However, the immunogenicity of this vaccine in humans against SARS-CoV-2 variants of concern remains unclear. Here we report humoral and cellular immune responses from 20 Ad26.COV2.S vaccinated individuals from the COV1001 phase I–IIa clinical trial2 against the original SARS-CoV-2 strain WA1/2020 as well as against the B.1.1.7, CAL.20C, P.1 and B.1.351 variants of concern. Ad26.COV2.S induced median pseudovirus neutralizing antibody titres that were 5.0-fold and 3.3-fold lower against the B.1.351 and P.1 variants, respectively, as compared with WA1/2020 on day 71 after vaccination. Median binding antibody titres were 2.9-fold and 2.7-fold lower against the B.1.351 and P.1 variants, respectively, as compared with WA1/2020. Antibody-dependent cellular phagocytosis, complement deposition and natural killer cell activation responses were largely preserved against the B.1.351 variant. CD8 and CD4 T cell responses, including central and effector memory responses, were comparable among the WA1/2020, B.1.1.7, B.1.351, P.1 and CAL.20C variants. These data show that neutralizing antibody responses induced by Ad26.COV2.S were reduced against the B.1.351 and P.1 variants, but functional non-neutralizing antibody responses and T cell responses were largely preserved against SARS-CoV-2 variants. These findings have implications for vaccine protection against SARS-CoV-2 variants of concern.The Ad26.COV2.S vaccine Analysis of the immunogenicity of the Ad26.COV2.S vaccine against the B1.351 and P.1 SARS-CoV-2 variants of concern shows reduced neutralization antibody titres, but comparable T cell responses and antibody-dependent effector functions. Such variants of concern may reduce the efficacy of current vaccines and natural immunity from SARS-COV-2 strains that were prevalent at the beginning of the pandemic. The mRNA-1273 and BNT162b2 vaccines have been reported to induce lower neutralizing antibody titres against the B.1.351 variant than against the original WA1/2020 strain7. Additional SARS-CoV-2 variants include the B.1.1.7 variant that was first identified in the UK8, the P.1 and P.2 variants that were first identified in Brazil9, and the CAL.20C variant that was first identified in California10.SARS-CoV-2 variants that partially escape from neutralizing antibodies to the WA1/2020 strain have emerged, including the B.1.351 variant that was first identified in South Africa11 that expresses a pre-fusion stabilized SARS-CoV-2 spike protein12 from the Wuhan 2019 strain, which is identical to the spike protein in the WA1/2020 strain. Ad26.COV2.S demonstrated protective efficacy against SARS-CoV-2 challenges in hamsters and non-human primates13 and showed safety and immunogenicity in humans14. In the phase III ENSEMBLE trial, a single dose of 5 × 1010 viral particles of Ad26.COV2.S resulted in 72%, 68% and 64% protection against moderate to severe COVID-19, and 86%, 88% and 82% protection against severe or critical COVID-19 in the US, Brazil and South Africa, respectively, by day 28 after vaccination1. In this trial, 69% of sequenced viruses from confirmed COVID-19 cases in Brazil were the P.2 variant, and 95% of sequenced viruses from confirmed COVID-19 cases in South Africa were the B.1.351 variant, which demonstrates that Ad26.COV2.S provided robust protective efficacy against these SARS-CoV-2 variants.Ad26.COV2.S is a replication-incompetent human adenovirus type 26 (Ad26) vector10 or 1 × 1011 viral particles administered intramuscularly as single-shot or two-shot vaccine schedules, 56 days apart, in healthy adults (NCT04436276)14. Cohort 1b enrolled 25 adults 18–55 years of age from 29 July 2020 to 7 August 2020 at a single site at Beth Israel Deaconess Medical Center (BIDMC), Boston, Massachusetts, for exploratory immunogenicity studies2. The study was approved by the BIDMC Institutional Review Board, and all participants provided written informed consent. Participants were randomly allocated to one of five experimental groups (n = 5 per group): (1) 5 × 1010 viral particles of Ad26.COV2.S on days 1 and 57 (low-dose–low-dose); (2) 5 × 1010 viral particles of Ad26.COV2.S on day 1 and placebo on day 57 as a single-shot vaccine (low-dose–placebo); (3) 1 × 1011 viral particles of Ad26.COV2.S on days 1 and 57 (high-dose–high-dose); (4) 1 × 1011 viral particles of Ad26.COV2.S on day 1 and placebo on day 57 as a single-shot vaccine (high-dose–placebo); or (5) placebo on days 