Many evidences indicate that Human T-cell leukemia virus type 1 (HTLV-1) is not completely silent in vivo. However, signals that trigger HTLV-1 latency-reactivation switching remain poorly understood. Here, we show that aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, plays a critical role in HTLV-1 plus-strand expression. Importantly, HTLV-1 reactivation could be tunably manipulated by modulating the level of AHR ligands. Mechanistically, activated AHR binds to HTLV-1 LTR dioxin response element (DRE) site (CACGCATAT) and drives plus-strand transcription. On the other hand, persistent activation of nuclear factor kappa B (NF-κB) pathway constitutes one key prerequisite for AHR overexpression in HTLV-1 infected T-cells, setting the stage for the advent of AHR signaling. Our findings suggest that HTLV-1 might achieve its reactivation in vivo when encountering environmental, dietary, microbial and metabolic cues that induce sufficient AHR signaling.Establishing latent infection but retaining the capability to reactivate in certain circumstance is an ingenious tactic for retroviruses to persist  in vivo because viral products were rarely detected in freshly isolated PBMCs of infected individuals. However, the existence of strong HTLV-1-specific immune response in most infected individuals suggests that the virus should not be completely silent in vivo. Since viral gene expression plays a critical role in cell transformation and de novo infection, a novel insight into where and how HTLV-1 achieves its reactivation in vivo is essential for developing new therapeutic approaches. AHR is a ligand-activated transcription factor that regulates intricate transcriptional programs in response to environmental, dietary, microbial and metabolic cues. It has been reported that AHR is constitutively overexpressed in HTLV-1-infected T-cells. Nevertheless, the functional role of AHR in HTLV-1 pathogenesis is still obscure. In this study, we show that activated AHR can directly bind to HTLV-1 LTR DRE site (CACGCATAT) and drive HTLV-1 plus-strand transcription. Importantly, HTLV-1 latency-reactivation-latency switching could be manipulated in MT-1 cells by adding and removing additional kynurenine (a well-known AHR ligand). Moreover, we explicate that the persistent NF-κB activation is critical for AHR overexpression in HTLV-1-infected T-cells. These results imply that constitutive AHR overexpression in infected T-cells endues HTLV-1 the potential to reactivate from latency when the level of AHR ligands reaches a certain threshold. Accordingly, we propose that HTLV-1 might achieve its reactivation in certain parts of the body that are prone to accumulate AHR ligands.HTLV-1 is considered largely latent  Unlike HTLV-1-transformed T-cell lines expressing abundant amounts of viral products in vitro, HTLV-1 is considered largely latent in vivo because virions and viral proteins were rarely detected in freshly isolated peripheral blood mononuclear cells (PBMCs) of infected individuals. However, the presence of high titles of HTLV-1-specific cytotoxic T-lymphocytes (CTLs) and antibodies in most infected individuals suggests that the immune system is frequently stimulated by newly synthesized HTLV-1 antigens  triggered HTLV-1 plus-strand expression [Various stimuli—such as extracellular antigens, inflammation cytokines, cytoplasmic oxidative stress and nuclear DNA damage—are known to activate NF-κB. Therefore, potential linkage between these factors and HTLV-1 plus-strand expression is established (when AHR ligands are sufficient). It has been reported that several stress-inducing agents [12-O-tetradecanoylphorbol-13-acetate (TPA), cisplatin, etoposide, taxol, 3-methylcholanthrene  and Hpression , 48, butpression , 50, we in vivo and address AHR as a potential target for prophylaxis and treatment of HTLV-1-related diseases.In summary, we here reveal a role of AHR in HTLV-1 plus-strand expression and its capability to reactivate HTLV-1 from latency. Our findings provide a novel insight into where and how HTLV-1 might achieve its reactivation MT-1, HPB-ATL-T, HPB-ATL-2, TL-Om1 are ATL-derived T-cell lines. MT-2 and MT-4 are HTLV-1-transformed T-cell lines. Jurkat, Molt-4 and CEM-T4 are HTLV-1-negative T-cell lines. All these T-cell lines were obtained from Prof. Masao Matsuoka  and cultured in RPMI1640 (Gibco) supplemented with 10% FBS (Gibco). HEK293 was purchased from Shanghai Institute of Cell Biology and cultured in DMEM (Gibco) supplemented with 10% FBS.CH-223191 was purchased from Selleck. L-kynurenine and BAY11-7085 were purchased from MedChemExpress. Antibodies were used as following: AHR , RelA , IκBα , phospho-IκBα , α-Tubulin , LaminB1 , HTLV-1 Tax , HTLV-1 gp46 , HTLV-1 p24 , HTLV-1 p19 , β-Actin , GAPDH .A78D were generated from pcDNA3.1-HA-AHR. pCG-M22  and pCG-M47  were generated from pCG-Tax. For site mutant, we used Mut Express II Fast Mutagenesis Kit V2 (Vazyme).pcDNA3.1-HA-AHR was constructed by cloning human AHR cDNA with HA tag into pcDNA3.1(-) vector (Invitrogen). pCG-Tax and pcDNA3.1-Myc-His-HBZ were described previously . 4xDRE-LTotal RNA extraction was performed with Trizol (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized using HiScript II Q RT SuperMix (Vazyme). Quantitative RT-PCR was performed using AceQ qPCR SYBR Green Master Mix (Vazyme).The following primers were used:18S rRNA forward 5’-GTTCTTAGTTGGTGGAGCGATTTG-3’;18S rRNA reverse 5’-TTGCTCAATCTCGGGTGGC-3’;AHR forward 5’-GCCAACATCACCTACGCCAGTC-3’;Human AHR reverse 5’-TCGGTCTCTATGCCGCTTGGAA-3’;Human RelA forward 5’-ATGTGGAGATCATTGAGCAGC-3’;Human RelA reverse 5’-CCTGGTCCTGTGTAGCCATT -3’;Human gag forward 5’-AGCCCCCAGTTCATGCAGACC-3’;HTLV-1 gag reverse 5’-GAGGGAGGAGCAAAGGTACTG-3’;HTLV-1 env forward 5’-CGTCCGCCGTCTAGCTTCC-3’;HTLV-1 env reverse 5’-ATTGTGAGAGTACAGCAGC-3’;HTLV-1 tax forward 5’-ACCAACACCATGGCCCA-3’;HTLV-1 tax reverse 5’-GAGTCGAGGGATAAGGAAC-3’;HTLV-1 HBZ forward 5’-ATGGCGGCCTCAGGGCT-3’;HTLV-1 HBZ reverse 5’-CTTCTAAGGATAGCAAACCGTCAAG-3’.HTLV-1 protease inhibitor cocktail (Thermo Scientific) at 4°C for 30 min. Lysates were cleared by centrifugation at 13,000 g for 10 min at 4°C. The extracts were subjected to SDS-polyacrylamide gel electrophoresis and further transferred onto nitrocellulose membrane (Thermo Scientific). The membrane was blocked in milk at 4°C overnight then probed with diluted specific antibody for 3 h at room temperature. After washing with 5% Tween–phosphate-buffered saline (PBS) for 10 min (3 times), the membrane was incubated in appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After that, the membrane was washed with 5% Tween-PBS for 10 min (3 times), and the protein band was visualized with BeyoECL Star (Beyotime Biotechnology).Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology) supplemented with For nuclear and cytosolic fraction separation, we used Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology) according to the manufacturer’s instructions.Lentiviruses were purchased from Genechem.The shRNA sequences were:AHR;5’-TTCTTTGATGTTGCATTAA-3’ targeting human RelA;5’-GATTGAGGAGAAACGTAAA-3’ targeting human HBZ;5’-ACAGCATAGTGCTAGGAAA-3’ targeting HTLV-1 5’-TTCTCCGAACGTGTCACGT-3’ as negative control.http://www.genechem.com.cn/index/supports/zaiti_info.html?id=83. Lentivirus transduction was performed by supplementation of 4 μg/ml polybrene. Transduced cells were selected with 0.5 μg/ml puromycin.The map of the lentivirus backbone vector GV493 is shown at Cell transfection was conducted using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. In brief, HEK293 cells were seeded to be 70–90% confluent at transfection. For 12-well plate/10 cm dish transfection, 1–1.5 μg/16 μg plasmids were used. The ratio of plasmids: p3000 reagent: Lipofectamine 3000 reagent is 1 μg: 2 μL: 3 μL.HEK293 cells were seeded on 12-well plate and transfected with the indicated plasmids when cells are 70–90% confluent. For each well, 20 ng renilla luciferase reporter plasmid pRL-TK (Promega) was used as an internal control. 48 h after transfection, luciferase activity was measured with Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.6 cells  were cross-linked by 1% formaldehyde (Sigma) for 10 min, and then quenched using glycine for 5 min. Cross-linked chromatin was digested into size around 150–900 bp using micrococcal nuclease. 10/200 μL sheared chromatin was used as common 10% input. ChIP (90 μL sheared chromatin for each) was performed with 2 μg HA antibody or equal normal rabbit IgG at 4°C overnight with rotation. Precipitated DNA was detected by semiquantitative RT-PCR under the following conditions: 3 minutes at 95°C for denaturation, 25 cycles of 15 sec at 95°C, 15 sec at 63°C, 10 sec at 72°C and 5 min at 72°C for final extension.ChIP assay was performed using Pierce Magnetic ChIP Kit (Thermo Scientific) according to the manufacturer’s instructions. In brief, HEK293 cells were seeded on 10 cm dish and transfected with the indicated plasmids when cells are 70–90% confluent. 48 h after transfection, 8 × 10The specific primers for HTLV-1 LTR containing DRE1:forward 5’-CAGGCGTTGACGACAACCC-3’;reverse 5’-GTGGATGGCGGCCTCAGGTA-3’.t-test or analysis of variance (ANOVA) using GraphPad Prism 5 (Graphpad software).Statistical significance was assessed by Student’s S1 FigHPB-ATL-T, MT-2, MT-4 and TL-Om1 cells were treated with CH-223191  or DMSO for 24 h, then subjected to analyze the level of cytosolic and nuclear AHR by immunoblot. α-Tubulin and LaminB1 were used as control for cytosolic and nuclear fraction respectively.(TIF)Click here for additional data file.


