase was set to 25,000,000 per run. The results were analyzed with cluster analysis, and the simulation models were shown using the BIOVIA Discovery Studio 4.5 Visualizer .To evaluate the interaction between small molecular inhibitors and CYP19A1, the molecular docking simulations were performed using AutoDock 4.2 with the Lamarckian genetic algorithm.2.22. E2 (β‐estradiol: E2758), 5‐FU (F6627), and ursolic acid (U6753) were purchased from Sigma‐Aldrich. Stock solution of 5‐FU (10 mM), ursolic acid (100 mM), and 17β‐estradiol (10 mM) was prepared in DMSO and 99.5% ethanol. Aliquoting these stock solutions and frozen them at −20°C. CYP19A1 plasmid (Cat. # HG15323‐UT) and its control (pCMV3 vector: Cat. # CV011) were purchased from Sino Biological. Plasmids were transformed to DH5α competent cells and amplified in LB medium containing ampicillin (100 μg/ml) overnight using a 37°C shaker with speed at 180 rpm. Then the bacteria were harvested and plasmids were purified using Invitrogen plasmid extraction and purification kit according to the procedure provided by the manufacture.The human gastric cancer cell lines AGS, SCM1, and MKN45 were purchased from the Food Industry Research and Development Institute in Hsinchu, Taiwan. AGS, SCM1, and MKN45 cells were cultured in RPMI 1640 medium (RPMI) (GIBCO) with 10% fetal calf serum and 1% penicillin/streptomycin (GIBCO). The cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2.35 cells were seeded in 6‐cm dish, and the transfection was performed next day. The transfection reagent is Lipofectamine 2000 (Invitrogen). The operations, respectively, according to manufacturer's instructions (Invitrogen or Promega). 3 microgram plasmid mixed with 20 microliter of Lipofectamine 2000 in 0.5 ml of Opti‐MEM reduced serum medium (Thermo Fisher). The DNA–lipofectamine mixture was incubated at room temperature for 15 min, then the mixture was added to the cells in a 6‐cm dish containing 1 ml of RPMI medium without fetal bovine serum and 1% antibiotics. After 6 h of transfection, the transfection medium was replaced by growth medium. Transfected cells were subjected to colony formation assay, cytotoxic assay, and western blotting 48–72 h later.2.5 × 102.43 cells/well were seeded in 96‐well plates with RPMI/10% fetal bovine serum (FBS) and were incubated for 24 h. The drugs were then added following previous work,The WST‐1 assay was used for assessing cell growth.2.5The method used for colony formation assay and standard cell number count was modified from a previous study.2.6The theory of linear and nonlinear regression analysis by Chou‐Talalay method was used to evaluate the synergy (CI < 1), additive (CI = 1), and antagonism (CI > 1) of the combination drug treatment.2.76 cells/well were seeded with RPMI in 10% FBS and were then incubated for 24 h. The cells were then treated with UA  and were incubated at 37°C for a further 48 h. Cells that had reached 80%–90% confluence in the 100‐mm dishes were then lysed with 1 ml of Trizol (Invitrogen). Next, phenol/chloroform was added, and the RNA‐rich layers were separated by centrifugation. With the addition of 2‐propanol, soluble RNA was then precipitated. Next, the RNA was rinsed with 75% ethanol and dried at room temperature before being dissolved in RNase‐free water. For first‐strand cDNA synthesis, 5 μg of total RNA was used to perform reverse transcription PCR with the PrimeScriptTM RT reagent kit (TAKARA Bio Inc.), while synthesized miRNA cDNA was produced using the Mir‐XTM miRNA First‐Strand synthesis Kit (Clontech). The synthesized cDNA and miRNA were produced following the manufacturers’ instructions.RNA was extracted from AGS, SCM1, and MKN45 cells as in a previous study.2.8TM SYBR FAST One‐Step qRT‐PCR Kit (KAPABIOSYSTEMS) were used according to the manufacturers’ instructions. Relative gene expression was determined by normalizing the expression level of the target gene to the expression level of a housekeeping gene (actin). Threshold value (Ct) dynamics were used (2−ΔΔCt) for the quantitation of gene expression. The qRT‐PCR primer sequences used were as followsA real‐time detection system  and the KAPA2.9Cells were lysed in RIPA buffer . Forty micrograms of protein per lane was prepared in 10% SDS gel for electrophoresis, and then the proteins were transferred onto PVDF membranes. These membranes were stained with anti‐actin  and anti‐aromatase  at 4°C overnight. Proteins were visualized and quantified using ChemiDoc system (Bio‐Rad Laboratories).2.10nuFoxnl/CrlNarl mice  were raised under specific pathogen‐free conditions . The xenograft tumor model used was modified from that presented in a previous study.7 of MKN45 cells were subcutaneous (s.c.) injected into two sites on the lower back of each mouse. When the tumor volume reached 200 mm3, the mice were randomly assigned to three groups  in order to identify compounds from the Taiwan Database of Extracts and Compounds (TDEC) that could potentially bind to the active site of aromatase. We ranked the top 10 compounds and compared them with known Ar inhibitors, for example, exemestane and letrozole, and found that UA was among the best in terms of performance in the fitting simulation. It showed that UA exhibited a better binding energy  than exemestane  and letrozole  Figure .The docking model showed that UA can successfully dock into the active site of aromatase, and that it then formed 16 hydrophobic alkyl–alkyl interactions with 9 amino acids, namely, Ile133 , Ala306 (3.26 Å), Val370 (3.92 Å and 5.00 Å), Leu372 (5.07 Å), Val373 (4.05 Å), Met374 (3.82 Å), Cys437 , Ala438 (3.22 Å), and Leu477 (4.71 Å and 5.46 Å). In addition, it also formed one hydrophobic pi–alkyl interaction with the amino acid Trp224 (4.79 Å). Figure .Exemestane can also successfully dock into the active site of aromatase. It formed five hydrophobic alkyl–alkyl interactions with two amino acids, namely, Val370  and Val373 (4.28 Å). In addition, it also formed three hydrophobic pi–alkyl interactions with three amino acids, Phe134 (5.32 Å), Phe221 (5.05 Å), and Trp224 (5.10 Å). Furthermore, it formed two electrostatic hydrogen bond interactions with two amino acids, Arg115 (2.38 Å) and Met374 (1.97 Å). Likewise, its major structure was docked into the location of the co‐crystal exemestane binding space in the active site of aromatase Figure . On the In order to test whether UA exerts a GCa suppression effect, in vitro tests were performed. As shown in Figure The data shown in Figures 3.2p = 0.2623).Moreover, we compared the tumor suppression efficacy of 5‐FU and UA Figure  and evalhttps://www.cancer.gov/tcga) database was introduced to observe Ar expressions in a clinical setting. As shown in Figure In order to survey Ar expression in GCa patients, the TCGA  decreased Ar expression in endometriotic cells through ERRK1/2 activation.As for the second hypothesis, there have also been reported examples of such regulation. For example, one study showed that a COX selective inhibitor (sulfonanilide) was surprisingly found to generate selective aromatase modulation activity, in which an aromatic ring was formed with two hydrogen bond acceptors and there was a hydrophobic function of Ar which suppressed Ar mRNA expression.4.2Hedyotis diffusa Willd. It was previously reported that Hedyotis diffusa Willd, a well‐known Chinese herbal medicine also known as Bai‐Hua‐She‐She‐Ca and as Oldenlandia diffusa (Willd), can be used to treat chronic inflammation caused by conditions such as metabolic syndrome, hepatitis, cancer‐related disease.Hedyotis diffusa Willd has also been used for patients with gastric cancer, and the overall survival analysis showed that Hedyotis diffusa Willd improves the outcome of patients with gastric cancer.Hedyotis diffusa Willd is the most commonly prescribed Chinese herbal medicine for patients with gastric cancer.Hedyotis diffusa Willd, such as triterpenes, flavonoids, and anthraquinones, have been reported to have anticancer activities.Hedyotis diffusa Willd were found to be able to suppress GCa cell growth in a dose‐dependent manner. As was noted in the results section, 4% UA was detected in the ethanol extracts of Hedyotis diffusa Willd. With regard to industrial application, UA can be easily concentrated for the manufacturing health‐promoting products.In this study, we demonstrated that CYP19A1 might be a target for natural products containing ursolic acid, a derivative natural compound from Hedyotis diffusa Willd with no identified target, might bind to Ar and, consequently, silence Ar gene transcription and ablate GCa cell growth.In conclusion, the current study revealed that UA, the anticancer agent from Wen‐Lung Ma and Ning Chang executed most of the experiments, and drafted the manuscript. Guan‐Yu Chen and Wei‐Chung Cheng are responsible for computational biology. Yang‐Chang Wu is responsible for acquiring the nature product. Ching‐Chia Li and Wei‐Chun Chang advised the translational study and edited the manuscript. Wei‐Chun Chang and Juan‐Cheng Yang supervised the entire project, supported this study, and final approval of the manuscript.None.This study was not human sample related, therefore, approval from an institutional review board or ethics committee can be waived.