1 and 57 (placebo–placebo).COV1001 is a multicentre, randomized, double-blind, placebo-controlled phase I–IIa trial to evaluate safety, reactogenicity and immunogenicity of Ad26.COV2.S at 5 × 1016, the median psVNA titres were 169, 142, 102, 80, 60 and 51 against the WA1/2020, D614G, B.1.1.7, CAL.20C, P.1 and B.1.351 strains, respectively, on day 57  assay 57 Fig. . The medOn day 57, median receptor binding domain (RBD)-specific binding antibody enzyme-linked immunosorbent assay (ELISA) titres were 1,772, 1,364, 486 and 392 against the WA1/2020, B.1.1.7, P.1 and B.1.351 variants, respectively Fig. . On day 18 was also used to evaluate spike- and RBD-specific binding antibody responses to WA1/2020, B.1.1.7, P.1 and B.1.351 351 Fig. . Similar19, including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), and antibody-dependent natural killer cell activation (ADNKA). Spike-specific ADCP, ADNP, ADCD and ADNKA responses against the B.1.351 variant were 1.5-, 2.9-, 1.6- and 1.1-fold reduced, respectively, compared with the WA1/2020 strain with the D614G mutation  assays on days 57 and 85  and depth (frequency of TCRs) of TCRs specific to either spike or non-spike SARS-CoV-2 proteins were determined, although these analyses may have underestimated total T cell responses. Higher breadth of spike-specific TCRs was observed in vaccine recipients compared with placebos   after vaccination, TCRβ sequencingved Fig. .Fig. 4TC7. By contrast, functional non-neutralizing antibody responses and CD8+ and CD4+ T cell responses were largely preserved against SARS-CoV-2 variants of concern.SARS-CoV-2 variants have emerged with several mutations in targets of neutralizing antibodies, such as the E484K mutation. Median pseudovirus neutralizing antibody titres induced by Ad26.COV2.S were 5.0-fold lower against the B.1.351 variant and 3.3-fold lower against the P.1 variant as compared with the original WA1/2020 strain, which is a comparable reduction of psVNA titres that has been reported for other vaccines1, Ad26.COV2.S was evaluated in the USA, Latin America including Brazil, and South Africa. In South Africa, 95% of sequenced viruses from COVID-19 cases were of the B.1.351 variant, and in Brazil, 69% of sequenced viruses from COVID-19 cases were of the P.2 lineage. Protective efficacy of Ad26.COV2.S against severe or critical disease was similar in all geographic locations by day 28, and protective efficacy against moderate to severe disease was only slightly reduced in South Africa compared with the USA. Although the mechanistic correlates of protection for COVID-19 are not yet known, the robust protective efficacy in these regions despite reduced neutralizing antibodies raises the possibility that functional non-neutralizing antibodies and/or CD8+ T cell responses may also contribute to protection. Indeed, TCRβ sequencing revealed substantial breadth of T cell responses in individuals vaccinated with Ad26.COV2.S. Alternatively, it is possible that low levels of neutralizing antibodies are sufficient for protection. In a non-human primate model, adoptive transfer of purified IgG was sufficient for protection against SARS-CoV-2 if titres of psVNA exceeded a threshold of approximately 50, but CD8+ T cells also contributed to protection if antibody titres were subprotective23.In the phase III ENSEMBLE trialIn conclusion, neutralizing antibody responses elicited by Ad26.COV2.S were reduced against the B.1.351 and P.1 variants, but other functional antibody responses and T cell responses were largely preserved against these variants. The relevance of these immune parameters to mechanistic correlates of vaccine efficacy remains to be determined.No statistical methods were used to predetermine sample size. The study was randomized, blinded, and placebo controlled. Investigators were blinded to allocation during experiments and outcome assessment.23. In brief, the packaging plasmid psPAX2 (AIDS Resource and Reagent Program), luciferase reporter plasmid pLenti-CMV Puro-Luc (Addgene), and spike protein expressing pcDNA3.1-SARS CoV-2 SΔCT of variants were co-transfected into HEK293T cells  using lipofectamine 2000 (ThermoFisher). Pseudoviruses of SARS-CoV-2 variants were generated by using the WA1/2020 strain , D614G mutation, B.1.1.7 variant (GISAID accession ID: EPI_ISL_601443), CAL.20C (GISAID accession ID: EPI_ISL_824730), P.1 (GISAID accession ID: EPI_ISL_792683), or B.1.351 