Streptomycesalbus strain CAI-21 has been previously reported to have plant growth-promotion abilities in chickpea, pigeonpea, rice, and sorghum. The strain CAI-21 and its secondary metabolite were evaluated for their biocontrol potential against charcoal rot disease in sorghum caused by Macrophomina phaseolina. Results exhibited that CAI-21 significantly inhibited the growth of the pathogen, M. phaseolina, in dual-culture , metabolite production (74% inhibition), and blotter paper (90% inhibition) assays. When CAI-21 was tested for its biocontrol potential under greenhouse and field conditions following inoculation of M. phaseolina by toothpick method, it significantly reduced the number of internodes infected  and length of infection  over the positive control (only M. phaseolina inoculated) plants. Under greenhouse conditions, scanning electron microscopic analysis showed that the phloem and xylem tissues of the CAI-21-treated shoot samples were intact compared to those of the diseased stem samples. The culture filtrate of the CAI-21 was purified by various chromatographic techniques, and the active compound was identified as “organophosphate” by NMR and MS. The efficacy of organophosphate was found to inhibit the growth of M. phaseolina in the poisoned food technique. This study indicates that S.albus CAI-21 and its active metabolite organophosphate have the potential to control charcoal rot in sorghum. Sorghum bicolor (L.) Moench] is a nutrient-rich staple food crop for millions of rural communities in the semiarid tropics [Macrophomina phaseolina (Tassi) Goid., is an important disease of postrainy sorghum. It affects more than 500 plant species including legumes, vegetables, and cereals and devastates up to 100% of the crop [Sorghum [ tropics . It is g tropics . The prothe crop .M. phaseolina is a soil-borne root pathogen that naturally forms heat-tolerant sclerotia [M. phaseolina deeply penetrate the host crop in search of nutrients, which leads to structural damage and death of the infected plants [M. phaseolina produces a metabolite, phaseolinone, which causes vascular blockage that leads to the death of the whole plant [clerotia . The patclerotia . Germinaclerotia . The popclerotia . The inod plants . M. phasle plant ,9. This le plant ; therefoM. phaseolina-free) seed, solarization of soil, and treatment with thiram and carbendazim are some of the control measures usually employed to manage charcoal rot disease but with limited success [Bacillus, Pseudomonas, Enterobacter, Trichoderma, Acinetobacter, Amycolatopsis, and Streptomyces [M. phaseolina interactions and identifying more potent biocontrol strategies that could be combined with a moderate level of tolerance in the host to manage this disease.The biggest drawback in managing charcoal rot in sorghum is the lack of a high level of host-plant resistance in currently cultivated cultivars. Control of charcoal rot in sorghum is challenging, as no single control measure is fully effective. Advanced sowing date, use of pathogen-free  abilities in chickpea, pigeon pea, rice, and sorghum [M. phaseolina in the in vitro studies [S. albus CAI-21 for its biocontrol potential against charcoal rot disease in sorghum by dual-culture assay, secondary metabolite production assay, blotter paper assay, and greenhouse and field assays and to finally purify and identify the active secondary metabolite of CAI-21 responsible for the inhibition of M. phaseolina. sorghum ,21,22,23 studies . TherefoM. phaseolina in both dual-culture and secondary metabolite production assays. In the dual-culture assay, CAI-21 caused a 15 mm inhibition of M. phaseolina  and hydrocyanic acids (HCN) [S. albus CAI-21 has been previously reported to produce siderophore and HCN [S. albus CAI-21 may produce one or more secondary metabolites capable of inhibiting M. phaseolina.The strain CAI-21 inhibited aseolina . This inby 73.8% . Inhibitds (HCN) ,25. Plands (HCN) ,27. The  and HCN . TherefoM. phaseolina-inoculated control, high disease severity with score 4 and 100% root infection were observed. The negative control plants were found to be healthy as these were without any disease symptoms or root infection  and lesser root infections (10–20%) were observed in CAI-21-treated sorghum roots, whereas in the nfection . FurtherS. albus CAI-21 was evaluated for its biocontrol potential against M. phaseolina infection, the charcoal rot disease severity was reduced significantly compared to a positive control (only M. phaseolina was inoculated). The charcoal rot infection was observed in only 1 internode (75% less) in the CAI-21-treated plants when compared to the positive control, where up to 4 internodes were found to be infected. Further, the length of charcoal rot infection was only 3 cm (75% less) in the CAI-21-treated plants when compared to the positive control, where it was 12 cm , whereas in the positive control, the infection was observed in 5.6 internodes on average. The length of charcoal rot infection was only 10 cm (51% less) in CAI-21-treated plants when compared to the positive control, where it was 19.5 cm  was found to be damaged , Actinovate (by S. lydicus WYEC 108), blasticidin-S (by S. griseochromogenes), validamycin (by S. hygroscopicus), kasugamycin (by S. kasugaensis), oxytetracycline (by S. rimosus), polyoxins (by S. cacaoi var. asoensis), Mycostop (by Streptomyces sp. K61), azalomycin B (by Streptomyces sp. HAAG3-15), abamectin/avermectins (by S. avermitilis), emamectin benzoate (by S. avermitilis), polynactins (by S. aureus), natamycin , the diketopiperazine cyclo(Tre-Phe) (by Streptomyces sp. SAI-25), a novel fatty acid amide derivative (by Streptomyces sp. CAI-155), and milbemycin (by S. hygroscopicus subsp. aureolacrimosus), which are widely reported as crop protection agents [S. albus CAI-21 may be used in biological control of M. phaseolina.ptomyces . Some ofn agents ,45,46,47Streptomyces albus CAI-21, previously identified by whole-genome sequencing and reported to have the capacity for PGP traits such as shoot mass, shoot volume, and root mass in sorghum [One strain of  sorghum ,22, was S. albus CAI-21 was tested for its inhibitory activity against M. phaseolina , Patancheru, India) by dual-culture assay as described earlier [M. phaseolina) of 6 mm diameter was placed on one edge (1 cm from the corner) of the starch casein agar plate, and CAI-21 was streaked on the other edge of the plate (1 cm from the corner), followed by incubation at 28 ± 2 °C for 5 days. Inhibition of fungal mycelium  around the CAI-21 was noted as positive, and the inhibition zone (in mm) was measured. Positive control plates contained only M. phaseolina, while negative control plates contained only sterile water. For the secondary metabolite production assay, CAI-21 was grown on starch casein broth  at 28 ± 2 °C for seven days. After seven days of incubation, the cell-free supernatant was collected, and its secondary metabolites were extracted using ethyl acetate by the solvent partitioning method [M. phaseolina for their growth inhibition ability by the modified poisoned food technique [M. phaseolina was bored and placed at the center of the plate. After five days of incubation at 28 ± 2 °C, the fungal growth was measured and compared with the positive control plates, where only 0.1 mL of methanol was added, and chemical control plates, where only chlorpyriphos (20%) was added. Negative control plates contained only sterile water. Both the dual-culture and the secondary metabolite production assays were repeated thrice to confirm the results. earlier . In brieg method . The pelechnique . In brieM. phaseolina on two-week-old seedlings of susceptible sorghum line 296B using a blotter paper assay [M. phaseolina inoculation; positive control, only M. phaseolina inoculation; and negative control, only sterile water) were tested. Fifteen plants were used per replicate, and 3 replications were made for each treatment. The symptoms of charcoal rot (black-colored infection and microsclerotia on the root surface) were recorded on a 0–4 rating scale  control was also calculated. The assay was repeated twice to confirm the results.CAI-21 was evaluated for its antifungal activity against er assay . In brieymptoms; ). The peM. phaseolina inoculation; positive control, only M. phaseolina inoculation; and negative control, only water) were tested with 10 replications. The trial was conducted in a completely randomized design. A mixture of 2 kg of Vertisols, sand, and farmyard manure (at 3:2:1 ratio) was filled in 8-inch plastic pots. Sorghum seeds (line 296B) were surface-sterilized with 2.5% sodium hypochlorite solution in water for 3 min and rinsed several times with sterilized distilled water. The surface-sterilized seeds were soaked in CAI-21 suspension  or in sterilized water (for a negative control) for one hour. The treated seeds (three per pot) were sown immediately at 3 cm depth and thinned to one plant per pot after germination. Booster doses of CAI-21 (5 mL per seedling) were applied by the soil drench method at 15 and 30 days after sowing (DAS). Ten days after flowering, all the plants (except the ones in the negative control) were artificially inoculated by inserting a toothpick infested with the inoculum of M. phaseolina into the second internode of the stalk [CAI-21 was evaluated for its biocontrol potential against the charcoal rot of sorghum under greenhouse conditions. This was performed with the toothpick inoculation method ,50. In bAt harvest, the stem samples were also examined by scanning electron microscopy (SEM) analysis for colonization of CAI-21 and for any morphological changes that might have occurred ,51. In bM. phaseolina inoculation; positive control, only M. phaseolina inoculation; and negative control, only water) were evaluated. Just before sowing, the seeds of 296B were treated with CAI-21 (containing 108 CFU mL−1) for 50 min and sown by hand. CAI-21  was also applied every 15 days in the soil close to the plant until the flowering stage. Plants were artificially inoculated with the M. phaseolina, by inserting a toothpick infested with M. phaseolina into the second internode of the sorghum stalk 10 days after 50% flowering. The growth of the M. phaseolina on toothpicks was achieved as described earlier [M. phaseolina), the disease severity was recorded by measuring the length of infection (in cm) and the number of internodes infected.CAI-21 was also evaluated for its biocontrol potential against charcoal rot of sorghum under field conditions at International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, India, during