In HAM/TSP, the increase of HTLV-1 provirus induces immune dysregulation to alter inflammatory milieu, such as expansion of HTLV-1-specific CD8+ T cells, in the central nervous system of the infected subjects, which have been suggested to underlie the pathogenesis of HAM/TSP. Factors contributing to the conversion from asymptomatic carrier to disease state remain poorly understood. As such, the identification and tracking of HTLV-1-specific T cell biomarkers that may be used to monitor the progression from primary infection to immune dysfunction and disease are of great interest. T cell receptor (TCR) repertoires have been extensively investigated as a mechanism of monitoring adaptive T cell immune response to viruses and tumors. Breakthrough technologies such as single-cell RNA sequencing have increased the specificity with which T cell clones may be characterized and continue to improve our understanding of TCR signatures in viral infection, cancer, and associated treatments. In HTLV-1-associated disease, sequencing of TCR repertoires has been used to reveal repertoire patterns, diversity, and clonal expansions of HTLV-1-specific T cells capable of immune evasion and dysregulation in ATL as well as in HAM/TSP. Conserved sequence analysis has further been used to identify CDR3 motif sequences and exploit disease- or patient-specificity and commonality in HTLV-1-associated disease. In this article we review current research on TCR repertoires and HTLV-1-specific clonotypes in HTLV-1-associated diseases ATL and HAM/TSP and discuss the implications of TCR clonal expansions on HTLV-1-associated disease course and treatments.Human T lymphotropic virus 1 (HTLV-1) is a human retrovirus identified as the causative agent in adult T-cell leukemia/lymphoma (ATL) and chronic-progressive neuroinflammatory disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 is estimated to infect between 5-20 million people worldwide, although most infected individuals remain asymptomatic. HTLV-1 infected persons carry an estimated lifetime risk of approximately 5% of developing ATL, and between 0.25% and 1.8% of developing HAM/TSP. Most HTLV-1 infection is detected in CD4 Two viral proteins, Tax and HBZ, play critical roles in HTLV-1 oncogenesis and chronic inflammation. Tax is a transforming and transactivating protein of HTLV-1 and induces the expression of a variety of cellular genes by activation of the NF-κB pathway and the modulation of the epigenetic machinery to induce cellular proliferation and transformation (Human T lymphotropic type 1 (HTLV-1) is a retrovirus known to be an etiological agent of an aggressive mature T cell malignancy termed adult T cell leukemia/lymphoma (ATL) and the chronic, progressive inflammatory neurologic disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) . Althougormation . HBZ is ormation , 10. HBZormation . Accumul+ T cells containing integrated HTLV-1 provirus, typically associated with T cell receptor (TCR) gene rearrangements  which plays a crucial role in immunity against HTLV-1 to secrete various factors that suppress viral replication and kill infected target cells  due to rapid progression . ATL is ngements , 13. Theal TCRVβ . Previoual TCRVβ , 16. IntTL cells , 17. HBZTL cells , 19. Botet cells –24. Howeet cells , 23, 24.et cells .+ T cells can induce the production of proinflammatory cytokines and proliferation of CD8+ T cells  . HTLV-1  T cells –34. CD8+artments , 35–39.  T cells . It rema T cells –43. Seve T cells , degener T cells –50, and  T cells , 52. Thedrocytes .In both HAM/TSP and ATL, sequencing of TCR repertoires has been a useful tool in parsing the drivers of disease. As such, identification of biomarkers for an HTLV-1-associated disease outcome is a central focus in the field. Advances in high throughput and single cell RNA sequencing technology have made characterization of TCR repertoires more efficient, economical, and accessible in recent years, and offer unmatched resolution of T cell populations. Unique molecular identifier (UMI) based single cell sequencing techniques have been used to identify unique molecular reads and remove bias introduced by PCR amplification. This technique has allowed highly accurate quantitation of T cell clonal expansions in disease states. Additionally, sequencing of the complementary determining region 3 (CDR3), responsible for peptide recognition has been used to determine HTLV-1 and disease-specific motifs in both HAM/TSP and ATL. In this review, we will compare the differing TCR repertoire profiles of HAM/TSP and ATL and discuss differences in profiles that may contribute to differing disease outcomes.15 to 1020 in PBMCs  to another (Vβ 13.1), corresponding to an increase in somatic mutations associated with upregulation of genes downstream of the TCR pathway . Further+ T cells   in CDR3 of the TCR-β chain  , 85. In -β chain –88.+ cells by enhanced antibody-dependent cellular cytotoxicity and has shown substantial anti-ATL activity, even in relapsing or chemotherapy-resistant disease  which can provide short-term survival of about one year , major T disease , 91. Mog T cells . Thus, TIn HTLV-1-associated disease, identification of conserved TCR sequences or motifs maybe useful in predicting disease-specific outcomes in early-stage HTLV-1 infection. While it is still challenging to identify shared TCR sequences between individuals and disease-specific TCRs across patients , sequenc+ T cells and Tax11-19-specific CD8+ T cells in HAM/TSP patients  demonstrating that PDR amino acid sequence motif was conserved in CDR3β of Tax301-309-specific CD8+ T cells regardless of clinical subtype in HTLV-1 infection  mice inoculated with an HLA-A*24:02+ HTLV-1-infected cell line (MT-2) resulted in significant decreases in size and eventual eradication of tumors, compared to uncontrolled tumor growth and eventual death of non-genetically modified PBMC treated and control mice (In addition, treatment with Tax301-309-specific PDRrol mice . These rrol mice , 98, furin vivo TCR repertoire in ATL and HAM/TSP, particularly in compartments such as CSF for HAM/TSP in which large numbers of cells are difficult to obtain and which may be more closely reflective of events in the CNS rather than T cells in the peripheral blood. Lastly, TCR dynamics, tracking and mRNA single cell sequencing in local tissues would offer a valuable tool to discover antigen specificity, transcriptional profiling, and the molecular mechanisms of T cell plasticity to understand the heterogeneity and complexity of HTLV-1-associated diseases.Analysis of TCR repertoires associated with a wide range of cancers, autoimmune, and inflammatory diseases in various compartments such as peripheral blood and CSF will greatly contribute to our understanding of the role that T cells play in these disorders and may lead to identification of markers of disease and even potential therapeutic targets. While understanding of TCR repertoires in HTLV-1-associated disease has made great strides in recent years due to advances in sequencing technology, large gaps remain in the literature regarding the progression of the TCR repertoire from HTLV-1-infected asymptomatic carrier to diseased individuals. Increasing evidence indicates T cell clonal profiles and motif sequences may be unique to different HTLV-1-associated diseases. Timing of progression from asymptomatic carrier to disease may improve our ability for early diagnosis and intervention. Longitudinal studies following asymptomatic carriers through disease development, treatment and remission or disease progression will be invaluable in this effort. Further studies utilizing new techniques in high throughput single cell sequencing are warranted to better characterize the AC and YE-A contributed to paper writing. SJ supervised and contributed to discussion and writing. All authors contributed to the article and approved the submitted version.This research was supported by the Intramural Research Program of the NIH, NINDS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We found that UA and ejection fraction (EF) were significantly correlated , but not EF and eGFR or eGFR and UA. A multiple regression analysis adjusted for age, body mass index, eGFR, and UA, showed that UA was significantly associated with a reduced EF . The UA cut-off indicative of an EF < 40% was 6.69 mg/dL . Among drugs known to effect UA concentrations, we found that only diuretics were used more frequently in patients with high UA (above the median) than in patients with low UA . Among patients that used diuretics, UA remained significantly correlated with EF. Thus, we showed that reduced EF was associated with UA in patients with AF and normal renal function, independent of eGFR and diuretic use.Hyperuricemia is associated with the risk of developing atrial fibrillation (AF) and heart failure. However, coexisting chronic kidney disease and certain cardiovascular drugs make it difficult to determine whether hyperuricemia is a risk factor or merely a marker of pathology. We retrieved data from the Polish Atrial Fibrillation (POL-AF) registry, which included consecutive patients hospitalized with AF from January to December, 2019. We included 829 patients (mean age: 72.7 ± 11.1 years) with data on serum uric acid  and estimated glomerular filtration rate (eGFR) ≥ 60 mL/min/1.73 m Our primary aim was to investigate whether the relationship between UA and EF in patients with AF might be independent of renal function. Our secondary aim was to assess the effects of selected drugs on that relationship.There is no doubt that atrial fibrillation (AF) and heart failure (HF) are two of the most serious challenges in cardiology in the 21st century ,4,5. TheThe Polish Atrial Fibrillation (POL-AF) Registry study was a multicenter, prospective, observational study that included patients with AF from 10 cardiology centers. The study was registered in ClinicalTrials.gov (study ID: NCT04419012). The data were collected for 2 full weeks each month, from January to December 2019. Eligible patients were over 18 years old, were diagnosed with AF, documented with an electrocardiographic examination or medical report, and were hospitalized for either urgent or planned reasons. No clear exclusion criteria were