variant (GISAID accession ID: EPI_ISL_712096). The supernatants containing the pseudotype viruses were collected 48 h after transfection, and then were purified by centrifugation and filtration with a 0.45-µm filter. To determine the neutralization activity of the plasma or serum samples from participants, HEK293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1.75 x 104 cells/well overnight. Three-fold serial dilutions of heat-inactivated serum or plasma samples were prepared and mixed with 50 µl pseudovirus. The mixture was incubated at 37 oC for 1 h before being added to HEK293T-hACE2 cells. Forty-eight hours after infection, cells were lysed in Steady-Glo Luciferase Assay (Promega) according to the manufacturer’s instructions. SARS-CoV-2 neutralization titres were defined as the sample dilution at which a 50% reduction in relative light unit (RLU) was observed relative to the average of the virus control wells.The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated in an approach similar to that described previously17. One day before the assay, Vero E6 USAMRID cells were plated at 20,000 cells per well in clear-bottom black-walled plates. Cells were inspected to ensure confluency on the day of assay. Serum samples were tested at a starting dilution of 1:20 and were serially diluted threefold up to nine dilution spots. Serially diluted serum samples were mixed in equal volume with diluted virus. Antibody–virus and virus-only mixtures were then incubated at 37 °C with 5% CO2 for one hour. After incubation, serially diluted sera and virus only controls were added in duplicate to the cells at 75 plaque-forming units at 37 °C with 5% CO2. Twenty-four hours later, the cells were lysed, and luciferase activity was measured via Nano-Glo Luciferase Assay System (Promega) according to the manufacturer specifications. Luminescence was measured by a Spectramax M3 plate reader (Molecular Devices). Virus neutralization titres were defined as the sample dilution at which a 50% reduction in RLU was observed relative to the average of the virus control wells.Full-length SARS-CoV-2 WA1/2020, B.1.351 and B.1.1.7, viruses were designed to express nanoluciferase (nLuc) and were recovered via reverse genetics−1 RBD proteins (provided by F. Krammer) in 1× DPBS and incubated at 4 °C overnight. After incubation, plates were washed once with wash buffer (0.05% Tween 20 in 1× DPBS) and blocked with 350 μl casein block per well for 2–3 h at room temperature. After incubation, block solution was discarded and plates were blotted dry. Serial dilutions of heat-inactivated serum diluted in casein block were added to wells and plates were incubated for 1 h at room temperature, before three further washes and a 1 h incubation with a 1:4,000 dilution of anti-human IgG HRP (Invitrogen) at room temperature in the dark. Plates were then washed three times, and 100 μl of SeraCare KPL TMB SureBlue Start solution was added to each well; plate development was halted by the addition of 100 μl SeraCare KPL TMB Stop solution per well. The absorbance at 450 nm, with a reference at 650 nm, was recorded using a VersaMax microplate reader. For each sample, ELISA endpoint titre was calculated in Graphpad Prism software, using a four-parameter logistic curve fit to calculate the reciprocal serum dilution that yields a corrected absorbance value (450–650 nm) of 0.2. The log10-transformed endpoint titres are reported.WA1/2020, B.1.1.7 and B.1.351 RBD-specific binding antibodies were assessed by ELISA. In brief, 96-well plates were coated with 2 μg mlECLA plates  were designed and produced with up to nine antigen spots in each well. The antigens included were WA1/2020, B.1.1.7, P.1 and B.1.351 S and RBD. The plates were blocked with 50 μl of blocker A (1% BSA in MilliQ water) solution for at least 30 min at room temperature shaking at 700 rpm with a digital microplate shaker. During blocking, the serum was diluted 1:5,000. The plates were then washed three times with 150 μl of the MSD kit Wash Buffer, blotted dry, and 50 μl of the diluted samples were added in duplicate to the plates and set to shake at 700 rpm at room temperature for at least 2 h. The plates were again washed three times and 50 μl of SULFO-Tagged anti-Human IgG detection antibody diluted to 1× in Diluent 100 was added to each well and incubated shaking at 700 rpm at room temperature for at least 1 h. Plates were then washed three