the postrainy 2017−2018 cropping season by toothpick inoculation method, as described in greenhouse trials. During the cropping season, a minimum nighttime temperature range of 11–17 °C and a maximum daytime temperature range of 28–38 °C were recorded. The trial was conducted in a randomized complete block design (RCBD) with three replications. The plot size was 3 rows of 2 m long with a row-to-row spacing of 75 cm and a plant-to-plant spacing of 10 cm. Sorghum seeds (line 296B) were sown in the field at a depth of 5 cm to achieve an estimated plant stand density of 60 plants per replication. A total of three treatments  was collected. The culture filtrates (10 L) were partitioned three times against ethyl acetate , and the resultant organic (EtOAc) and aqueous fractions were evaporated and collected in methanol (10 mL). Both organic and aqueous fractions were tested against M. phaseolina by a modified poisoned food technique [CAI-21 was grown on SCB at 28 °C for 7 days in an orbital shaker at 120 rpm. At the end of the 7-day incubation, the culture was centrifuged at 12,000× echnique .M. phaseolina by the poisoned food technique [The organic fraction, which showed antagonistic potential, was subjected to C18 open column chromatography. The column (23 × 3.6 cm) was packed with C18  one day prior to allow the C18 to settle in the column. After conditioning the C18 column, the bioactive fraction was loaded and eluted with a gradient of methanol in Milli-Q water . All the collected fractions were evaluated for their antagonistic activity against echnique .18 analytical column, 5 µm, 250 × 4.6 mm, flow rate 0.5 mL min−1; gradient method with acetonitrile/water) and subjected to structural identification studies. Removal of impurities in the purified sample was performed with a preparative HPLC Agilent 1260 Infinity instrument, Santa Clara, USA equipped with two G1361A 1260 Prep Pumps, a G2260A 1260 Prep ALS, G1316A 1260 TCC Switching Valve, a G7114A 1260 Variable Wavelength Detector, and a G7102 1290 Evaporative Light Scattering Detector. An Agilent 5 Prep-C18  column was used with a flow of 20 mL/min. Samples were dissolved in pure methanol and eluted isocratically in methanol/water 80:20 for 4.5 min and then isocratically in pure methanol for 8.5 min. The fraction at tR = 11.64 min was collected in a G1364B 1260 FC-PS fraction collector and evaporated in a smart evaporator under a stream of nitrogen.The most bioactive open-column chromatography fraction was further purified on HPLC . Samples were ionized using an ESI (Electrospray ionization) probe in positive mode. No chromatographic separation was used previously for the mass analysis. The calculation of the exact mass and spectral processing was performed using Waters™ MassLynx V4.1 SCN871 software.3) were measured at 300 K, using TMS signal as a reference. The following 1D and 2D pulse sequences from the Bruker User Library were used for the NMR experiments: 1H 1D (600 MHz), 13C 1D (150 MHz), 2D heteronuclear single quantum coherence (HSQC) with multiplicity editing, 2D heteronuclear multiple bond correlation (HMBC) with threefold low-pass J-filter to suppress one-bond correlations, and 2D H–H-correlation spectroscopy (COSY) with gradient pulses for selection.NMR data were acquired using a Bruker Avance III HD spectrometer, operating at a proton frequency of 600.18 MHz, with a 5 mm triple-resonance cryogenic CP-TCI probe, and equipped with a z-gradient. The samples containing a solution of 15 mg of the substance in chloroform-d  in a completely randomized design (CRD) for greenhouse and randomized complete block design (RCBD) for field studies to evaluate the efficiency of Streptomyces strain to be reported to manage the charcoal rot of sorghum in both greenhouse and field experiments. Furthermore, the active metabolite responsible for the inhibition of M. phaseolina was identified as an organophosphate. In the absence of a high level of genetic resistance in high-yielding varieties, CAI-21 and its metabolite could be effective in managing charcoal rot disease in sorghum. Thus, this strain may be used as an important tool in managing charcoal rot in sorghum. Further experiments are needed to determine the effectiveness of the CAI-21 and the organophosphate, as an effective antifungal compound, under different field conditions and using diverse sorghum cultivars. The biosafety issues related to the organophosphate secreted by the CAI-21, such as risk assessment for human and domestic pet health and environmental contamination, also need to be assessed before it is released in the environment.It is concluded from the results of this study that CAI-21 may be the first


Neuronal survival in multiple sclerosis (MS) and other demyelinating diseases depends on the preservation of myelin and remyelination of axons. Myelin protection is the main purpose to decrease myelin damage in the central nervous system (CNS). Ursolic acid (UA) as a natural product in apple is suggested to protect neural cells. This study is the first to demonstrate an effect for UA on CNS myelin loss induced by cuprizone toxin. In the current study, we hypothesized that daily treatment with UA in drinking water (1 mg/mL) prevents myelin damage by 6 weeks administration of CPZ in mice pellet which lead to corpus callosum axonal demyelination. We assessed the myelin content and the number of myelinating cells in corpus callosum by FluoroMyelin and luxol fast blue staining as well as by immunostaining against MBP and Olig2. Our finding indicated that UA could decrease the extent of demyelination area and enhanced myelin stain intensity within CC and protected oligodendrocyte lineage cells against cuprizone toxin. We could conclude that myelinated structures could be protected by UA in corpus callosum, which provide favorable evidence for the possibility of application of UA in demyelinating diseases and traumatic injuries.  Myelin, a protective coating around the nerve fibers facilitates action potential conduction in the nervous system. It is involved in salutatory conduction of neural impulses and accelerates the conduction velocity by 20-100-fold compared to non-myelinated axons of the same diameter . TherefoAxonal survival depends on myelin therefore myelin protection and remyelination of axons contributes to axonal survival and prevent transition to progressive phase of MS , 14. Moset al. (Ursolic acid (3β-Hydroxy-12-ursen-28-ic acid) is a natural pentacyclic terpenoid  which iset al.  demonstret al. . UA alsoet al. . In braiet al. , althougIn the current study, we hypothesized that UA may protect myelin in CNS in the context of demyelinating conditions. To examine the protective effects of UA, mice received daily treatment of UA in drinking water when fed with cuprizone (CPZ) to induce demyelination in corpus callosum (CC). We showed myelin protection using different myelin staining methods including LFB, FluoroMyelin and immunohistofluorescence studies.AnimalsAdult male C57BL/6 mice weighing 20-25 g (8–10 weeks) were purchased from Pasteur Institute . The animals were maintained three per cage under 12 h light/dark cycles under controlled temperature (23 ± 2 °C). Food and water were freely available. All experiment procedures were designed based on international guidelines for animal studies and approved by the ethical committee for animal research, Tarbiat Modares University, Tehran. Efforts were made to minimize both the number of animals used and their suffering.InterventionsFor induction of demyelination, the male C57BL/6 mice were fed with a diet containing 0.2% cuprizone ; Sigma-Aldrich Inc., St. Louis, MO, USA) mixed into their normal chow for 6 weeks. The mice were divided into three groups including control with normal water, cuprizone diet with normal water, and cuprizone diet with water containing UA (1 mg/mL) (Enzo Life Sciences). The animals’ brains were collected at 6 weeks post cuprizone. The animal’s weight and food intake were recorded every other day. Y-maze behavioral testY-maze test was performed to confirm the presence of behavioral impairment due to demyelination in their brains. Spontaneous alternations within the Y-maze were calculated prior to induction of demyelination and 6 weeks after feeding with cuprizone diet. The Y-maze arms were labeled as A, B, and C. The number of overlapping entrance sequences  was defined as the number of spontaneous alternations. The percentage of alternations was calculated as follows.Processing and sectioning of brain tissues The animals were perfused with phosphate buffer saline. The brains were removed and the hemispheres were dissected. The right hemispheres were post-fixed in 4% paraformaldehyde 24 h, then in sucrose 30% (2-3 days), and embedded in optimal cutting temperature (OCT) for long term preservation in -80 °C. Ten micrometer thick sections were cut using a cryostat  and mounted on superfrost plus slides for later evaluations. The sections were stored at -20 °C until final staining. Luxol Fast Blue and Cresyl Fast Violet stainingTo evaluate the myelination intensity and the extent of demyelination in corpus callosum, the sections with 10-μm thick were selected for myelin staining. For FluoroMyelin (FM) staining, the sections were incubated with red FM (Invitrogen F34652) for 20 min at room temperature. FM solution were prepared by diluting FM 1:300 in PBS. For Luxol fast blue (LFB) staining the sections were stained with 0.1% LFB solution  at 60 °C for 1 h. Adequate contrast was made by rapid immersion of preparations in 0.05% lithium carbonate followed by several changes of 70% alcohol. The sections were washed with distilled water, and then counterstained with 0.1% Cresyl Fast Violet  for 4 min. After washing with distilled water, the sections were dehydrated in a graded series of alcohols, cleared in xylene, coverslipped, and screened for demyelination and subsequent quantitative analysis. For each section, the extent of demyelination was assessed by using Image J software as the percentage of whole corpus callosum. The extent of demyelination was averaged for 8 sections obtained from each animal and then averaged for each animal group (n = 3 for each data point).Immunostaining2 and MBP. Afterward, the sections were washed three times with PBS and incubated with corresponding secondary antibody for 1 h at room temperature. The list of antibodies used in this experiment is provided in To perform the immunofluorescence staining against myelin antigens, the sections were washed three times with