defined; however, patients admitted for an ablation due to AF were excluded. The study was approved by the Ethics Committee of the Swietokrzyska Medical Chamber in Kielce (104/2018). The Ethics Committee waived the requirement for informed consent from the patients.n = 14), with malignant diseases (n = 77), and with eGFR < 60 mL/min/1.73 m2 . Thus, 829 patients were included in the final analysis, including 366 women . We also collected data on pharmacotherapy regimens, including the use of beta-blockers (BB), angiotensin converting enzyme inhibitors (ACEi), angiotensin receptor blockers , and diuretics . The data from the registry indicated the use of a given group of drugs, but lacked information on doses.Data were collected on baseline characteristics, including demographics, medical history, diagnostic test results, and pharmacotherapy. Impaired kidney function was defined as eGFR < 60 mL/min/1.73 mPrevious studies demonstrated a relationship between UA and EF <40% in patients with AF. However, pharmacotherapy regimens that are standard for HF with reduced EF (HFrEF), particularly diuretics, can affect the concentration of UA. Because the UA cut-off point identifies patients with EF < 40%, we could expect a significant difference in EF between patients treated and those not treated with standard HF pharmacotherapy regimens, particularly diuretics. However, if patients were divided according to the objective UA value into two equal groups , the influences of UA on the EF and the frequency of pharmacotherapy regiments would no longer be clear. In the present our study, we aimed to evaluate the relationship between UA and EF, independent, as much as possible, from the potential impact of pharmacotherapy. To that end, we used the median concentration of UA as the criterion for dividing the patients into groups, instead of a UA cut-off point for EF < 40%.t-test or the Mann–Whitney U test, for continuous variables, and the chi-squared test for categorical variables. We performed Pearson’s correlation analyses to investigate relationships between variables. For logistic regression models, we report the odds ratios (OR) with 95% confidence intervals (95% CI). We built a multiple regression model with backward stepwise calculations. The model included age, body mass index (BMI), eGFR, and UA concentration. In the logistic regression model, we took into account the fact of using particular groups of drugs and the diagnosis of permanent AF. To determine the predictive value of variables, we performed receiver-operating characteristic (ROC) curves, and set cut-off points according to the Youden index. Two-tailed p-values < 0.05 were considered significant. Missing data were removed case-wise. All statistical analyses were performed with Statistica 13 .Continuous data are expressed as the means and standard deviations and categorical data are expressed as frequencies and percentages. We performed parametric tests to analyze continuous variables with a normal distribution; and we performed non-parametric tests for variables with non-normal distributions. Group comparisons were performed with the Student’s p = 0.0436). The mean EF was 49.9 ± 12.5%. Among the comorbidities, arterial hypertension was the most common (86% of patients), and EF < 40% was found in 16% of the cohort. The general characteristics of the study population are presented in The cohort had a mean age of 72.7 ± 11.1 years, 366 were women (44% of the study population), and the mean UA was 6.56 ± 1.78 mg/dL. The general norms of UA are different for men and women; however, in our study population, there was no significant difference in the mean UA concentrations between women (6.520 mg/dL) and men  in our study population.In the multiple backward stepwise regression analysis model, we included age, BMI, eGFR, and UA ; we founp = 0.001), and the predictive ability showed approximately 60% sensitivity and 60% specificity. According to the univariate logistic regression analysis, patients with a UA ≥ 6.69 mg/dL were about twice as likely to have an EF < 40% than those with a UA < 6.69 mg/dL .Based on the ROC curve , we founTo minimize the impact of the standard HFrEF pharmacotherapy regimen on our results, as described in Patients with high UA concentrations were distinguished by a lower mean EF and a higher frequency of EF < 40% compared to those with low UA concentrations. In the context of pharmacotherapy, these groups only differed significantly in the use of diuretics .2 vs. 73 mL/min/1.73 m2, respectively, for UA < 6.69 mg/dL and for UA ≥ 6.69 mg/dL; p = 0.071). It is worth noting that, with that division criterion, we found significant differences in the mean concentrations of HDL and TG . It should also be noted that the type of AF did not have a significant effect on whether the UA was above or below 6.69 mg/dL. In a univariate logistic regression analysis, permanent AF was not associated with UA concentration ≥ 6.69 mg/dL . The results of this univariate logistic regression are presented in The difference in mean eGFR values requires an additional comment. This difference resulted from adopting the median UA concentration as the criterion for dividing the studied population. In an additional analysis, when the division criterion was UA = 6.69 mg/dL (according to the ROC for EF < 40%), the mean eGFR values did not differ significantly between groups (75 mL/min/1.73 mp = 0.012), and increased by 69%, with the use of any diuretic . The results of univariate logistic regression are presented in We performed a logistic regression model to analyze all groups of drugs used by the included patients. However, only the use of diuretics was significantly associated with a UA above the median. The probability of having a UA concentration equal to or above the median value increased by 43% with the use of ARA . The results of this analysis are presented in 2, hyperuricemia is a marker of reduced EF and the relationship between UA and EF is independent both of renal function and the use of drugs typical for treating heart failure. These three issues need to be discussed.In the prospective Chronic Renal Insufficiency Cohort study, the prevalence of AF was 18% in patients with CKD . MoreoveIt is known that the concentration of UA increases with deteriorating kidney function. On the other hand, an increasing number of studies have shown that, in patients with normal kidney function, high UA levels may lead to kidney damage ,28. Morep = 0.004) in a multivariate analysis [p = 0.01) [p = 0.04, in multivariate cox proportional hazard model) [To our knowledge, our observations are the first of this type described in an AF patient population. Moreover, our results were consistent with observations from other studies regarding the importance of hyperuricemia in assessing the prognosis of patients with HFpEF. Palazzuoli et al. showed that hyperuricemia, but not CKD, was an independent predictor of hospitalization due to HF or death in a HFpEF subpopulation  . In theiIn the second part of the analysis, we assessed the extent to which hyperuricemia might be related to the use of top-class drugs for HFrEF pharmacotherapy. A previous study by Shimizu and colleagues showed that ACEi, beta-blockers, and diuretics were used more frequently in the group with hyperuricemia, compared to a group without hyperuricemia . It is wUltimately, it remains controversial whether high UA alone leads to impaired cardiac function or whether it is merely a marker of HF status. However, one of our results may shed new light on this issue. Although it can be assumed that permanent AF is the form of arrhythmia that most significantly impairs cardiac systolic function, in our population, permanent AF occurred with a similar frequency in the subgroups divided by the median UA concentration. Moreover, permanent AF had no significant effect on whether patients had UA levels above the median. Evidence that emerged in the last year showed that drugs that lowered UA concentrations had positive effects on heart function and structure and reduced the risk of HF symptoms ,35. TakiOur study had some limitations. First, the registry protocol did not provide tools for performing a prospective follow-up. Second, no information was available on the use of drugs for reducing UA. Moreover, there were no data on the doses of the drugs that we included in the regression analysis or on the specific molecules used (apart from ARA). In addition, the correlations demonstrated in this study, despite their statistical significance, were weak ,37. TherThis study demonstrated an association between reduced EF and UA levels in patients with AF that had normal renal function. Moreover, we found that this association was independent of eGFR. Higher UA concentrations indicated lower EF values, and the UA concentration cut-off point for predicting an EF < 40% was 6.69 mg/dL. We also showed that the use of drug classes known to affect UA levels, including diuretics, did not affect the relationship between EF and UA in patients with AF and a normal eGFR.HighlightsIn patients with AF, hyperuricemia is a marker of reduced EF.2, the relationship between UA and EFIn patients with AF and eGFR >60 mL/min/1.73 m(a)Is independent of renal function; and(b)Is independent of the use of drugs typical for treating heart failure


