Omics Analyses of Gut Microbiota in a Circadian Rhythm Disorder

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Bioactive Constituents, Metabolites, and Functions

Omics analyses of gut microbiota in a circadian rhythm disorder mouse model fed with oolong tea polyphenols Tongtong Guo, Dan Song, Chi-Tang Ho, Xin Zhang, Chundan Zhang, Jinxuan Cao, and Zufang Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03000 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 23, 2019

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Journal of Agricultural and Food Chemistry

Omics Analyses of Gut Microbiota in a Circadian Rhythm Disorder Mouse Model Fed with Oolong Tea Polyphenols Tongtong Guo†, Dan Song†, Chi-Tang Ho‡, Xin Zhang†, § ,*1, Chundan Zhang§, Jinxuan Cao†, Zufang Wu† †Department

of Food Science and Engineering, Ningbo University, Ningbo 315211,

P.R. China ‡Department

of Food Science, Rutgers University, New Brunswick, New Jersey

08901, United States §Key

Laboratory of Animal Protein Deep Processing Technology of Zhejiang

Province, Ningbo University, Ningbo 315211, P.R. China



Corresponding author: Xin Zhang; E-mail address: [email protected] 1

ACS Paragon Plus Environment


1

ABSTRACT: Microbiome has been revealed as a key element involved in

2

maintaining the circadian rhythms. Oolong tea polyphenols (OTP) has been shown to

3

have a potential prebiotic activity. Therefore, this study focused on the regulation

4

mechanisms of OTP on host circadian rhythms. After 8 weeks of OTP administration,

5

a large expansion in the relative abundance of Bacteroidetes with a decrease in

6

Firmicutes was observed, which reflected the positive modulatory effect of OTP on

7

gut flora. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways

8

of ATP-binding cassette (ABC) transporters, two-component system and the

9

biosynthesis of amino acids enriched the most differentially expressed genes (DEGs)

10

after OTP treatment. Of the differentially expressed proteins (DEPs) identified, most

11

were related to metabolism, genetic information processing and environmental

12

information processing. It underscores the ability of OTP to regulate circadian rhythm

13

by enhancing beneficial intestinal microbiota and affecting metabolic pathways,

14

contributing to the improvement of host micro-ecology.

15

KEYWORDS: oolong tea polyphenols, circadian clock, modulatory effect, intestinal

16

microbiota

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Introduction

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Tea is widely consumed around the world. Oolong tea is a unique semi-fermented tea

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containing various ingredients, and the putative effective compounds is attributed to

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polyphenols.1 Oolong tea polyphenols (OTP) have been shown to have a potential

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prebiotic activity.2 The main phenolic compounds in OTP include (-)-epigallocatechin

22

gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and

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(-)-epicatechin (EC).3 Furthermore, as the O-methylated form of EGCG,

24

(-)-epigallocatechin 3-O-(3-O-methyl)gallate (EGCG3″Me) shows a satisfied effect

25

on host intestinal micro-ecology by modulating gut microbiota.4

26

The rotation of the earth around its axis generates a cycle of light and darkness.

27

Circadian rhythm is an endogenous oscillation of physiology synchronizes many

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biological processes with changes in environmental factors.5 Therefore, almost all

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living organisms have evolved circadian clock to adapt and respond to the physical

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environment.6 The mammalian circadian rhythm is generated by a central clock that

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receives direct light input from the retina and synchronizes other central and

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peripheral tissues phases.7 In a peripheral clock system, the clock in the liver is

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critical because it affects many physiological processes.8 Therefore, natural products

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have been noted as potential functional components with circadian modulatory

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activity. In recent years, the relationship between circadian rhythm and intestinal flora

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has aroused widespread concern.9

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In the gut, the microbiota has been revealed as a critical element affecting host

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health,10 and the disturbance of microbiota composition has been demonstrated to be 3



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related to the development of certain metabolic syndromes.11 Circadian clocks and

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metabolism are inseparable, both central and liver circadian clocks coordinate