PBS containing 0.05% Tween 20, then incubated in blocking solution for 1 h at room temperature. Blocking solution consisted of 0.1% Triton X-100 , 1% bovine serum albumin, and 5% normal serum in PBS. Then, the samples were incubated at 4 °C for overnight with primary antibodies against OligData Analysisp-value was less than 0.05. Each animal group included 3-6 mice. Statistical analyses were conducted using GraphPad Prism . One-way analysis of variance ANOVA, followed by Tukey post-test was used for comparing the experimental groups. The results were expressed as mean ± SEM. The differences were considered significant when Ursolic acid effects on CPZ feeding and water drinkingTo identify the UA effect on the normal appetite of in CPZ-fed mice, we used UA as a dietary supplement in drinking water during 6 week after CPZ-exposure. The results indicated no significant difference between the food intake, the amount of drinking water, and the average animals’ weight after 6 weeks. . The same amount of food intake stands for the same amount of CPZ delivery to the UA treated and non-treated mice. Therefore, the changes in the myelination and lesion extent in these two groups will be due to the UA treatment.Ursolic acid improved the spatial recognition memoryp < 0.05 compared to the baseline; p < 0.01 compared to the age-matched controls  and at week 6 post CPZ-exposure . The rescontrols . TherefoUrsolic acid protected myelin in corpora callosa of CPZ-fed mice p <0.01). UA treatment for 6 weeks during the course of demyelination induction could reduce the amount of demyelinated area in corpus callosum when compared to CPZ group without treatment (p < 0.05). There was no significant difference between this group and the control one with the same age which may imply the complete protection of myelin. All quantification and statistical comparisons are presented in Demyelinated regions within the corpora callosa could be clearly discovered following FluoroMyelin (FM) staining as a selective fluorescent dye for myelin . The losp < 0.01, p < 0.05). To confirm the difference in the extent of demyelination within the corpus callosum, luxol fast blue (LFB) staining was performed at the end of 6 weeks CPZ feeding. Myelination intensity in the control group of the same age was considered as 100% myelination.Mature OLs and fully myelination OLs express MBP as key protein in myelin compaction. Therefore, myelin staining using specific antibodies provides a more reliable method for detecting the changes in myelination. Immunohistofluorescent study was used to assess the myelin levels in corpus callosum of the animal groups . p < 0.01), the level of myelin in CPZ+UA group was significantly higher than that in the control group , UA administration in drinking water led to observing a higher number of OL lineage cells within the CC (p < 0.01) that suggests UA has the capability for preserving the oligodendrocyte lineage.To examine the preservative effect of UA in cellular level we performed immunostaining against Olig2 a marker of OL lineage cells (including both progenitors (OPCs) and mature OLs). The results demonstrated a reduced numbers of Olig2+ cells in CPZ-treated mice with implication of possible cell loss by CPZ toxicity as well as a preservation of OLs and/or OPCs by UA . Quantifet al.  glycan  glycan . They deAlthough UA promoted the regeneration of injured sciatic nerve and increased the myelination following peripheral nerve injury , our stuIn addition, UA-treated mice showed a significantly increased number of OL lineage cells stained with Olig2 antibody. Olig2 is expressed in both OPCs and mature oligodendrocytes and measuring the number of Olig2+ cells give us a general estimation on the population of cells that myelinate or differentiate into myelinating cells. Therefore, we suggested that UA reduced demyelination via protecting the related cells following CPZ administration. However, the mechanisms of myelin protection remain to be clarified. The higher number of Olig2+ cells may be also due to generation of new Olig2+ cells and therefore enhanced repair, a possible mechanism which need to be studied in future works. OLs generation is a multiple-step process that is included the proliferation, migration, and differentiation of oligodendroglial progenitor cells into mature oligodendrocytes . The numThe impact of UA on the brain is not limited to the cellular level. Xu and colleagues  showed tTherefore, our results demonstrate that UA prevents demyelination in corpus callosum when administrated during the course of demyelination induction with cuprizone. We suggest that UA could activate a neuroprotective strategy and improve the myelin and myelinating cells survival in demyelinated corpus callosum axons in cuprizone model of MS.In the current study, we showed that the groups of animals which received daily treatment UA in drinking water, when simultaneously were challenged with a demyelinating insult by CPZ, could protect the myelin and the OL lineage cells. The results obtained in the present study indicate that UA therapy protected myelination as measured by FM and LFB staining and MBP staining. Although the molecular mechanisms responsible for the therapeutic effects of UA on demyelination remain unclear, we suggest that UA may modulate neuroimmune and inflammatory processes and provide a favorable external environment for neural cells and their protection.


Rab GTPases play key roles in various cellular processes. They are essential, among other roles, to membrane trafficking and intracellular signaling events. Both trafficking and signaling events are crucial for proper embryonic development. Indeed, embryogenesis is a complex process in which cells respond to various signals and undergo dramatic changes in their shape, position, and function. Over the last few decades, cellular studies have highlighted the novel signaling roles played by Rab GTPases, while numerous studies have shed light on the important requirements of Rab proteins at various steps of embryonic development. In this review, we aimed to generate an overview of Rab contributions during animal embryogenesis. We first briefly summarize the involvement of Rabs in signaling events. We then extensively highlight the contribution of Rabs in shaping metazoan development and conclude with new approaches that will allow investigation of Rab functions in vivo. Membrane trafficking regulates molecule and protein transport between intracellular compartments and the extracellular milieu. Defects in membrane trafficking events are linked to a plethora of human diseases . One impHere, we compiled data highlighting Rab GTPases’ involvement in metazoan embryogenesis. Although, numerous studies have detailed the phenotypic contributions of Rabs in embryonic development, a limited number have described their molecular roles in this context. For that reason, we first discuss specific examples of Rabs in developmentally relevant signaling events. This with the goal of offering a potential hypothesis on putative Rab molecular roles during embryogenesis. Then, we highlight the requirement of Rabs during the embryonic development of various metazoan model organisms. Finally, we describe novel available techniques that will bring new insights on Rabs’ roles in signaling and their molecular contribution during embryogenesis.2+.Depending on their cellular localization, activated receptors can differently affect signaling pathways, resulting in distinctive cellular responses. Intrinsically, membrane trafficking plays a central role in signaling regulation. Multiple studies have demonstrated the influence of Rab GTPases in signaling events. We will highlight a few of these studies in this section and discuss how Rabs are involved in whole-cell homeostasis by integrating and regulating intra- and extra-cellular signals such as amino acids, growth factors and CaDrosophila S2 cells revealed that knockdown of Rab1, Rab5, or Rab11 reduced the phosphorylation of S6K by mTORC1 . . 101]. Aproteins . When APEscherichia coli biotin ligase)-fused Rabs to mitochondria /CRISPR-associated protein 9 (Cas9) to generate null or knock-in alleles, it will be significantly easier to perturb Rab function and analyze its impact on cell function. Lower model organisms like rganisms . Moreove


Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label‐free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC‐derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.Cardiac ischaemia‐reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re‐perfused post‐ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post‐anoxic heart mitochondria. However, post‐ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up‐regulated upon I/R such as the  Indeed, application of S1QELs protected the perfused mouse heart from I/R injuries.To test this assumption, a number of mitochondrial‐targeted antioxidants have been designed. MitoQ, one prominent example, consists of ubiquinol (reduced quinone) targeted specifically to mitochondria by a covalently fused lipophilic triphenyl phosphonium (TPP) cation.Ciona intestinalis  is catalytically active under various experimental conditions. In human cells, AOX conferred resistance against the ETC inhibitor cyanidePlants and many other organisms, but not insects or vertebrates, harbour a by‐pass mechanism to protect from ETC‐mediated respiratory stress conditions, the alternative oxidase (AOX).Here, we tested how maintaining electron flow impacts on remodelling in the post‐ischaemic mouse heart. We show that AOX expression supports ETC function and decreases mitochondrial ROS levels, yet impairs adaptive cardiac remodelling. This indicates an essential role for ETC‐derived stress signals in post‐ischaemic cardiac adaptation.22.1Ciona intestinalis AOXMice with ubiquitous expression of 2.2Details of the procedure have been published elsewhere.2.3s.c. prior to anaesthesia to minimize postoperative pain. For anaesthesia induction, mice were exposed to 4.5 Vol% isoflurane in ambient air for approximately 3 minutes in an air‐tight box. Anaesthesia was maintained after intra‐tracheal intubation and ventilation using 1.5 Vol% isoflurane using the rodent MiniVent ventilator  adjusted to 225 hubs per minutes with a tidal volume of 250 µL. During the procedure, animals were kept in a supine position on a heat‐controlled plate at 37°C. A left anterolateral thoracotomy was performed between the second and third rib to visualize the mouse heart and lay open the LAD. The LAD was ligated in a proximal