Smad3 might be the target gene ofmiR-140-3p. These findings illuminate the inhibitory effects of miR-140-3p onthe proliferation and migration of macrophages and contribute to the cognitionof the essential roles of miRNAs during peripheral nerve regeneration.Macrophages benefit myelin debris removal, blood vessel formation, and Schwanncell activation following peripheral nerve injury. Identifying factors thatmodulate macrophage phenotype may advantage the repair and regeneration ofinjured peripheral nerves. microRNAs (miRNAs) are important regulators of manyphysiological and pathological processes, including peripheral nerveregeneration. Herein, we investigated the regulatory roles of miR-140-3p, amiRNA that was differentially expressed in injured rat sciatic nerves, inmacrophage RAW264.7 cells. Observations from EdU proliferation assaydemonstrated that elevated miR-140-3p decreased the proliferation rates ofRAW264.7 cells while suppressed miR-140-3p increased the proliferation rates ofRAW264.7 cells. Transwell-based migration assay showed that up-regulated anddown-regulated miR-140-3p led to elevated and reduced migration abilities,respectively. However, the abundances of numerous phenotypic markers of M1 andM2 macrophages were not significantly altered by miR-140-3p mimic or inhibitortransfection. Bioinformatic analysis and miR-140-3p-induced gene suppressionexamination suggested that  Peripheral nerves, unlike central nerves, exhibit a remarkable spontaneousregeneration capacity after nerve injury. The successful regeneration of injuredperipheral nerves largely depends on the reprogramming of Schwann cells, theattraction and recruitment of macrophages, and the activation of the intrinsicgrowth capacity of affected neurons . FollowiEmerging studies have demonstrated that non-coding RNAs, especially microRNAs(miRNAs), play essential roles following peripheral nerve injury . miRNAs Sequencing data of injured rat sciatic nerves showed that miR-140-3p, a miRNApreviously annotated as miR-140* according to miRbase database  using Lipofectamine RNAiMAX transfection reagent.The murine macrophage cell line RAW264.7 was purchased from the Typical CulturePreservation Commission . RAW264.7 cells werecultured in DMEM  supplemented with 10%heat-inactivated FBS  in a 5% COTM miRNA qRT-PCR Starter Kit (RiboBio) orHiScript III RT SuperMix for qPCR (+gDNA wiper) . A StepOne Real-time PCR System  was used to determine the Ct values of miR-140-3p, miRNA internal controlU6, Cd86, Tnf,Gpr18, Egr2, Myc,Il-10, Smad3, and mRNA internal controlGapdh. The comparative 2-ΔΔCt method was appliedto calculate relative gene expressions. Bulge-loopTM miRNA qRT-PCRPrimer Sets specific for miR-140-3p was designed and synthesized by RiboBio andprimer sets for coding RNAs were synthesized by Sangon Biotech . Primer sequences  and reverselytranscribed using Bulge-Loopequences  weredesCell counting kit-8 (CCK-8) proliferation assay kit  was used to determine the effect of miR-140-3p on the viabilityof cells according to the manufacturer’s instructions. Briefly, RAW264.7 cellswere transfected with miR-140-3p mimic, mimic control, miR-140-3p inhibitor, orinhibitor control for 36 hours, respectively. Then cells were seeded onto a96-well cell culture plate and treated with 10 μl CCK-8 solution for 2 hours(TM EdU DNA Cell Proliferation Kit (RiboBio) was applied toexamine the effect of miR-140-3p on the proliferation rate of cells aspreviously reported  while FBS containing complete cell culture medium was addedto the lower chamber. After 36 hours of culture, RAW264.7 cells left on uppersurface of the upper chamber were scraped away with a cotton swab while invadedcells on the bottom surface were fixed in 4% paraformaldehyde, stained with 0.1%crystal violet, and observed under a DMR inverted microscope (LeicaMicrosystems). The relative migration ability of RAW264.7 cells was calculatedby measuring crystal violet-stained areas in randomly selected fields. Inaddition, cell migration was confirmed with a second assay usingfibronectin-coated transwells. The procedure was the same as above except thatthe surface of the transwell chamber was pre-coated with a layer fibronectin before cells plantation, aspreviously reported  terms and Kyoto Enrichment of Genes and Genomes (KEGG)pathways were analyzed using the ClueGo plug-in in the Cytoscape software  test using GraphPad Prism 5.0 . Differences with a p-value less than 0.05were considered statistically significant.Statistical analysis was conducted by the Student's Cultured macrophage RAW264.7 cells were transfected with miR-140-3p mimic, mimiccontrol, miR-140-3p inhibitor, or inhibitor control to determine the functionaleffects of miR-140-3p on macrophages. Compared with RAW264.7 cells or cellstransfected with mimic control, RAW264.7 cells transfected with miR-140-3p mimicexhibited robust elevation of miR-140-3p expression . In contThe effect of miR-140-3p on macrophage proliferation was then investigated.Considerable cells were observed to be existed in a proliferating state inRAW264.7 cells transfected with mimic control or inhibitor control. Comparedwith cells transfected with mimic control, less amount of RAW264.7 cells werelabeled as EdU-positive cells. Rather, the number of EdU-positive RAW264.7 cellswas obviously higher in cells transfected with miR-140-3p inhibitor as comparedwith cells transfected with inhibitor control . The inhTranswell migration assay was applied to observe the effect of miR-140-3p onmacrophage migration. Crystal violet staining of the bottom surface of the upperchamber of transwell showed that RAW264.7 cells obtain certain ability tomigrate across transwell. However, following the transfection of miR-140-3pmimic, crystal violet-stained areas of migrated cells reduced to about 60% ascompared with stained areas in the control group. In contrast, RAW264.7 cellstransfected with miR-140-3p inhibitor had enlarged crystal violet-stained areas. AdditioQuantificational results showed that miR-140-3p mimic significantly increased theexpression level of miR-140-3p (over 300 fold). So, we wondered if miR-140-3pmimic could reverse the effect of the inhibitor when RAW264.7 cells weretransfected with both. PCR results showed that miR-140-3p inhibitorsignificantly reduced the expression level of miR-140-3p which was consistentwith the above results. However, miR-140-3p mimic reversed the effect ofinhibitor and elevated the abundance of miR-140-3p when RAW264.7 cells weresimultaneously transfected with miR-140-3p inhibitor and mimic . CellulaCd86and Tnf or M2 macrophage markers Egr2,Myc, and Il10. For M1 macrophage markerGpr18, although the mRNA expression ofGpr18 seemed to be decreased after miR-140-3p inhibitortransfection, the expression of Gpr18 was not increased aftermiR-140-3p mimic transfection (Pro-inflammatory (M1) and anti-inflammatory (M2) macrophages execute uniquefunctions in diverse biological activities. The switch of M1 and M2 phenotypesof macrophages is also involved in the regeneration of injured peripheralnerves. Therefore, the expression levels of M1 and M2 macrophage markers inmiR-140-3p-transfected RAW264.7 cells were evaluated to identify whethermiR-140-3p would affect the M1/M2 polarization of macrophages. RT-PCR resultsshowed that neither miR-140-3p mimic nor miR-140-3p inhibitor significantlyaltered the abundances of mRNAs coding for M1/M2 markers sfection .These oAsp1,Smad3, Cxcl12, and Hdac4,were identified to be potential target genes of mmu-miR-140 (Asp1 was not found to be significantlyrelated with GO terms or KEGG pathways and thus was not displayed in theinteraction network. The constructed network showed that Smad3was associated with activities enriched in functional groups of mineralcorticoidreceptor binding, osteoblasts development, and positive regulation of positivechemotaxis, Hdac4 was associated with osteoblasts development,positive regulation of positive chemotaxis, and regulation of myotubedifferentiation, while Cxcl12 was associated with regulation ofmyotube differentiation, response to ultrasound, and positive regulation ofpositive chemotaxis. Considering that Smad3 was linked to alarge number of essential biological processes, the regulating effect ofmiR-140-3p on Smad3 mRNA expression was further examined.RT-PCR results demonstrated that the mRNA expression of Smad3was reduced after miR-140-3p mimic transfection but was increased aftermiR-140-3p inhibitor transfection, suggesting that miR-140-3p negativelyregulates the abundance of Smad3 mRNA (Potential target genes of miR-140 in macrophage RAW264.7 cells were discoveredusing Cytoscape bioinformatic analysis software in view of the fact that miRNAsregulate biological activities via binding to and suppressing their targetgenes. A total of four coding genes, i.e., -miR-140 . Downstr-miR-140 . Asp1 waad3 mRNA .Functional recovery of injured peripheral nerves requires the joint work of neurons,Schwann cells, endothelial cells, macrophages, fibroblasts, and other types ofcells. miRNAs extensively regulate the biological functions of various types ofcells and execute considerable functions during peripheral nerve regeneration. Byperforming deep sequencing, the expression patterns of miRNAs in the proximal nervestumps after rat sciatic nerve injury were explored and many dysregulated miRNAswere screened, including miR-140-3p (Although the regulatory roles of miR-140-3p in Schwann cells have not been reported,a previous study examined the effect of miR-140-5p, a miRNA that originates from theopposite arm of the pre-miR-140, on Schwann cells and showed that miR-140-5pinhibits the differentiation and myelination of Schwann cells (Gpr18 and novel murine M2macrophage markers Egr2 and Myc were determined(Besides cellular proliferation and migration, the polarization of macrophages andchanges of M1 and M2 macrophages are highly associated with the regeneration process(Smad3, a well-known intracellular signaltransducer of TGF-β signaling pathway, was identified as a candidate target gene ofmiR-140-3p. TGF-β to trigger theactivation of its downstream Smad3 effector. Therefore,Smad3 is an important factor affecting cell migration.Cytoscape analyses-identified Smad3-associated GO terms and KEGGpathways are also closely connected with tissue development and regeneration. Forinstance, mineralcorticoid receptor activation adjust the pro-inflammatory signalingand regulate the injury response of cardiac tissue (Smad3 mRNA expression, indicate that miR-140-3p may participatein macrophage phenotype modulation and peripheral nerve regeneration via negativelyregulating Smad3, which remains to be confirmed by our furtherstudy.The potential downstream target genes and biological implications of miR-140-3p werefurther analyzed. Notably, Collectively, our results reveal functional effects of miR-140-3p on macrophages andsuggest that overexpression and silencing of miR-140-3p inhibits and promotes theproliferation rate and migration ability of macrophages, respectively. Thesefindings contribute to a better understanding of the essential regulatory roles ofmiR-140-3p in multiple cell types and emphasize the functional involvement of miRNAsin peripheral nerve repair and regeneration.