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metabolic events in response to the awakening-sleep cycle.12 The gut microbiome is

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important in maintaining the host's circadian rhythm. Despite the existence of light

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and dark signals, sterile mice exhibited impaired circadian clock gene expression,

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whereas the intestinal microbiota of the normal mice showed different diurnal

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variation depending on dietary composition.13 Dietary tea polyphenols have been

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reported to ameliorate memory impairment via a circadian clock-related mechanism.14

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In addition, plant polyphenols could effective regulate the expression and rhythm of

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circadian clock genes as well as the internal environment.15 Our previous studies have

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also shown that tea polyphenols are beneficial for the stability of gut flora, especially

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in environmentally induced microbial imbalances.16,17

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Prebiotics not only modulate gut flora, but also improve the integrity of intestinal

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tight junctions.18 Tea catechins have been showed prebiotic activity and generated

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short-chain fatty acids (SCFAs) in our previous study.19 In addition, we have

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identified KEGG pathways enriched the most DEGs after OTP intervention.16 At

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present, the research on the regulation of tea catechins on intestinal flora is an

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emerging field, and there is still a lack of systematic understanding. Tea catechins can

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affect the circadian rhythm of peripheral clock systems, but the molecular regulation

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mechanisms are still unclear. Therefore, investigating the inter-relationship between

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intestinal flora and host circadian rhythm is a breakthrough in promoting the theory of

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circadian rhythm regulation. 4


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Materials and Methods

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Chemicals. Polyamide resin was purchased from Ocean Chemical Co., Ltd.

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(Qingdao, China). Standards of tea catechin monomers were prepared according to

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the method we reported.19 All other chemicals were of analytical grade.

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Preparation of OTP. Oolong tea was obtained from a local tea plantation in

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Ningbo. The OTP preparation was performed according to our reported method.16

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Briefly, the tea was extracted with hot water at a ratio of 1:16 (m: v) at 96 °C for 40

68

min, and purified by a polyamide column. The elutions were analyzed by HPLC,16

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and the desired fractions containing OTP were concentrated and lyophilized.

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Animals and experimental design. We initially colonized young adult (6 weeks

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old) male C57BL/6J mice by a microbial community present in freshly stool samples

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from 5 healthy volunteers (3 females and 2 males, 25-30 years old). Fresh human

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stool samples were diluted in 10 mL of PBS under anaerobic conditions, and 0.2 mL

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of the vortexed stool suspension was introduced by gavage into each sterile recipient.

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The mice were acclimated to the environment for 7 days in constant darkness, and

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then randomly divided into 3 groups: a 12 h light-dark cycle group (control), constant

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darkness group (CD) and constant darkness with OTP group (CD-OTP) fed with 0.1%

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(w/w) OTP. The body weight, food and water consumption of each animal was

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recorded weekly, and faecal samples were collected from the CD-OTP group after 0

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(OTP-0), 2 (OTP-2), 4 (OTP-4) and 8 weeks (OTP-8). After 8 weeks, the laparotomy

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was performed under pentobarbital anesthesia, and liver and epididymal fat were

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isolated and immediately weighed. 5



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Analysis of intestinal microbiota. DNA was extracted using E.Z.N.A. ®Stool

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DNA Kit by the manufacturer's instructions.16 Prior to sequencing, the above 16S

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rDNA V3-V4 region of each sample was amplified with a set of primers targeting the

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16S rRNA gene region. Sequences were analyzed using QIIME (Quantitative Insights

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Into Microbial Ecology) (version 1.2.8) software package.20 High quality reads were

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clustered into operational taxonomic units (OTUs) using CD-HIT software (version

89

4.6.1). The OTUs reached 97% nucleotide similarity level were used for richness

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(Chao1), alpha diversity (Shannon and Simpson).21 Phylogenetic beta diversity

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measures unweighted UniFrac distance metrics analysis and principal coordinate

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analysis (PCoA) were performed using OTUs.