position using a 7‐0 prolene suture. Pale discoloration of ventricular tissue demarcated the region of ischaemia. After ligation, animals received 5 IU of Heparin‐Natrium s.c. and the open wound was covered using cheese cloth soaked with 0.9% NaCl solution. After 45 minutes of ischaemia, the ligation was opened and cardiac reperfusion confirmed by visual control. The wound was closed using absorbable, synthetic 5‐0 vicryl (polyglactin 910) sutures. Weaning from ventilation took several minutes and was ended when spontaneous respiration became evident.Male WT and AOX littermates received 0.1 mg/kg bodyweight buprenorphine 2.42PO4 1.2, KCl 2.5, MgSO4 0.8, NaHCO3 25, glucose 5, C3H3NaO3 1.9, CaCl2 2.5). Perfusate flow was adjusted to a perfusion pressure of 70 mm Hg. To determine left ventricular pressure, a balloon was inserted into the left ventricle and inflated at 10 mm Hg. Ischaemia was initiated by flow stop.Hearts were isolated from mice, and procedures to measure cardiac functions were performed as previously described.2.52PO4, pH 7.2; Sigma, P5405, E3889, H3375, P5655) along with 30 μL of 1 mg/mL superoxide dismutase , 30 µL of 1 mg/mL horseradish peroxidase , 30 µL of 10 mg/mL fatty acid‐free‐BSA  and 5 µL of Amplex Red Reagent  for hydrogen peroxide detection. Mitochondrial respiration was initiated by the addition of 10 mmol/L succinate . To confirm AOX activity, 1 mmol/L cyanide  was added to both WT and AOX mitochondria, prior to AOX inhibition by 100 μM n‐propyl gallate . To assess hydrogen peroxide production as the consequence of RET, mitochondrial respiration was stimulated by the addition of succinate and respiration allowed to diminish all oxygen present within the chamber. Once anoxic, mitochondria were left for 20 or 30 minutes to simulate ischaemia, after which oxygen was allowed back into the chamber and oxygen consumption and ROS production measured. To assess sole AOX activity, cyanide was added at the start of the experiment.Mitochondria were isolated from cardiac tissue as described previously.2.6Western blots were performed essentially as described elsewhere.2.72PO4 , 60 mmol/L KCl, 5 mmol/L MgCl2, pH7.4. Substrates used for cI‐driven respiration (PGM plus ADP): 10 mmol/L pyruvate , 10 mmol/L glutamate , 2 mmol/L malate  and adenosine 5′‐diphosphate . Substrates used for cIV measurements: 2 mmol/L (+)‐sodium L‐ascorbate  and 0.5 mmol/L N,N,N′,N′‐tetramethyl‐p‐phenylenediamine . Respiratory inhibitors used for control: 1.5 µmol/L rotenone , 5 mmol/L sodium azide , 1 mmol/L cyanide  and 100 µmol/L n‐propyl gallate .Mitochondrial respiratory activity upon in vivo I/R was measured using skinned fibre isolation (3‐weeks I/R) as described in detail elsewhere2.8−ΔΔCt. B2m (β2 microglobulin) served as a reference housekeeping gene.Total RNA was extracted from left ventricles by RNeasy Micro Kit (Qiagen). The extracted RNA was reverse‐transcribed (RT) to cDNA using iScript cDNA Synthesis Kit (Bio‐Rad). qPCR was done using iTaq SYBR Green Supermix with ROX (Bio‐Rad) and Mx3000P qPCR System (Agilent Technologies). All measurements were carried out in duplicate. Melting curves confirmed desired products formation. The calculation of ΔCt values was done by subtracting the Ct values of the target gene from the endogenous control [ΔCt = Ct (endogenous control) − Ct (target)]. Transcript expression levels as fold change were calculated from 22.9en bloc in half‐saturated uranyl acetate, dehydrated in an ascending ethanol series and embedded in Agar 100 (Agar Scientific). Ultra‐thin sections were cut using an ultramicrotome  and examined in a transmission electron microscope (Zeiss EM 902). Digital images were captured with a slow‐scan 2K CCD camera .A small piece of tissue from the heart apex was kept in storage buffer containing 1.5% glutaraldehyde, 1.5% formaldehyde, 0.15 mol/L HEPES/KOH (pH 7.4) at 4°C until embedding and sectioning. For epoxy resin embedding, samples were fixed in 1% osmium tetroxide, stained 2.104HCO3  and protease inhibitor mix  were added. Homogenization was done with five cycles of 30 seconds 6 m/s pulses and had 5 minutes break on ice in between the cycles. After homogenization, the protein concentration was measured using NanoDrop, and a volume corresponding to 200 µg protein was used for further analysis. Proteins were reduced with dithiothreitol , alkylated with iodoacetamide  and in‐solution digested with LysC  and trypsin .Mouse heart samples were homogenized using a FastPrep‐24 5G High Speed Homogenizer . Tissue pieces were transferred to 2 mL tubes with Lysing matrix D, and 750 µL 8 mol/L urea  in 50 mmol/L NHm/z) of 400 to 1200 with the resolution R = 70 000 at m/z 200 after accumulation to a target of 3 000 000 ions in the quadruple. For MS/MS, the ten most abundant multiple‐charged ions were selected for fragmentation on the high‐energy collision dissociation cell at a target value of 100 000 charges or maximum acquisition time of 100 ms The MS/MS scans were collected at a resolution of 17 500. Target ions already selected for MS/MS were dynamically excluded for 30 seconds.The resulting peptides were desalted and concentrated before mass spectrometry using µC18 tip (Pierce C18 Tips). The peptides were eluted with 0.1% trifluoroacetic acid  in 60% acetonitrile , dried and solubilized in 7 μL 0.1% formic acid  for mass spectrometry analysis. Each peptide mixture was analysed on an Easy nLC1000 nano‐LC system connected to a quadrupole Orbitrap mass spectrometer  equipped with a nano‐electrospray ion source . For the liquid chromatography separation of the peptides, an EasySpray column capillary of 25 cm bed length was employed. The flow rate was 300 nL/min, and the peptides were eluted with a 2%‐30% gradient of solvent B in 240 minutes. Solvent A was aqueous 0.1% FA and solvent B 0.1% FA in acetonitrile. The data‐dependent acquisition automatically switched between MS and MS/MS mode. Survey full scan MS spectra were acquired from a mass‐to‐charge ratio 2.11P value <.05 was considered being statistically significant. All data are shown as mean. Error bars represent standard error of the mean (SEM). Proteome analysis was done using Perseus software (version 1.6.1.3).t test was done with a P value of <.05 being considered statistically significant.Statistical analyses, except for proteome, were performed using GraphPad Prism . 1way or 2way ANOVA and post hoc analyses were used as indicated for comparisons of at least n = 3 independent experiments, and a 33.1Succinate accumulation during cardiac ischaemia3.2One possible explanation for the observed failure of protection could be insufficient AOX expression or its catalytic impairment in the post‐ischaemic heart. To test this possibility, we isolated mitochondria from WT and AOX mouse hearts and measured oxygen consumption and ROS production in vitro, and, importantly, confirmed a robust expression of AOX by Western blot Figure A. As exp3.3We hypothesized that the restoration of electron flux by AOX and supposed decrease in RET‐induced ROS, despite the lack of a measurable advantage during the acute phase, might yet be beneficial for the post‐ischaemic remodelling process. We tested this assumption in WT and AOX mice 3 weeks after a transient (45 minutes) ischaemic insult followed by restoration of blood flow (reperfusion).atrial natriuretic peptide (Anp), which is recognized as both a biomarker for the diagnosis of heart failure as well as a prognostic marker for cardiovascular risk.Anp was significantly increased by I/R in WT hearts but, importantly, this increase was blunted in AOX animals  and 3 (Col3a1), which we found specifically increased in the AOX I/R group  is a mitochondrial protein that detoxifies superoxide generated as by‐product of OXPHOS, by converting it to hydrogen peroxide and molecular oxygen. Sod2 is induced as part of the cell‐inherent ROS stress response,Sod2 appeared down‐regulated in AOX hearts compared with WT, irrespective of any intervention  has previously been shown to be instrumental for protection against pressure overload‐induced right heart failure.Ucp2 specifically up‐regulated in the AOX I/R group , ex vivo functional measurements revealed a significant decrease in P syst  and used GOrilla, an online tool designed to discover and visualize enriched gene ontologies in ranked lists4In the present study, we tested how respiratory restoration by AOX affects the development of I/R injury and adaptive remodelling in the mouse heart. Our data demonstrate that AOX, despite being sufficiently expressed and catalytically active in the healthy and diseased mouse heart,One fundamental difference between the use of antioxidants targeted to mitochondria and genetically expressed AOX is that the former are not normally replenished beyond the acute post‐ischaemic phase. In addition, antioxidants, unlike AOX, may exhibit antioxidant effects during transit to the mitochondria and may therefore not be entirely specific for mitochondrial ROS. An obvious follow‐up study to test this idea would be the use of an inducible AOX strain to enable transient AOX expression during different phases of the I/R procedure. The use of AOX as a therapeutic, in the form of an mRNA mimetic or recombinant protein, may also address this issue. Both approaches would nevertheless suffer other shortcomings such as time‐ and/or organ‐unspecific expression of relevant drivers, unwanted side effects of the same drivers in case of the genetic approach, and immune and inflammatory responses in case of the therapeutic use of AOX, necessitating cumbersome controls.Saccharomyces cerevisiae serves as an NADH dehydrogenase, greatly decreases I/R injury and infarct size.Despite its ability to maintain respiration and dampen RET‐induced ROS in isolated mitochondria, we found that AOX not only fails to provide cardioprotection but actually promotes post‐ischaemic maladaptation. An interesting aspect of our study is that the presence of AOX in the post‐ischaemic heart restores cI‐driven respiration. In fact, cI may be both, a ROS‐producing complex for instance under conditions as seen in the post‐ischaemic heartThe present findings have some similarity with another study in which AOX was found to disrupt skeletal muscle remodelling upon knock‐out of cIV assembly factor COX15, a protein involved in the biosynthetic pathway of mitochondrial haem A, a prosthetic group of cIV.Anp at the transcript level. ANP was originally found to be up‐regulated in the failing heart, irrespective of the underlying causeCol1a1 and Col3a1 in AOX hearts, despite the blunted Anp