However, cancer therapy targeting CD276 may be effective in the treatment of slow proliferating tumor cells.The cell surface molecule CD276 (B7-H3) is an immune checkpoint antigen. The elevated expression of CD276 on tumors contributes to the suppression of anti-tumor T-cell responses and correlates with poor prognosis. Methods: The expression of CD276 was explored in vitro on eight urothelial carcinoma cell lines (UM-UC) in comparison to eight normal urothelial cells (NUCs) by RT-qPCR, Western blotting, and flow cytometry. Cell proliferation was enumerated over consecutive passages. The expression of cancer stem cell markers CD24 and CD44, cytokeratins, and vimentin was investigated by immunofluorescence. The expression of CD276 in bladder tumor samples and metastases was explored by immunohistochemistry. Results: Expression of CD276 on cell surfaces was elevated on UM-UCs when compared to NUCs. In UM-UCs, CD276 transcripts correlated moderately positive with CD276 protein expression (ρ = 0.660) and strongly positive with CD276 surface-expression (ρ = 0.810). CD276 mRNA expression (ρ = −0.475) and CD276 protein expression (ρ = −0.417) had a significant negative correlation with proliferation, while a significant correlation between proliferation and cell surface expression was not observed in UM-UCs. Conclusion: The expression of CD276 on UM-UC bladder tumor cell surfaces is elevated. Slow proliferating UM-UC cells express more CD276 mRNA and protein than fast proliferating cells. In patients, slow proliferating CD276 Urothelial cell carcinoma (UCC) is the most common malignancy of the urinary system . It is aIn bladder cancer tissue elevated expression of immune checkpoint antigens such as programmed cell death protein-1 , its ligand, PD-L1 (=CD274), anti-cytotoxic T-lymphocyte associated antigen-4 , and B7-H3 (=CD276) was observed ,9. TheseThe elevated expression of CD276 was reported on a variety of cancer cells, including bladder cancer cells ,17,18. INormal human urothelial cells (NUCs) and human bladder cancer cell lines of the UM-UC series were expanded in vitro. The cultures presented with quite distinct features. Upon seeding, NUCs and UM-UC-13 attached as individual cells and proliferated to yield confluent cultures A,F. In ct-test: p = 0.015). The normal distribution of CD276 transcripts was tested using Shapiro–Wilk test with p = 0.37 confirming normal distribution . The expression of total CD276 protein in NUCs and UM-UC lines followed at large the transcript amounts. (n = 5), UM-UC-10 expressed significantly more CD276 . Overall, the UM-UC lines investigated presented more CD276 on cell surfaces  than NUCs . Taken together, the highest cell surface expression was observed on UM-UC-15 cells , the expression of AE1/AE3 antigens  and total protein (ρ = –0.417) expression. This indicated that the elevated expression of CD276 was not a selection marker in the UM-UC lines investigated nor enhanced during extended in vitro culture. It rather confirmed the elevated expression of this immune checkpoint antigen in bladder cancer-derived cells . A signiHowever, knowledge of the cellular distribution of CD276 in cancer cells is of interest as a recent study provided evidence that cytoplasmic expression of CD276 was associated with shorter disease-specific survival of cancer patients . This wolow tumor cells which could become more accessible to anti-tumor immune responses. However, for its function as an immune-modulatory antigen, elevated cell surface expression of CD276 is relevant [For the transport of CD276 to the cell surface, proteins must be encapsulated by coated vesicles, which shuttle cargo between the endoplasmatic reticulum and the Golgi apparatus. From Golgi, three classical export routes using secretory vesicles, a direct route, or endosomes may transport CD276 to the cell surface . Additiorelevant . When anIn recent studies, the expression of CD276 in bladder cancer tissue samples was investigated and a wide variety of CD276 staining intensities was found in both the pathologically benign-appearing samples from bladder cancer patients (score 0 to approx. 150) in comparison to pathologically define malignant areas score 0–300) confirming previous studies ,50. This0 confirmThe elevated expression of CD276 was considered a bladder cancer stem cell marker. We therefore investigated the expression levels of other BC stem cell markers in UM-UCs as well. Comparably high expressions of ALDH1-A1 and CD24 were recorded in UM-UC-15. However, CD44 was detected at comparably moderate levels in these cells. UM-UC-15 expressed CD276 transcript levels in the range of NUCs, and a simultaneous and significant upregulation of the four BC stem cell markers investigated in this study was not found. In the other UM-UC lines variable expression patterns of CD276, ALDH1-A1, CD24, and CD44 were observed, supporting the notion that evidence for a concurrent activation of stem cell marker expression is lacking in the UM-UC cells.pos bladder cancer stem cells are not enriched in metastases of lymph nodes, but CD276 is found on both sides. The knock-down of CD276 by a lentiviral vector decreased cell proliferation and induced apoptosis in progenitor cells. The silencing of CD276 of such cells arrested mitosis in the G0/G1 stage of the cell cycle [The knock-down of CD276 expression in melanoma cells decreased their metastatic potential. It therefore was considered a promotor of metastases ,21,53. Cll cycle , while ill cycle . This sull cycle ,56. We tll cycle ,45, but ll cycle . The patn = 8) were obtained from pathologically healthy-appearing sites of surgical samples from patients after informed and written consent. NUCs were prepared, expanded, and characterized as described recently [Normal urothelial cells , were srecently . At abouThe quantification of gene expression on mRNA transcript levels was performed as described recently . In briev/v) Tween20 in PBS (PBS-T) to remove SDS, blocked by 5% dry milk (w/v) in PBS-T , slow shaker), washed again in PBS-T and incubated overnight with anti-CD276 antibody  in PBS-T, 4 °C, slow shaker). The primary antibody solution was removed, the blot was washed , and reacted with HRP-labelled rb- anti-mo IgG , 1:10,000 in PBS-T, 1 h, AT, slow shaker). The blot was washed 3× with PBS-T at AT [For extraction of total protein, cells were washed twice with PBS, detached by the aid of Accutase, aspirated, washed again, counted, and sedimented by centrifugation . 5E05 cells were resuspended in 100 μL RIPA+ buffer . Samples were homogenized by vigorous pipetting and stored at −80 °C. For further analyses, samples were thawed on wet ice and mixed by vortexing. To remove insoluble debris, samples were centrifuged  and aliquots of the supernatant were transferred to fresh tubes and stored at −80 °C. The protein concentration in the precleared supernatant was determined by spectrophotometry as suggested by the supplier . For Western blotting, samples were mixed with Lämmli protein gel loading buffer to yield 50 μg of protein per lane and separated by SDS-PAGE as described . The pro-T at AT . To visuThe expression of CD276 on cell surfaces was enumerated by flow cytometry (FC) as described recently . In brieTo detect the expression of cell surface proteins on cultured cells, immunocytochemistry (ICC) was performed. Cells were washed 3× from 2 min with PBS, fixed , and washed twice again with PBS. The cells were permeabilized by saponin  and washed twice by PBS again. Unspecific peroxidase activity and antibodies binding were blocked as recommended , and samples were washed again. Antibodies to cytokeratins detected by AE1/AE3  as epithelial and urothelial markers or anti-vimentin  as mesenchymal antigen were added and incubated in a humidified box . The primary antibodies were washed off , and detected by the HRP or AP polymer reagent (IHC ZytochemPlus). Samples omitting the primary antibodies served as controls. The detection reagents were washed off  and the samples were counterstained by HE, covered , and recorded by microscopy .In addition, for UM-UC-13 cells immunofluorescence (IF) was employed. Antibodies AE1/AE3 (see above), anti-vimentin (see above), and anti-CD276  were added to the cells, incubated, and washed as described above. The binding of primary antibodies was detected by Cy-3 labeled gt-anti-mo affinity-purified antibodies  or AlexaFluor 488-labeled gt-anti-rb antibodies . The incubation of samples with mouse  or rabbit  IgG1 antibodies served as isotype controls. Samples omitting the primary antibodies served as secondary antibody controls. Cell nuclei were labeled by DAPI. The staining of cells was visualized and recorded by fluorescence microscopy .To determine the expression of cytokeratins, CD276, CD24, and CD44 on lymph node metastases from bladder cancer patients and on the primary tumor of the bladder, immunohistochemistry (IHC) was performed. Microsections (5 μM) were generated from paraffin-embedded samples from lymph node metastases of 5 bladder cancer patients and from 3 bladder cancer tissue samples . The paraffin sections were rehydrated