times and 150 μl of MSD GOLD Read Buffer B was added to each well and the plates were read immediately after on a MESO QuickPlex SQ 120 machine. MSD titres for each sample was reported as RLU, which were calculated as sample RLU minus the blank RLU for each spot for each sample. The limit of detection was defined as 1,000 RLU for each assay.19. Biophysical profiling was performed using a custom Luminex based assay where individuals bar-coded beads were coated with spike (S) or (RBD) variants by carboxy coupling. The D614G, B.1.1.7 and B.1.351 variants (provided by E. Ollman Saphire and F. Krammer) were profiled. The overall levels of IgG1, IgG2, IgG3, IgA, IgM and FcγR2a, FcγR2b, FcγR3a and FcγR3b binding were assessed. Functional profiling included the assessment of ADCP, ADNP, ADCD and ADNKA. In brief, for the ADCP, ADNP and ADCD assays, fluorescent beads (LifeTechnologies) were coupled via carboxy-coupling, and plasma was added, allowing immune complex formation, excess antibodies were washed away, followed by the addition of THP1 monocytes, primary neutrophils, or guinea pig complement, individually, respectively. The level of phagocytosis and complement deposition was assessed by flow cytometry. For ADNKA, ELISA plates were coated with antigen, followed by the addition of plasma. Excess antibodies were washed away following by the addition of primary natural killer cells. Natural killer cells were treated with Golgi Stop (BD) and brefeldin A (Sigma Aldrich) and were stained for the surface markers CD56, CD16 and CD3 and for activity markers CD107a (BD) and MIP-1b (BD). Fluorescence was determined by flow cytometry. Natural killer cells were classified as CD56+CD16+CD3−.Both the biophysical and functional quality of polyclonal vaccine induced SARS-CoV-2 antibodies were profiled using systems serologyl-glutamine, and 0.1% of 55 mM 2-mercaptoethanol) for 2–4 h at 37 °C. SARS-CoV-2 pooled spike peptides from WA1/2020, B.1.351, B.1.1.7, P.1 and CAL.20C  were prepared and plated at a concentration of 2 μg per well, and 100,000 cells per well were added to the plate. The peptides and cells were incubated for 15–20 h at 37 °C. All steps after this incubation were performed at room temperature. The plates were washed with ELISPOT wash buffer and incubated for 2–4 h with biotinylated mouse anti-human IFNγ monoclonal antibody from MabTech (1 μg ml−1). The plates were washed a second time and incubated for 2–3 h with conjugated Goat anti-biotin AP from Rockland (1.33 μg ml−1). The final wash was followed by the addition of Nitor-blue Tetrazolium Chloride/5-bromo-4-chloro 3′ indolyphosphate p-toludine salt (NBT/BCIP chromagen) substrate solution for 7 min. The chromagen was discarded and the plates were washed with water and dried in a dim place for 24 h. Plates were scanned and counted on a Cellular Technologies Limited Immunospot Analyzer.ELISPOT plates were coated with mouse anti-human IFNγ monoclonal antibody from MabTech at 1 μg per well and incubated overnight at 4 °C. Plates were washed with DPBS, and blocked with R10 medium  were re-suspended in 100 μl of R10 medium supplemented with CD49d monoclonal antibody (1 μg ml−1) and CD28 monoclonal antibody (1 μg ml−1). Each sample was assessed with mock , pooled S peptides from WA1/2020, B.1.351, B.1.1.7, P.1 and CAL.20C  (2 μg ml−1), or 10 pg ml−1 phorbol myristate acetate and 1 μg ml−1 ionomycin (Sigma-Aldrich)  and incubated at 37 °C for 1 h. After incubation, 0.25 μl of GolgiStop and 0.25 μl of GolgiPlug in 50 μl of R10 was added to each well and incubated at 37 °C for 8 h and then held at 4 °C overnight. The next day, the cells were washed twice with DPBS, stained with aqua live/dead dye for 10 min and then stained with predetermined titres of monoclonal antibodies against CD279 , CD4 , CD27 , CD8 , CD45RA  for 30 min. Cells were then washed twice with 2% FBS/DPBS buffer and incubated for 15 min with 200 μl of BD CytoFix/CytoPerm Fixation/Permeabilization solution. Cells were washed twice with 1× Perm Wash buffer (BD Perm/Wash Buffer 10× in the CytoFix/CytoPerm Fixation/Permeabilization kit diluted with MilliQ water and passed through 0.22-μm filter) and stained with intracellularly with monoclonal antibodies against Ki67 , IL-21 , CD69 , IL-10 , IL-13 , IL-4 , TNF , IL-17 , IFNγ , IL-2 , IL-6 , CD3 , for 30 min. Cells were washed twice with 1× Perm Wash buffer and fixed with 250 μl of freshly prepared 