response. Whilst collagen decreases wall stress, it also impairs contractile function due to increased stiffness, and accompanies the development of heart failure.To provide some indication of the mechanism whereby AOX leads eventually to a maladaptive cardiac remodelling, we studied the expression of a small set of marker genes. Although the findings do not provide a definitive explanation, they reveal valuable clues that should be followed up by a more detailed characterization of the physiological and molecular changes in the AOX I/R model, as now discussed. First, AOX abrogated the up‐regulation of Anp signalling, shown previously in rat pulmonary arterial smooth muscle cells, is the inhibition of TGFβ‐induced extracellular matrix components.Sod2, but up‐regulation of Ucp2, previously observed in conditions of cardiovascular stress.One consequence of In conclusion, we infer that mitochondrial respiratory restoration and the presumed decrease of mitochondrial ROS by AOX in the post‐ischaemic heart are not sufficient to confer cardioprotection. Instead, AOX expression interferes with adaptive organ remodelling leading to contractile failure, implying an essential role for ETC‐derived stress signals in this process.Ciona intestinalis AOX. All other authors declare no competing interests.MSz has financial interests in developing therapeutics based on MSz, HTJ, K‐DS, FNG, TK and MPM conceived and designed experiments and supervised parts of the work. MSz, RS, ZG, ED, MW, PKD, GD‐V, JH, IW, TAN, UG, ARH, VP and CV performed experiments and analysed data. MSz, IW, TK, MPM, TB, FNG, K‐DS and HTJ interpreted data and drafted the manuscript. All authors read and revised the manuscript and contributed substantially to the work. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Microorganisms efficiently coordinate phenotype expressions through a decision-making process known as quorum sensing (QS). We investigated QS amongst distinct, spatially distributed microbial aggregates under various flow conditions using a process-driven numerical model. Model simulations assess the conditions suitable for QS induction and quantify the importance of advective transport of signaling molecules. In addition, advection dilutes signaling molecules so that faster flow conditions require higher microbial densities, faster signal production rates, or higher sensitivities to signaling molecules to induce QS. However, autoinduction of signal production can substantially increase the transport distance of signaling molecules in both upstream and downstream directions. We present empirical approximations to the solutions of the advection–diffusion–reaction equation that describe the concentration profiles of signaling molecules for a wide range of flow and reaction rates. These empirical relationships, which predict the distribution of dissolved solutes along pore channels, allow to quantitatively estimate the effective communication distances amongst multiple microbial aggregates without further numerical simulations. Microorganisms preferentially reside on solid surfaces, which often leads to a closer proximity of neighboring cells than when in a planktonic form . At elevIndividual microbial cells synthesize and release signaling molecules at a basal rate. At low population densities, the concentration of signaling molecules remains low as it degrades both biotically and abiotically . At a suQS induction is affected by mass transport characteristics controlling the spatial distribution of signaling molecules. In a confined space, even a single microbial cell can be QS induced if the signaling molecules accumulate to sufficiently high concentration . HoweverThe signaling molecules transported either via advection or diffusion can induce QS in neighboring cells . BecauseHere, we evaluate the effect of combined diffusive and advective transport on QS processes in environmentally relevant conditions using a reactive transport modeling approach. The advection–diffusion–reaction equation was nondimensionalized to capture the characteristic properties of QS systems  and used to formulate empirical expressions describing concentration profiles of signaling molecules under various flow conditions. Using these relationships, we evaluate calling distances and threshold biochemical conditions for QS induction of a single microbial aggregate under various flow conditions. Then, we investigate QS interactions between heterogeneously distributed microbial aggregates. Finally, we demonstrate the importance of autoinduction for coordinated microbial behaviors in advection-dominated environments. This study quantifies the effect of flow velocities, autoinduction, and relative position of microbial aggregates to calling distances in a 2D flow channel.f:fi travel in the direction i with the lattice velocity ci , c1 = , c2 = , c3 = , c4 = , c5 = , c6 = , c7 = , c8 = ) to a new position r + ciΔt after a time step Δt. The relaxation time (τ) was described by the commonly used Bhatnagar–Gross–Krook collision operator , cs is a lattice dependent constant , c1 = , c2 = , c3 = , c4 = , c5 = ) corresponding to the lattice weights ωi , and Qi : F represents a multiplication factor which was set to either 0 or 10 to reflect the magnitude of autoinduced signal production (We used the Lattice Boltzmann (LB) method to implement a numerical model for the transport of signaling molecules due to diffusion and advection. The LB method is a mesoscopic approach solving the Boltzmann equation across a defined set of particles which recovers the macroscopic Navier–Stokes equation (NSE) and advection–diffusion–reaction equation (ADRE) . First, operator  and the 2(τ−Δt2) . Once th2(τ−Δt2)  and the 2(τ−Δt2) :(3)\docoduction , \documebehavior , which iWith the imposed flow field from olecules , limitinTo describe the characteristic properties of a microbial system across various flow and reaction conditions, Da, either due to high k’ , high B , or low This nondimensionalized ADRE is fully characterized by the Péclet number, expressing the magnitude of advective flow relative to diffusion Da  while Da was fixed at 5. For the 2D simulations in a straight channel  and no-gradient An important property of Da . For exa channel , the flox = 0) and outlet (x = 4) with no flow boundaries in both normal and tangential direction at the bottom (y = 0) and top (y = 2) of the domain resulting in parabolic Poiseuille flows. Simulations were carried out under low Mach numbers (Ma = u/cs ≪ 1) to ensure incompressible flow conditions  produced from a single microbial aggregate assumed a source constrained to a single grid cell located at x = 1 was investigated under various Pe conditions (0.5 ≤ Pe ≤ 10) while Da was fixed at 5 ) decreasing with increasing Pe : Amax decreased from 1.68 (Pe = 1) to 1.35 (Pe = 5) and 1.21 (Pe = 10). However, Amax of all of the simulations with Da = 5 exceeded 1 , where Amax = 1, can easily be computed using the linearity of the nondimensionalized ADRE in Da , higher signal production rate constant (k’), or lower QS induction threshold , 6 cells/mL which largely agree with the experimental observation of 2.69~6.23 × 106 cells/mL where signal concentration starts to show a strong spike (Table S1 in The increasing dvection  and matcdvection . Equatio0 nmol/L ) in a fl−10 m2/s . Our resAmax, advection also influenced the spatial distribution of signaling molecules. We define the “transport distance” (d) as the distance between the point of production (x0) and the point (x1) where the signal concentration reaches a certain value A* , distinguishing it from the “calling distance” between two QS induced microbial cells or aggregates. If the signal transport occurred only through diffusion, transport distances would be isotropic (dup) are much shorter than the downstream distances (ddn). Moreover, fast advective flows  reduced overall transport distances which are illustrated in A = 0.1 are dup = 0.08 and ddn = 0.62 at Pe = 1 and Da = 5 ; 0 ≤ x ≤ 1) and downstream (Adn(x); 1 < x ≤ 4) signal concentration profiles. Among the tested regression models, the exponential (A(x) > 0.001 were used to improve the fitting quality and the signal concentration at x = 1 was fixed as 1. The additional regression analysis was then carried out for the coefficients  obtained from simulated profiles at 24 Pe conditions to construct a relationship between the coefficients and Pe  were tested to the upstream  could underestimate the actual concentration because the confined channel width would push the produced chemical further upstream and downstream.These analytical expressions are applicable not only to QS but also to other chemical processes subject to zero-order production reactions e.g., . The equF = 0; Da = Daθ) and with  enhanced signal production. The transport distances from a single microbial aggregate under various Pe were then calculated using x.QS often involves autoinduction which substantially increases signal production rates. The effect of autoinduction on transport distances was investigated by using Pe = 1, 5 and 10. The concentration ratios (0.1 ≤ A/Amax ≤ 0.9) were used instead of absolute concentrations to generalize transport distances for various Da conditions. For example, the transport distance (dA) for A/Amax = 0.5 indicates that A(x0 + dA) = 0.5 if Da = Daθ while A(x0 + dA) = 0.05 when Da = 0.1Daθ. The consequence of the enhanced signal production was the significant increase of dup and ddn. Without the enhanced signal production, dup and ddn for A/Amax = 0.4 at Pe = 1 were estimated as 0.021 and 0.024, respectively , xi is the location of ith aggregate, x0 is the reference location (x0 = 1), Dai is the Da calculated only with the density of ith microbial aggregate , and Aup and Adn are Daθ consist of four aggregates (1–4) located at x1 = 0.4, x2 = 1, x3 = 1.096 and x4 = 1.7 with the evenly distributed microscopic iDa (i.e. Da1 = Da2 = Da3 = Da4 = 0.8Daθ) was tested. In using Dai= Da* that does not consider autoinduction (F = 0). Then, if there is an aggregate with A(xi) ≥ 1, the profile was recalculated with updated Dai = (1 + F) × Da* until all Dai with A ≥ 1 were updated.QS processes of multiple aggregates were investigated by constructing the concentration profiles using F = 0) illustrates the crucial importance of relative positioning of microbial aggregates for QS induction with respect not only to each aggregate but also to the flow direction . But due to transport, the local concentration at 2 reached 0.879, receiving A of 0.048 and 0.031 from 1 and 3, respectively. 