and incubated with primary antibodies to AE1/AE3, CD276, CD24  and CD44  as described above. HE-staining was employed to visualize the histology of the tissue sections. The microsections were counterstained and recorded as described above. The study was approved by the local Ethics Committee (file number 8041/2020/BO2).t-test with independent variables. Correlation analyses were computed by Spearman’s rank-order correlation. Correlations were displayed as values from −1 (linear negative correlation) to +1 (linear positive correlation) for ρ (rho). Differences in data sets with p-values smaller than 0.05 were considered statistically significant and indicated in the artwork where aprorpiate .Experimental results were processed by a spreadsheet program (Excel) and imported to SPSS statistics software  for further analyses. We tested for normal distributions using the Shapiro–Wilk test. Differences in mean values were computed using either a Mann–Whitney test or a double-sided Student’s We conclude that the individual UM-UC lines seem to represent a cohort of bladder cancer cell clones with different; quite variable CD276 expression levels. The same applies—at least on transcript levels–to the expression of the stem cell markers ALDH1; CD24; and CD44 as well. Our recent studies provided evidence that elevated CD276 expression is recorded in all stages of bladder cancer . We corr


The study showed that the Phage Display technique is effective for the identification of HTLV-1-related peptides. Clone B6 indicated to be a good marker for bioprospecting diagnostic test for HTLV-1 infection and could be used as a possible vaccine candidate for future studies.Human T-lymphotropic virus 1 (HTLV-1) is endemic worldwide and the infection results in severe diseases, including Adult T-cell Leukemia (ATL) and HTLV-1 associated myelopathy (HAM). There are some limitations of employing the present commercial serological assays for both diagnostic and epidemiological purposes in different geographical areas of the Brazil, such as the Amazon Region. Currently, methods for diagnosis are usually expensive to adapt for routine use. The aim of this work was to identify and characterize specific ligands to IgG that mimic HTLV-1 epitopes through the Phage Display technique, which could be used for diagnosis and as future vaccine candidates. Initially, IgG from 10 patients with HTLV-1 and 20 negative controls were covalently coupled to protein G-magnetic beads. After biopanning, genetic sequencing, bioinformatics analysis and Phage-ELISA were performed. The technique allowed the identification of 4 clones with HTLV-1 mimetic peptides, three aligned with gp46, A6 (SPYW), B6 (SQLP) and D7 (PLIL), and one with the protease and Tax, A8 (SPPR). Clones A6 and B6 showed higher values of accessibility, antigenicity and hydrophilicity. The reactivity of the clones evaluated by the Receiver Operating Characteristic (ROC) curve showed that the B6 clone had the highest Area Under Curve (0.83) and sensitivity and specificity values (both were 77.27 %;  Infection with human T-lymphotropic virus 1 (HTLV-1) can cause diseases such as adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy (HAM) . It is eIn Brazil, the diagnosis of HTLV infection presents great difficulties, since the tests for the virus are not fully available in the basic health care for all people. The test is requested for specific groups or situations, such as individuals with clinical manifestations compatible with diseases associated with HTLV-1, the differential diagnosis of myelopathies, during pregnancy and for blood donors. The diagnosis follows the algorithm of the Ministry of Health, which is still quite complex, expensive, and not available to the entire population , 6. As aAlthough serological tests have been improved in recent years , the lacThe phage display technique consists of the presentation of peptides on the surface of filamentous bacteriophages. These peptide sequences can interact with a wide variety of target molecules, resulting in a physical link between the displayed peptide and the target molecule . The selPhage display is a low-cost method that yields relatively fast results, characteristics that have favored the use of the technique in the identification of peptides for use in different areas of health –15. The To select peptides to screen, serum samples were taken from people living with HTLV-1 from the Clinic of Infectious Diseases  of the Center for Tropical Medicine, Institute of Health Sciences, Federal University of Pará , and an HTLV-negative control group taken from the general population of Belém  as well as blood donors from the state of Pará.n = 10). Samples of individuals older than 18 years old, without restriction to ethnicity, social status, or skin color, who were not using corticosteroids were included in the study. Samples of individuals with HTLV-1 who were coinfected with HIV, HBV, or HCV, who had autoimmune diseases, who had an indeterminate diagnosis, or who had a low cut-off value in the enzyme-linked immunosorbent assay  were excluded from the study to minimize potential errors during data analysis and improve the sensitivity and specificity.Forty individuals participated in the study; 10 individuals were diagnosed with HTLV-1 infection, and 20 individuals in the control group were seronegative for anti-HTLV-1/2 and anti-HIV-1 antibodies. To increase the specificity of the selected peptides, a subtractive selection step was performed using pooled serum from individuals seropositive for HIV-1 (The project was approved by the Human Research Ethics Committee of the Haemotherapy and Hematology Center of Pará (HEMOPA Foundation) under registration number 0011.0.324.000-11. Individuals who agreed to participate in the study signed an informed consent form.Initially, the pool of immunoglobulin G (IgG) in the different groups was linked to magnetic beads  according to the manufacturer's recommendations. Next, the IgG coupled to magnetic microspheres was incubated with a library of random peptides (Ph.D.-C12 Phage Display (PD) Peptide Library Kit; New England Biolabs, Ipswich, MA, USA). The Ph.D.-12 library is a combinatorial library of random 12-mer peptides fused to a minor coat protein (pIII) of M13 phage.11 phage particles of the PD library) first with IgG from HTLV-seronegative individuals for 30 min at room temperature. Then, the supernatant containing unbound phages was added to IgG of HTLV-2-seropositive individuals with incubation for 30 min at room temperature. The next step consisted of positive selection, in which the final supernatant of the subtractive step was incubated with purified IgG from HTLV-1-positive individuals for 30 min at room temperature. Three biopanning cycles were performed for the selection of HTLV-1 peptides.The biopanning procedure was performed as described with some modifications . The bioThe phages bound to the magnetic microspheres of the positive selections were recovered by acid elution and subjected to steps of amplification, titration, supernatant production, DNA extraction, and phage DNA sequencing .E. coli strain ER2738. The medium was incubated under agitation at 37°C until the early-log phase (OD600–0.3). Upon reaching this stage, the bacterial culture was inoculated with the phages and incubated at 37°C overnight under strong agitation. The next day, the material was centrifuged to remove the bacteria, PEG/NaCl was added to the supernatant, the mixture was centrifuged, and the phage pellet was washed with 1× sterile phosphate-buffered saline (PBS).Amplification consisted of phage multiplication in Luria-Bertani (LB) culture medium containing tetracycline (20 mg/mL) and −1 to 10−5) and amplified eluate (diluted from 10−1 to 10−12) were used. E. coli ER2738 was cultured in LB medium, to which dilutions containing the phages were added. The samples were plated on LB agar containing tetracycline and isopropyl-beta-d-thiogalactopyranoside/5-bromo-4-chloro-3-indolyl-α-d-galactoside with the addition of Top agar and incubated at 37°C for 24 h.The titration was performed to determine the amounts of input and output of viral particles during the biopanning cycles. Eluate containing phages from each selection cycle that were not amplified  containing DNA was extracted from the phage supernatant using iodide buffer. The procedure consisted of viral particle lysis, protein precipitation, DNA precipitation, and DNA hydration. The presence of single-stranded DNA was verified by electrophoresis in a 0.8% agarose gel containing HydraGreen Safe DNA Stain 20,000X  by visual comparison with purified DNA from single-stranded standard M13mp18 tape .For the sequencing reaction, phage DNA, primer −96 gIII (5'-OH CCC TCA TAG TTA GCG TAA CG-3') (New England Biolabs), and Premix  were used. The reaction was performed in a thermocycler . The sequenced DNA was precipitated and resuspended in dilution buffer and read in the ABI PRISM 3130 Genetic Analyser .in silico using the ExPASy Translate Tool 6.0 (http://web.expasy.org/translate/), and the alignment of the selected peptides was generated using