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Construction of a gut metagenome reference. Firstly, we performed taxonomic

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assignments and functional annotations using the Non-Redundant (NR), KEGG, and

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Gene Ontology (GO) databases. 16 In addition to setting the E value was set to 10-5, all

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genes were searched against integrated microbial genomes (IMG, version 3.4) using

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default parameters. The taxonomic association of a gene was determined by the

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lowest common ancestor of all taxonomically annotated results.

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Protein extraction, digestion and LC-MS/MS measurements. Faecal samples

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were suspended with PBS and rotated on a Thermo shaker (MSC-100) overnight at

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4 °C. The precipitates were collected by centrifugation and re-suspended in 90%

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pre-cooled acetone. The extract was centrifuged, then the supernatant was transferred

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to a new tube and the protein was precipitated by methanol chloroform.

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Protein samples were digested according to previously reported method.22 The 6


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peptides were dried under vacuum, re-suspended in 2% acetonitrile and 0.1% TFA

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and desalted with Sep-Pak C18 (Waters, WAT023590). Peptides were analyzed on a

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Q Exactive mass spectrometer coupled with Easy-nLC 1200. The peptides were

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loaded onto a C18-reversed phase column and eluted with a gradient of 5-80%

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acetonitrile + 0.1% formic acid at a flow rate of 300 nL/min.

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Taxonomic analysis of peptides and identification of proteins. The MS/MS

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spectra were searched according to the bacterial database from the Uniprot database

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(http://www.uniprot.org/) and the decoy database.23 Protein quantification was

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performed in Peaks Studio 8 software based on MS1 peak intensity, and functional

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analysis

115

(http://www.genome.jp/kegg/pathway.html).

of

identified

proteins

were

assigned

to

the

KEGG

database

116

Statistical analysis. Data obtained were analyzed by SAS and expressed as mean

117

± standard deviation (SD). Comparisons between groups were performed using

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one-way analysis of variance (ANOVA) and Duncan’s multiple-comparison test. All

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results were considered statistically significant at P < 0.05.

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Results

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The tea catechin contents in oolong tea. The HPLC chromatogram of oolong tea

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infusion in Fig. S1 shows that peaks 1-13 are gallic acid, (-)-gallocatechin (GC),

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theobromine,

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(-)-gallocatechin-3-gallate (GCG), EGCG3″Me, ECG, and (-)-catechin gallate (CG).

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It indicated that the EGCG content is the highest (Table S1), making it the main tea

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catechin, while the content of EGCG3″Me is high.

EGC,

(-)-catechin

(C),

theophylline,

7


EGCG,

caffeine,

EC,


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The effect of OTP on body and organ weight of the mouse model. The increase

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in body weight after 2 weeks was significantly higher (P < 0.05) in the CD group than

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in the CD-OTP group (Fig. 1), which showed the direct weight-reducing activity of

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OTP. In Fig. S2, the values for the liver and epididymal weight in the CD group were

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significantly higher than those of the control (P < 0.05), while the weight gain of the

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CD-OTP group was significantly reduced (P < 0.05). The results of food (Fig. S3A)

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and water consumption (Fig. S3B) showed no significant difference (P > 0.05) in all

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groups.

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The effect of OTP on bacterial diversity of the mouse model. The estimator of

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Chao1 richness for total bacterial community diversity was increased during OTP

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treatment (Table S2). The functional role of OTP in improving species richness and

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diversity was further supported by a significantly higher Shannon and lower Simpson

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indexes (P < 0.05). A Venn diagram showed that there were only 243 of the total

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richness of 1,029 OTUs were shared among all samples, demonstrating that >23% of

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the OTUs observed after OTP treatment were the same as in the initial treatment.