3 received slightly less signaling molecules from 1 (A = 0.044) due to the longer distance of 3 than 2 from 1. However, 2 provided much more signaling molecules (A = 0.157) to 3 than was provided by 3 because of advective flows favoring downstream transport of signaling molecules (3 exceeded the QS threshold (A(x3) = 0.044 from 1 + 0.157 from 2 + 0.8 from 3 + 0 from 4 = 1.001 > 1) while the upstream located 2 did not. The QS induction of 3 demonstrates the importance of transport distances. QS induction was achieved because of the upstream aggregates located within the transport distance of 0.696. However, the calling distance would have been estimated as the length of a grid voxel (0.002) because only 3 was QS induced. Therefore, considering only the calling distance could lead to the wrong conclusion that the local Da condition at 3  is a sufficient condition for QS induction. Although 4 did not reach the QS induction threshold, it received A from all the other aggregates resulting in a concentration (A(x4) = 0.029 + 0.044 + 0.048 + 0.8 = 0.921) that was higher than at 2 despite the longest separation distance from other aggregates.The signal concentration profile produced by four aggregates without the enhanced signal production (irection . The micolecules  and 4. AF = 10) increased the transport distances and hence induced other aggregates  and provided more signaling molecules to 2. As a result, A(x2) exceeded the QS threshold (0.048 + 0.8 + 0.335 + 0 = 1.183). The QS induction of 2 and 3 resulted in the final signal concentrations of A(x2) = 9.183 (= 0.048 + 8.8 + 0.035 + 0) and A(x3) = 10.569 (= 0.044 + 1.725 + 8.8 + 0). While 4 still did not contribute signaling molecules to any of upstream aggregates, enhanced contribution from 2 and 3 QS induced 4, A(x4) = 9.839 (= 0.029 + 0.48 + 0.53 + 8.8). Despite increased transport distances by QS induction, 1 was still too far away from the other aggregates thus the signal concentration at 1 remained unchanged A(x1) = 0.8. As a result of the QS induction of 2-4, DaT had increased from the initial 3.2Daθ (= 0.8Daθ × 4) to 27.2Daθ (= 0.8Daθ + 3 × 11 × 0.8Daθ).Accounting for QS induction  provided more signaling molecules to its adjacent microbial aggregates and resulted in the QS induction of neighboring aggregates, 2 and 4. It may be counterintuitive that the upstream-located 2 was also QS-induced by the contribution from 3 despite the contracted upstream transport distances under the presence of advective flows. This result shows that the enhanced signal production can overcome the influence of advection and promote QS induction, and provide a way to provoke upstream microbial aggregates, for example, to slow down the substrate consumption to ensure efficient resource utilization in crowded environments  is required for QS induction. Reduced upstream delivery of signaling molecules under advective flow limits propagation of QS; it can be relaxed if autoinduction increases signal production rates. Our study results highlight the importance of relative positioning of microbial aggregates with respect to flow directions and the role of autoinduction to overcome advection for upstream signal propagation.This study has demonstrated that advection and the enhanced signal production can determine the spatial extent of QS induction. Reactive transport simulation results reveal that fast flow conditions dilute signaling molecules and thus higher The simulations focused on the effect of various flow conditions on QS and assumed that microbial aggregates have a negligible impact on flow fields, which is a reasonable approximation for low microbial density conditions. However, it may not hold when large aggregates producing extracellular polymeric substances (EPS) perturb flows substantially. In such a case, estimating signal transport requires fully resolving nonlinear feedback between cell activity and fluid flow , includiA > 1  = 1.05) would have been QS-induced, independent of the local cell density. This could lead to detrimental impacts on a microbial population, unless there are other counteracting mechanisms such as differential QS induction sensitivity to signal concentration even in within a clonal population (Although QS mediated gene expression has been understood as evolutionarily beneficial collective behaviors, long transport distances observed in this study suggests that it may not be always true. The transport of signaling molecules, especially in downstream direction, combined with enhanced signal production, suggests that QS induction can be decoupled from microbial density. In the above example , any micpulation  or biofipulation . Future


At present, the association between uric acid (UA) and brachial-ankle pulse wave velocity (baPWV) has not been well clarified. This study is the second analysis based on a cross-sectional study. 912 participants (average age is 51.5 ± 9.6 years) who underwent medical health examinations were included in this study, UA levels and baPWV were measured. Participants were divided into four groups according to UA levels , and the differences of baPWV between the four groups were compared. Univariate analysis showed a positive correlation between UA and baPWV . After adjusting for confounding factors, the positive correlation between UA and baPWV still exists . Furthermore, curve fitting showed that UA and baPWV had a non-linear positive correlation. In conclusion, elevated UA were associated with baPWV, suggesting that UA could be used as a predictor of atherosclerosis. As is known to all, cardiovascular disease has the clinical characteristics of high incidence, high mortality and high disability rate6. Once cardiovascular disease occurs, it will bring a huge economic burden to patients and national health security system. Therefore, early identification of cardiovascular risk factors and timely intervention is the focus of clinicians and medical and health institutions.With the continuous development of social economy and the change of people’s eating habits, cardiovascular disease has become the main cause of death worldwide9, but early atherosclerosis is lack of specific manifestations, and patients do not have obvious clinical symptoms, thus atherosclerosis is easy to be ignored in its early stages10. Therefore, how to identify arterial stiffness early is an important strategy to prevent cardiovascular diseases. With the gradual development of medical equipment and the continuous improvement of people’s attention to cardiovascular diseases, the technology of evaluating arterial stiffness by non-invasive method has become the focus of clinicians12. Brachial-ankle pulse wave velocity (baPWV)16 is the measurement of pulse wave velocity between brachial artery and ankle artery, which mainly reflects the stiffness of the vascular wall of peripheral large and middle arteries17. The larger the baPWV value, the higher the degree of arterial stiffness, and then the greater risk of cardiovascular diseases. At present, baPWV has been used as a non-invasive and important index to judge the degree of arterial vascular wall damage.Previous studies have shown that atherosclerosis is an important pathological of cardiovascular disease20. Previous studies have shown that UA may promote oxidative stress22, which might be related to the increase of oxygen free radical, nitric oxide and myeloperoxidase. In recent years, a series of reports have advocated that elevated UA are key hazard factors for the existence and development of cardiovascular diseases24. A prospective cohort study included 466 patients with ST-segment elevation myocardial infarction (STEMI) and UA level was measured. The results showed that patients with elevated UA had higher in-hospital mortality 25. A meta-analysis of 33 studies involving 427 917 participants showed that elevated UA was significantly associated with heart failure, all-cause mortality and cardiovascular mortality26. Similarly, on another meta-analysis, the authors discovered that higher UA was closely related to atrial fibrillation in both cross-sectional and cohort studies27.Uric acid (UA) is a feeble organic acid, which is the final decomposition product of purine nucleotides in blood circulation29, and it is not clear whether they share some common mechanisms in the process of arterial stiffness. An investigation in South Korea achieved that UA level was associated with baPWV in males (p < 0.001) and in females (p = 0.04)28. In contrast, another study noticed that UA level was not connected with baPWV29. Therefore, our study targeted to inspect the association between UA and BAPWV based on a cross-sectional study.However, the association between UA and baPWV is still controversialhttps://datadryad.org/stash). The data used in this study are from a cross-sectional study of Japanese population , Data from: Association between serum γ-glutamyltranspeptidase and atherosclerosis: a population-based cross-sectional study, Dryad, Dataset, 10.5061/dryad.m484p)30. Briefly, 912 participants who underwent health check-ups in Murakami Memorial Hospital from March 2204 to December 2012 were included. Exclusion criteria: (1) participants treated with hormones; (2) participants using oral contraceptives; (3) participants who tested positive for hepatitis B virus; (4) HCV-positive participants; (5) participants in pregnancy (women); (6) ankle-brachial index (ABI) value less than 0.95. Current research is the secondary analysis based on a previous study, thus it is not necessary to provide participants’ informed consent and approval by the ethics committee.All data used in this research can be downloaded freely and without restriction from Dryad Digital Repository  to measure the values of baPWV and ABI. This non-invasive method uses a cuff oscilloscope suitable for the arm and ankle to measure the pulses of the brachial and posterior tibial arteries of the participants. Before baPWV measurements, participants had to rest in a supine position at room temperature of 25 °C for 5 minutes. The electrodes were placed on both wrists of the participants, and the heart sound microphone was placed on the left edge of the sternum of the participants. Variables and formulas for calculating BaPWV (cm/s) have been described in Fukuda et al. studies28.It is necessary to point out that Fukuda and his colleagues completed the entire process of data collection , body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), Log2glutamyltranspeptidase (Log2GGT), fasting blood glucose (FBS), total cholesterol (TC), triglycerides, high density lipoprotein (HDL), low density lipoprotein (LDL), uric acid (UA), alcohol consumption, estimated glomerular filtration rate (eGFR), ankle brachial index (ABI), sex, smoking status, Ex-Smoker, regular exerciser, fatty liver and menopausal status. According to Fukuda and his colleagues, venous blood was taken from the anterior elbow veins of the participants after 8 hours of fasting. The samples were gathered in a silicified glass tube containing sodium fluoride. After the venous blood was collected, the plasma and serum samples were centrifuged and stored at −80 °C. Professionally trained technicians performed abdominal examinations with Aloka SSD-650CL . The diagnosis of fatty liver was based on the results of abdominal ultrasound. In addition, standardized questionnaires were used to investigate participants’ basic information, including age, sex, smoking, drinking, exercise and menopausal status.2 transformed, namely log2GGT.In this study, participants were divided into four groups according to the level of UA, , and the differences of baPWV between the four groups were compared. The assumption of normality of distribution was examined using Shapiro-Wilk test. Considering the partial distribution of glutamyltranspeptidase (GGT), we convert GGT into logFor continuous data with normal distribution, variables are represented as mean ± standard deviation, and least significant difference (LSD) variance analysis was conducted to evaluate the differences between groups. For categorized data, variables are represented as number and percentages  and chi-square test was conducted to evaluate the differences between groups. We compared the difference with odds ratio (OR) and 95% confidence intervals (CI).32 regression analysis to screen covariates. In the subsequent model, we only retain the covariates that have more than 10% influence on the UA regression coefficient. In addition, LASSO regression is characterized by feature selection and complexity adjustment while fitting the generalized linear model. Consequently, whether the target variable is a continuous variable or a classification variable, it could be demonstrated and forecasted by LASSO regression. Feature selection refers to not setting variables into the model for matching, but discriminatingly placing variables into this pattern to acquire appropriate variables.In univariate analysis, we use baPWV as dependent variable and other variables as independent variable to explore the relationship between them. In addition, in order to better investigate the association between UA and baPWV, we used the variation of regression coefficient and least absolute shrinkage and selection operator (lasso)https://www.r-project.org/) and EmpowerStats (http://www.empowerstats.com/cn/) for statistical analysis. P < 0.05 indicated that there was statistical difference (two sides).Furthermore, we used baPWV as dependent variable, UA as independent variable, and selected variables as covariates in multivariate regression analysis to adjust and observe the real association between UA and baPWV. Hosmer and Lemeshow test were used to measure goodness-of-fit. Considering that there is not necessarily a linear relationship between UA and baPWV in the real world, we use curve fitting to observe the change trend of UA and baPWV. In this study, we used SPSS 22 statistical software , R version 3.6.1 , age , SBP , DBP , AST , ALT , Log2GGT , FBS , TC , triglycerides , HDL , LDL , fatty liver , eGFR , menopausal status  and UA  were associated with baPWV  are retained and adjusted as final covariates in multivariate regression analysis . In model 2, fatty liver and menopausal status were adjusted and the result showed that UA is associated with baPWV . Similarly, this association still exists in model 3   . In addition, in further curve fitting, we also found that the relationship between UA and baPWV is not a simple linear relationship, but a nonlinear-positive correlation. Our results suggest that UA might be a useful predictor of atherosclerosis.35. A study included 1116 patients with suspected coronary heart disease (CHD) and divided them into four groups. The results showed that with the increase of UA level, the incidence of coronary atherosclerotic plaque and stenosis increased significantly (P < 0.05). In addition, elevated UA was associated with coronary artery calcification score36. Another study assessed the relationship between UA and subclinical atherosclerosis in Korean men. The results showed that after adjusting the known variables, the relationship between UA and coronary artery calcification (CAC) score and carotid intima-media thickness (cIMT) was OR = 1.101 (P = 0.046) and OR = 1.266 (P = 0.002), respectively37. Similarly, the CARDIA study intended to identify the association between UA and subclinical atherosclerosis in healthy adults. They found elevated UA was not related to BMI, but was related to the risk of coronary atherosclerosis. It is concluded that UA is an early biomarker of subclinical atherosclerosis, and this relationship is independent of BMI38. However, some researchers hold the opposite viewpoint. A cross-sectional study used Mendelian randomization to speculate the causal relationship between UA and atherosclerosis. It was found that the body mass index (BMI) alone could explain the changes in UA levels in women and men. After BMI or eGFR were adjusted, the relationship between UA and cIMT was not exist. The results showed that there was essentially a causal relationship between BMI and UA, BMI is a powerful confounding factor between UA and cIMT, which indicating that there was no evidence that UA played a role in the early stage of atherosclerosis39. In addition, a study based on the NHLBI database aims to explore the relationship between UA and CAC. The results showed that there was no evidence of a correlation between UA and CAC in both male and female , and this relationship did not exist in subgroup analysis, indicating that there was no evidence to confirm the relationship between UA and CAC. It is suggested that UA may not promote atherosclerosis through the effect of CAC40. Similarly, a cross-sectional study in Korea29 also showed that UA was not associated with baPWV levels. It should be noted that the participants in JH Lim study29 are between 20 and 80 years old, which may be the reason why the results are different from our study. In addition, population differences may also be the cause of different results.Few studies have paid close attention to the relationship between UA and baPWV, lacking theoretical basis to guide clinical practice. On the contrary, a series of studies have focused on the relationship between UA and atherosclerosis indicators36 and Zhang Z37, we explored the relationship between UA and baPWV based on data from a cross-sectional study and found that UA was positively correlated with baPWV, which was independent of BMI or eGFR. In addition, curve fitting displayed that the association between UA and baPWV is a non-linear positive correlation, not a simple linear relationship. It should be noted that our result is somewhat contrary to some predecessor’s result which have emphasized that no significant different was observed between UA and atherosclerosis. The possible explanations are as follows. First, in Oikonen M study39, the subjects were young people, aged between 30 and 45 years old, and is a study based on Finland population. In NHLBI study40, the average age of the subjects was 58 years old, and the subjects were the American population. Unlike previous studies, the subjects of our study were Japanese population aged between 24 and 84 years old. As is known to all, the degree of atherosclerosis tends to increase with age, and different dietary habits and genes in different populations can also lead to differences in atherosclerosis. Secondly, different measurement indicators will lead to different results. In our study, the evaluation index is baPWV; while in previous studies, cIMT or CAC were the indicators of evaluation. In addition, different measuring instruments could also lead to different results.Consistent with the studies of Sun YThere are several aspects to explain the mechanism of uric acid and baPWV.43. Ox-LDL can not only promote the formation of foam cells, but also aggregate under the endothelium to form lipid stripes. Moreover, the apoptosis of intimal cells in coronary artery could be induced by activating multiple atherosclerotic pathways (ERK1/2 and bcl-2 pathways)46, and then leads to endothelial cell damage. In addition, oxygen free radicals and reactive oxygen species can also damage vascular endothelial function through leukocyte activation. Furthermore, urate crystals are deposited in the arterial wall, which induces inflammation, stimulates mast cells, induces lipid infiltration, and further damages the intimal cells of the arteries, thus blocking apolipoprotein B (apoB) metabolism, and ultimately leads to the aggravation of atherosclerosis48. Similarly, atherosclerosis can lead to glomerular hypoxia, and then increased lactic acid production, competitive excretion of uric acid, and increased UA levels.Hyperuricemia may form oxidized low-density lipoprotein (ox-LDL) by increasing oxygen free radicalsThis study has the following advantages. First of all, our study used univariate analysis, Lasso regression analysis and regression coefficient changes to screen covariates, and the results are more in line with clinical practice. Secondly, three regression models are used to adjust the potential variables in this study, and the results are consistent, which indicating that the results are stable. Thirdly, we use curve fitting to discover the real association between UA and baPWV. The result showed that the relationship between UA and baPWV is not a simple linear relationship, but a non-linear positive correlation, which is a remarkable difference from previous studies. It should be emphasized that this study has the following shortcomings. First, the current study is a second analysis based on a cross-sectional study, and cannot draw the causal relationship between UA and baPWV. Second, the participant of this study is the Japanese population, and the findings do not necessarily apply to other population. Third, we do not have access to the information about the participants’ medication or cardiovascular diseases, which might lead to mixed bias.Elevated UA were associated with baPWV, and this relationship is non-linear and positive correlation. suggesting that UA could be used as a predictor of atherosclerosis.