the program BioEdit 7.1.5.0  to analyse their antigenicity, hydrophilicity, and accessibility profile. The antigenicity profile was determined using the Kolaskar and Tongaonkar antigenicity scale, a semiempirical method that made use of the physicochemical properties of amino acid residues and their frequency of occurrence in experimentally known epitopes. This scale was developed to predict antigenic determinants in proteins. According to the authors, it can identify antigens with an accuracy of ~75% . The absorbance values were measured using a reading filter at a wavelength of 450 nm.Titration curves were generated to determine the most appropriate phage concentration and antibody dilution to be used, to optimize the tests. A microplate (Nunc-Immune Plate MaxiSorp Surface) was sensitized with 10 μg/well anti-M13 monoclonal antibody  diluted in 50 mM carbonate buffer, pH 9.6 (1:500), and incubated overnight at 4°C. After washing and blocking the plate, clones diluted in 1× PBS (1:100) were added to the plate and incubated for 1 h at 37°C. The plate was washed, and pools of HTLV-1-positive serum and negative controls were added. After washing, anti-human IgG diluted in 1× PBS/0.1% Tween (1:5000) was added, and the plate was incubated for 1 h at 37°C. Finally, tetramethylbenzidine was added, and the reaction was stopped by the addition of HA first phage ELISA was performed to evaluate the reactivity of all identified phage clones using the pool of HTLV-1-negative samples (control) and HTLV-1-positive samples. In the ELISA reaction, the samples positive for HTLV-1 were arbitrarily defined as those with an optical density value at least twice as high as the absorbance value of the control samples.The calculation of the area under the receiver operating characteristic (ROC) curve (AUC), sensitivity, specificity, and positive likelihood ratio (LR+) was performed using the program GraphPad Prism version 6.0 . The statistical test used was one-way ANOVA, with post Bonferroni correction test, which is a two-tailed test.After biopanning, a total of 42 selected peptides had different amino acid sequences and were tested for immunoreactivity by phage ELISA .After the screening performed in the first Phage ELISA, the phage clones containing the peptides that presented a ratio between the absorbances of the positive and control samples >2 were submitted to a second Phage ELISA. Although most of the peptides could discriminate individuals with HTLV-1 infection from seronegative control individuals, four peptides  stood out from the rest . In the n = 20) and positive individuals (n = 10), respectively, for each of the four most reactive clones. For the analysis, the ROC curve, sensitivity, specificity and likelihood ratio (LR) were used . It had an AUC equal to 0.83, in addition to having the highest LR+ value (3.4). Thus, this clone could be promising for use in diagnostic immunoassays , while the A8 peptide was aligned with the amino acid sequence of the HTLV-1 protease and Tax protein .For the antigenicity, hydrophilicity, and accessibility tests, only the Brazilian strain (AY563954.1) was used. In the accessibility profile scale, values above 1 denote regions accessible to antibodies. In the HTLV-1 gp46, the peptide that showed the highest accessibility value was clone A6 (score 2.732), corresponding to the region ranging from amino acids 132–135 (SPYW). Regarding the antigenicity profile, the D7 peptide was the most antigenic (score of 1.176), with a location corresponding to the region between amino acids 13 and 19 (PLIL) of gp46. Hydrophilicity was lowest for peptide D7 (-1.029). In The immunodiagnostic assays used for peptide detection have evolved with the use of tests such as ELISA and Western blot, although there are false-positive as a consequence of cross-reactions with other aetiological agents , 27 and Four peptides characterized as linear mimetopes corresponding to the envelope (gp46), Tax, and protease regions were identified. HTLV-1 envelope glycoproteins, especially gp46, play important roles in the infectious process and are exposed on the surface of viral particles and infectious cells, making them accessible to the immune system , 31. RecIt was not possible to analyse the 3D structure because the gp46 structure is not available in the RCSB Protein Data Bank, but accessibility, antigenicity, and hydrophilicity of the mimetopes were investigated. Clones A6 and B6 showed values above the cut-off point for all three analyses which is a good indication for using in diagnostic platforms.in vitro assays, but the clinical results have not been satisfactory , and the highest LR+ value (3.4), all due to its good accessibility, antigenicity, and hydrophilicity.The phage ELISA method was used to assess the reactivity of clones against the sera of HTLV-1 infected persons. Screening was initially performed, and the clones with the highest optical density and those with HTLV-1 consensus sequences were selected  for the evaluation of sensitivity and specificity by calculating the ROC. The mimetic peptide (KLDVFTKPLVFT) of clone D7 showed low accessibility and hydrophilicity, which may have hindered the interaction between the peptide and the anti-HTLV-1 antibodies. The best clone in the sensitivity and specificity tests was B6 (NNDPLQLRSQRY), with a sensitivity and specificity of 77.27%, an accuracy of 0.83  and one related to protease and Tax (A8). The analysis of accessibility, antigenicity, and hydrophilicity showed that clones A6 and B6 are potentially suitable for diagnostic use and that clone B6 has great potential for use in vaccine tests. The best reactivity, evaluated by phage ELISA, was that of clone B6, which showed good AUC, sensitivity, specificity, and LR. Thus, the successful testing of the B6 clone peptide would be relevant to diagnostic tests on platforms that allow rapid results and to the availability of more affordable testing for the overall population, and it would contribute to the prevention and control of HTLV-1 infection.50bp). The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The deposition of the raw data for this manuscript was not possible because of the following reasons: (i) these very small sequences are linked to a bacterial phage (inherent to the phage display methodology), and consequently are not purified; (ii) the sequences are already described as they overlap with gene sequences of glycoprotein 46 (gp46), tax and pro of HTLV-1, which are widely known and fully characterized proteins of the virus, consequently they cannot be registered as “new”; and (iii) the sequences do not reach the size required by the GenBank (The studies involving human participants were reviewed and approved by Ethics Committee in Research with Human Beings of the Hemotherapy and Hematology Center of Pará (HEMOPA Foundation), under registration number 0011.0.324.000-11. The patients/participants provided their written informed consent to participate in this study.LM, LF, MI, and RI: conceptualization. FS and LS: data curation. FS, LS, AS, and MQ: investigation and methodology. LM, LS, AV, and RI: formal analysis. LM, LF, and LS: writing—original draft. LM, MQ, AV, MI, and RI: writing—review and editing. RI: project administration. All authors contributed to the development of research, read, and approved the final manuscript.This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Pró-Amazônia Program (R.I. Proc. 23038000732/2013-09). LS received fellowships of CAPES, Finance Code 001, to develop the study. AV (#302935/2021-5), RI (#312979/2018-5) and LM (#314209/2021-2) are supported and acknowledge the Research Grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Publication of the article was supported by Public Notice PAPQ, PROPESP/FADESP of the Federal University of Pará.