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In each group, Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria were

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the most abundant phyla (Fig. 2). After 8 weeks of OTP treatment, a dramatically

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decrease in the relative abundance of Firmicutes with an increase of Bacteroidetes

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was observed. The corresponding Firmicutes/Bacteroidetes (F/B) ratio was decreased

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from 0.80 (OTP-0) to 0.41 (OTP-8), indicating that Firmicutes was largely inhibited

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by OTP. In the control group, the F/B ratio was also decreased. However, in the CD

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group the ratio showed an opposite trend, which suggested the circadian rhythm 8


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disorder may lead to imbalance of intestinal flora, and the result was similar to the

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consequences of obesity-induced gut dysbiosis.

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At the family level (Fig. 3A), it can be seen that both Prevotellaceae and

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Bacteroidaceae showed significant increasing trends (P < 0.05) during the treatment

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of OTP. Prevotellaceae was the predominant bacterium, and the relative abundance of

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which increased from 0.34 ± 0.02 (OTP-0) to 0.44 ± 0.02 at the 8th week. For

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Bacteroidaceae, it showed a similar trend, while Eubacteriaceae, Ruminococcaceae

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and Lachnospiraceae showed a decreasing trend after OTP intervention. Prevotella

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and Bacteroides accounted for the majority at the genus level (Fig. 3B), whose

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relative abundances were increased after OTP treatment, while Faecalibacterium,

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Mitsuokella and Ruminococcus were decreased. In addition, as shown in Fig. S4, the

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faecal flora in the OTP-fed group at 0, 2, 4 and 8-week were separated clearly by

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PCoA.

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The effect of OTP on the gut microbiome of humanized mouse. The imputed

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relative abundances of KEGG pathways in OTP-0 and OTP-8 were used to predict

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metabolic function changes within the microbiomes (Fig. 4). The KEGG pathways

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indicated that membrane transport, carbohydrate metabolism and signal transduction

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accounted for the top three. At the same time, the largest statistical differences

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between OTP-0 and OTP-8 were cell motility, membrane transport and environmental

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adaptation. After 8 weeks, the OTP-associated DEGs were enriched in various KEGG

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pathways involved in ABC transporters, two-component system, and biosynthesis of

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amino acids (Fig. S5). 9



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GO analysis of DEGs between OTP-0 and OTP-8 indicated that most genes in

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biological processes including regulation of transcription, DNA-templated, transport,

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translation, metabolic process and carbohydrate metabolic process; cellular

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components including cytosol, plasma membrane, cytoplasm, integral component of

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membrane and membrane; and molecular functions including transcription factor

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activity, sequence-specific DNA binding, ATP binding, structural constituent of

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ribosome, protein binding and catalytic activity (Fig. 5).

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Metaproteomic analysis was applied to study the proteins of the intestinal

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microbiota of the CD group fed with OTP. A total of 312,676 MS/MS spectra were

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generated from the faecal samples, and 65,535 peptides could be identified. The

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distribution of molecular weight (A), number (B) of peptides and sequence coverages

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(C) by label-free proteomics were shown in Fig. S6A-C. Of the differentially

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expressed proteins (DEPs) between OTP-0 and OTP-8 identified, most were related to

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metabolism, genetic information processing and environmental information

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processing (Fig. 6). After OTP intervention, DEPs enriched in the metabolism

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domains included carbohydrate metabolism, global and overview maps and amino

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acid metabolism; genetic information processing including translation, folding,

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sorting degradation and transcription; environmental information processing including

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membrane transport and signal transduction.

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Discussion

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The human gut is considered to be the main site for the interaction between OTP and

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intestinal flora.24 Meanwhile, evidence has showed an inextricable link between gut 10


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microbiota and metabolic syndromes. Previous studies underscored the ability of

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microbial-derived metabolites to alter circadian rhythms as well as the metabolic

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function of the host.14

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Sleep defects and circadian rhythm disruption are closely related to metabolic

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disorders, which may lead to obesity by regulating feeding time and amount of food

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intake.25 Studies have shown that tea polyphenols could modulate the relatively

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shallow daily oscillations of circadian clock gene expression in the liver caused by

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continuous darkness.15 People with persistent circadian rhythm disorders, such as

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insomnia and night shift workers, have higher incidence of hyperlipidemia,