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A fundamental question in cell biology underlies how nutrients are regenerated to maintain and renew tissues. Physiologically, the canonical Wnt signaling is a vital pathway for cell growth, tissue remodeling, and organ formation; pathologically, Wnt signaling contributes to the development of myriad human diseases such as cancer. Despite being the focus of intense research, how Wnt intersects with the metabolic networks to promote tissue growth and remodeling has remained mysterious. Our understanding of metabolism has been revolutionized by technological advances in the fields of chemical biology, metabolomics, and live microscopy that have now made it possible to visualize and manipulate metabolism in living cells and tissues. The application of these toolsets to innovative model systems have propelled the Wnt field into new realms at the forefront answering the most pressing paradigms of cell metabolism in health and disease states. Elucidating the basis of Wnt signaling and metabolism in a cell-type and tissue-specific manner will provide a powerful base of knowledge for both basic biomedical fields and clinician scientists, and has the promise to generate new, transformative therapies in disease and even processes of aging. The goal of the present review is to introduce this new field of research linking Wnt signaling, cell metabolism, and endocrine disease. Canonical Wnt signaling has profound effects on cellular metabolism that occur on the timescale of hours, through ß-catenin gene transcription, and minutes, as Wnt ligands induce rapid regulation of cellular homeostasis prior to the transcriptional programs. This flurry of discoveries highlights the enormous potential of Wnt-targeted therapies and the current gaps in knowledge to be addressed before the links between Wnt and metabolism can be fully exploited.Canonical Wnt signaling is a fundamental pathway during embryogenesis and in adult tissues , 2. The The canonical Wnt pathway is composed of a discrete set of proteins that lead to the transcriptional activity of ß-catenin . GSK3ß svia endosomal sorting complexes required for transport (ESCRT)-dependent machinery  and Zinc and Ring Finger protein 3 (ZNRF3), which ubiquitinate Wnt receptors to remove them ocytosis , 23. Secocytosis , 25. Thuocytosis  and scleocytosis . TogetheThe endocrine system consists of a network of glands that release hormones and control body functions. Canonical Wnt signaling has pleiotropic roles across endocrine systems during embryogenesis and in adults to regulate tissue homeostasis and remodeling . ContrarWnt signaling is a fundamental pathway in regulating bone tissues. Bone remodeling is orchestrated by the dynamic interplay of three types of bone cells. Osteoblasts are responsible for bone formation while osteoclasts degrade the bone matrix. Osteocytes represent 90% of bone cells and reside within the bone mineralized matrix . While tOsteocyte secreted factors and the multitude of functions have been recently reviewed . OsteocaFibroblast growth factor 23 (FGF23) is an osteocyte secreted factor that suppresses phosphate reabsorption and vitamin D synthesis in the kidney. The pathophysiological consequences of elevated FGF23 levels involve kidney and bone disease, among others , 33. FGFAdipose tissues integrate whole-body glucose homeostasis and energy balance. Autocrine and paracrine signals from Wnt ligands are famous for their pro-growth roles during embryogenesis and in cancer. This is in stark contrast to Wnt activities within adipocytes, whereby Wnt ligands function as key inhibitors of adipogenesis . LRP5 isEndocrine disorders come in all shapes and sizes. Overproduction of hormones by a gland leads to hormonal imbalance-driving disorders such as diabetes, acromegaly, and bone loss in chronic kidney disease. Underproduction of hormones is the basis of disorders such as hypothyroidism, hypopituitarism, and growth hormone deficiency. The roles of Wnt signaling in these contexts have recently come to light.Aberrant Wnt signaling contributes to several endocrine disorders. For example, diseases characterized by altered hormonal production in adrenal and thyroid glands have been linked to low Wnt activity .Wnt signaling is an essential regulator of bone; genetic mutations in the Wnt pathway proteins lead to either high or low bone density disorders. Loss-of-function mutations in sclerostin lead to pathologically dense bones in diseases such as osteosclerosis –55. LRP5in vitro studies using ACTH secreting glands isolated from Cushing’s patients displayed a downregulation of the Wnt inhibitor, secreted frizzed related protein 2 (SFRP2), to promote pituitary corticotroph adenoma . The role of Wnt signaling in Cushing’s syndrome was underscored by reports showing that patients have increased rates of fracture and elevated levels of sclerostin in circulation . In cont adenoma . Converssynthase . TogetheWnt pathway inhibitors link bone and thyroid signaling. Hyperthyroidism is driven by the upregulation of thyroxine (T4), which increases physiological metabolic rate and leads to weight loss . Mouse mDiabetes is a chronic condition that affects how the body converts food into energy. Obesity is a major concern of public health that is a result of genetic and environmental factors. In Type 1 Diabetes (T1D), an autoimmune attack renders pancreatic islet cells unable to produce insulin, increasing blood sugar. Type 2 Diabetes (T2D) is characterized by insulin resistance, a condition in which the body does not respond appropriately to insulin. Interestingly, genetic mutations in the Wnt pathway proteins were identified in T2D and obesity –72. In Tvia mTOR and TSC2 phosphorylation  . Since tBeyond these tantalizing reports linking Wnt signaling and cellular metabolic reprogramming of cancer, canonical Wnt also regulates tissues outside of cancer. For example, bone formation is driven by a subset of cells lining the calcified bone matrix known as osteoblasts. Wnt signaling is a well-established regulator of osteoblastic activity that leads to new calcified bone tissues. Interestingly, similar to its role in cancer, Wnt activity was found to increase glycolic enzymes through the Warburg Effect to generate energy in osteoblasts. This finding was shown using the LRP5 knockout mouse and resulted in low bone mass and low circulating serum lactate levels, which were linked to reduced mTOR activity and low glycolysis . Additio+-dependent isocitrate dehydrogenase (IDH1/2), a key TCA enzyme that converts isocitrate to α-KG. In myeloid malignancies and solid tumors, mutations in IDH1/2 changes the activity to generate 2-Hydroxyglutarate (2-HG), which competes with α-KG-dependent processes  provide specialized details to modulate Wnt responses. Beyond the well-established roles of protein phosphorylation, we now know there are a variety of modifications that are central to regulating the Wnt pathway. Notably, many of these modifications require cofactors that are sensitive to changes in nutrient availability based on diet (m) render protein methylation and acetylation modifications sensitive to nutrient levels  and acetyl-coA as cofactors, respectively. In contrast to protein phosphorylation, the intracellular concentrations of SAM and acetyl-coA relative to the Michaelis constant , a recently identified tumor suppressor, was shown to render cancer cells sensitive to methionine restriction by lowering SAM levels , 99. Simdiseases . Furtherdiseases . These rdiseases , 101.Amino acids are the building blocks of proteins. Protein catabolism also restores amino acids in cells through degradation in proteasomes or lysosomes. Lysosomes were originally thought to be simply the trash can of the cell. We now know that lysosomes are a central signaling hub and that their function is coopted during the progression of myriad diseases .GBA gene that encodes for Glucocerebrosidase (GCase) enzyme, which breaks down lipids inside the lysosome. Patients present with nutritional depletion, hypermetabolic states, insulin resistance, and hypoadiponectinemia. Recent advances in the fields studying LSDs have been made through the application of induced pluripotent stem cell (iPSC) models. In iPSC GD models, low Wnt signaling was linked to higher levels of Dkk1. Similarly, low Wnt signaling activity was also reported in iPSC models of Niemann-Pick disease, an LSD that causes dysregulated lipid and cholesterol metabolism  are a group of rare inherited diseases that are characterized by dysfunctional lysosomes and are associated with hormonal imbalances and metabolic disorders. One of the most prevalent LSDs is Gaucher’s disease (GD) . GD is ctabolism . In Niemtabolism . These rProtein catabolism is usurped to reprogram tumor cell metabolism. Lysosomes have emerged as an important target in cancer as they replenish nutrients that enable growth. Recent work highlights a central role for the Wnt signaling pathway in regulating lysosomal activity. First, Wnt ligand stimulation rapidly replenished nutrients by activating lysosomes . Second,Sclerostin is a secreted Wnt inhibitor that has emerged in the progression of metabolic disease and endocrine dysfunction. Normally, sclerostin is secreted from osteocytes, bone cells that are embedded within the bone matrix, and inhibits Wnt signaling in osteoblasts . sclerosNew strategies for disease management of obesity, diabetes, and cancer are emerging in the clinic. Caloric restriction delays aging processes and rejuvenates tissues , 116 andThe restriction of specific amino acids could help improve therapies in cancer. Many tumors rely on nutrients supplied by the host’s diet and can even become dependent on a single amino acid –121. EssMethionine is an essential amino acid obtained from specific foods, such as Brazil nuts and red meat. It is essential for methylation as it generates SAM, the universal methyl-donor of the cell, during one-carbon metabolism . Long-tein vitro, and do not accurately recapitulate whole-body metabolism. The use of stable isotope-labels in disease animal models have recently been used to map the metabolic fates of nutrients exchanged between organs in vivo  is an emerging field that has the potential to revolutionize modern medicine (medicine , 130. ThCF and LA wrote the manuscript. MN, CF, and LA designed and created the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.