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atherosclerosis, hypertension and other diseases.26 The occurrence of these chronic

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diseases was closely related to the human intestinal micro-ecological imbalance, and

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showed circadian rhythm oscillation similar to the intestinal flora.27 A common

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characteristic of these chronic diseases is that they appear to be accelerated by the

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inflammatory

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characteristic of circadian clock,29 which maintaining biological homeostasis through

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a number of specialized metabolic and signaling pathways.30 It has been demonstrated

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that environmental circadian rhythm disruption can lead to disorders of the gut flora,

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especially when under dietary stress.31

process.28

Most

intestinal

microbial

communities

showed

a

211

Tea catechins exhibited proliferative effects on certain beneficial bacteria in our

212

previous study; in addition, it promoted the production of total SCFAs.19 The

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production of SCFAs is the result of intestinal microbiota metabolism, which plays a

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vital role in regulating host metabolism and cell proliferation.25 In this study, an 11



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increase in the relative abundances of genera known to produce acetate and butyrate

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were observed in mice supplemented with OTP. Different strains of gut microbiota

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exhibited varying degrees of growth sensitivity to the metabolites of polyphenols.32

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The changes of microbiota populations after circadian rhythm disruption have been

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characterized by the expansion of pro-inflammatory bacteria and a decrease in

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putative anti-inflammatory, SCFAs-producing ones.33

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In the present study, circadian disorder leads to symptoms similar to obesity. After

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8 weeks of treatment, the body weight and epididymal fat were significantly increased

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(P < 0.05) in the CD group, while a decrease in the relative abundance of Firmicutes

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was observed in the circadian rhythm disorder group with OTP intervention. In

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addition, OTP increased the relative abundance of Bifidobacteria dramatically after 8

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weeks, which may enhance intestinal barrier function, stimulate the host immune

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system and regulate lipid metabolism, and considered to be important bacterial group

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associated with host health.34 An increased ratio of F/B has been observed in obese

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people.35 However, the generation of circadian rhythm disorder involves more

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complex issues, which cannot only be simply explained by an imbalance in the F/B

231

proportion.

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Although the relationships between intestinal flora and metabolic syndromes

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remain unclear, high-throughput sequencing offers the opportunity to explore

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taxonomy and genes through less biased and more comprehensive measurements.36

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The mechanism by which tea polyphenols regulate the flora in the gut includes the

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interactions with the basic development and metabolic aspects of bacteria, as well as 12


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interference with cell membrane function and bacterial energy metabolism.37 The

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effect of tea polyphenols on the gut microbiota depends on the structure of both the

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polyphenols and the microbial strain.38,39 Tea polyphenols can form hydrogen bonds

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with stacking interactions of nucleobases, which may explain their inhibition on

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bacterial DNA and RNA synthesis.40 In our study, enriched GO terms suggesting that

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OTP alleviated the negative consequences of circadian rhythm disorder by affecting

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cellular components. Moreover, tea polyphenols can influence bacteria through a

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variety of cellular targets. For instance, plant polyphenols can form complex with

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proteins via hydrogen bonding, covalent bonding and hydrophobic interactions.41

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KEGG analysis of DEGs showed the most enriched metabolic pathways between

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OTP-0 and OTP-8 in the present study. The ABC transporters are widely distributed,

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and involved in detoxification and transport processes.42 In addition, the results

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revealed by metaproteomic indicated that after 8 weeks, most DEPs were enriched in

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the metabolic domains, and that may be due to that the gut microbiota can regulate

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energy harvest of the host.43 To be more specific, intestinal microflora affects the

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host's energy metabolism system by regulating the efficiency of energy extraction

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from diet and the way of using or storing this harvested energy.

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EGCG has been reported to relieve diet-induced metabolic syndromes related to

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the circadian clock,44 and dietary tea polyphenols can ameliorate memory impairment

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through a circadian clock-related mechanism.14 However, due to the limited

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bioavailability of tea polyphenols in vivo, the majority ingested are metabolized into

258

various derivatives by intestinal microbiota. The activity of tea polyphenols depends 13



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to a large extent on their conversion in the intestine. It has been indicated that the

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disruption of circadian clock by changing the light/dark circadian cycle induced the

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changes in the intestinal microbiota, including composition and circadian rhythm

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oscillation.45 Recent studies have also demonstrated the ability of microbial metabolic

263

derivatives to regulate central and liver circadian rhythm as well as host metabolic

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function, which implies prebiotics may alleviate circadian rhythm misalignment.46

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Furthermore, this study will better assess the feasibility of tea polyphenols' microbial

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metabolites in the treatment of circadian rhythm disruption and the related metabolic

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syndromes.

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In conclusion, OTP ameliorated circadian rhythm disorder induced gut dysbiosis,

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and showed activities to maintain micro-ecology balance. For the DEPs identified by

270

metaproteomic analysis, most were related to metabolism, genetic and environmental

271

information processing. Finally, our results suggested that OTP has prebiotic activity

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for ameliorate metabolic syndrome associated with circadian rhythm disorders.

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Acknowledgment

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We thank LC-Bio Technology for the bio-information analysis.

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Supporting Information description

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Figure S1. Representative elution profiles of tea catechins, gallic acid, caffeine,

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theobromine and theophylline from oolong tea (1, gallic acid; 2, (-)-gallocatechin

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(GC); 3, theobromine; 4, EGC; 5, (-)-catechin (C); 6, theophylline; 7, EGCG; 8,

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caffeine; 9, EC; 10, (-)-gallocatechin-3-gallate (GCG); 11, EGCG3″Me, 12, ECG; 13,

280

(-)-catechin gallate (CG)). 14


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Figure S2. Effect of OTP on liver weight (A) and epididymal fat weight (B) of the

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circadian rhythm disorder mouse model. Different letters indicate significant

283

differences (P < 0.05) among different groups.

284

Figure S3. Effect of OTP on water intake (A) and food intake (B) of circadian rhythm

285

disorder mouse model.

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Figure S4. Principal coordinate analysis (PCoA) plot of the faecal microbiota based

287

on the unweighted UniFrac metric.

288

Figure S5. KEGG analysis of differentially expressed genes (DEGs) between OTP-0

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and OTP-8.

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Figure S6. The distribution of molecular weight (A), number (B) of peptides and

291

sequence coverages (C) by label-free proteomics.

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Table S1. Contents of tea catechins, gallic acid, caffeine, theobromine and

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theophylline in oolong tea.

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Table S2. The influence of OTP on the biodiversity of faecal microbiota in the

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circadian rhythm disorder mouse model.

15



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Funding

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This work was sponsored by Zhejiang Provincial Natural Science Foundation of

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China (LY19C200006), the Key Research and Development Project of Zhejiang

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Province (2017C02039 & 2018C02047) and K.C. Wong Magna Fund in Ningbo

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University.

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Figure captions

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Figure 1. Effect of OTP on the body weight of the circadian rhythm disorder mouse

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model (n=8). *indicates significantly differences (P < 0.05) between CD and CD-OTP

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group.

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Figure 2. The relative abundance of the top phylum from samples (n=3).

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Figure 3. Relative abundance analyses at the family (A) and genus (B) levels from

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the faecal microbiota of the circadian rhythm disorder mouse model.

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Figure 4. Imputed metagenomic differences between OTP-0 and OTP-8 in KEGG

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pathway maps. The relative abundance of metabolic pathways encoded in each

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imputed sample metagenome was analysed using STAMP.

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Figure 5. GO analysis of differentially expressed genes (DEGs) between OTP-0 and

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OTP-8.

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Figure 6. KEGG classification of differentially expressed proteins (DEPs) in the

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faecal microbiota of the circadian rhythm disorder mouse model between OTP-0 and

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OTP-8 by metaproteomic analyses.

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Omics Analyses of Gut Microbiota in a Circadian Rhythm Disorder

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