1.5% formaldehyde. Fixed cells were transferred to 96-well round bottom plate and analysed by BD FACSymphony system. Data were analysed with FlowJo v.9.9.Peripheral blood mononuclear cells 21. TCRs that react were further screened for enrichment in COVID-19-positive repertoires collected as part of ImmuneCODE compared to COVID-19-negative repertoires to remove TCRs that may be highly public or cross-reactive to common antigens. Individual response could be quantified by the number and/or frequency of SARS-CoV-2 TCRs seen post-vaccine. TCRs were further analysed at the level specific ORF or position within ORF based on the MIRA antigens. The breadth summary metric is calculated as the number of unique annotated rearrangements out of the total number of unique productive rearrangements, while depth summary metric corresponds to the sum frequency of those rearrangements in the repertoire. Sequences of known variants were obtained from GISAID (www.gisaid.org) and aligned to known MIRA antigen locations.Immunosequencing of the CDR3 regions of human TCRβ chains was performed using the immunoSEQ Assay (Adaptive Biotechnologies). Extracted genomic DNA was amplified in a bias-controlled multiplex PCR, followed by high-throughput sequencing. Sequences were collapsed and filtered to identify and quantitate the absolute abundance of each unique TCRβ CDR3 region for further analysis as previously describedFurther information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-03681-2.Reporting Summary


Enterococcus sp. F2 through ethanol precipitation, anion-exchange, and gel-filtration chromatography and characterize the physicochemical properties by spectral techniques. EPS-F2 was identified as a neutral homo-exopolysaccharide composed of only glucose with a high molecular weight of 1.108 × 108 g/mol. It contained →6)-α-D-Glcp--α-D-Glcp-(1→ branch chain). Moreover, EPS-F2 possessed excellent thermal stability (266.6°C), water holding capacity (882.5%), oil holding capacity (1867.76%), and emulsifying activity against various edible oils. The steady shear experiments exhibited stable pseudo plasticity under various conditions . The dynamic oscillatory measurements revealed that EPS-F2 showed a liquid-like behavior at a low concentration (2.5%), while a solid-like behavior at high concentrations (3.0 and 3.5%). Overall, these results suggest that EPS-F2 could be a potential alternative source of functional additives and ingredients and be applied in food industries.The present study sought to isolate a novel exopolysaccharide (EPS-F2) from  F2.A homopolysaccharide (EPS-F2) with high Mw was obtained from -It had a noteworthy thermal stability under high temperature.-Enterococcus sp. F2 were assessed.Functional properties of EPS-F2 from -The non-Newtonian pseudoplastic behavior was observed in EPS-F2 solutions.Exopolysaccharides (EPSs) are high molecular weight carbohydrate polymers secreted to the extracellular matrix by various microorganisms. EPSs are classified into two major groups based on their monosaccharide compositions: (1) homo-exopolysaccharides, comprising of a single type of sugar with repeating subunits; (2) hetero-exopolysaccharides, comprising of two and more types of monosaccharide subunits . EPSs haLactic acid bacteria (LAB) strains have attracted wide attention in the past decades due to their structural diversity and functional properties. Accumulating studies have described the structural characteristics, functional and rheological properties, and potential bioactivity of EPS produced by lactobacilli  bacteria  of the screened EPS were not elucidated by these studies. Therefore, this study aimed to isolate, purify, and characterize EPS-F2, and analyze its functional properties, including emulsifying activity (EA), oil holding capacity (OHC), water solubility index (WSI), and water holding capacity (WHC). Furthermore, the rheological properties influenced by concentrations, temperatures, pH values, and salts were assessed.The EPS-producing strain F2 was isolated from Guizhou fermented soya beans from Liupanshui city, Guizhou province, China. Fermented soya bean samples were homogenized and serially diluted in sterile distilled water. Each diluted sample was spread on an agar plate containing MRS medium and incubated at 30°C until single colonies appeared. The strain F2 was identified by cell morphological and 16S rDNA sequence analyses . The phyg at 4°C for 20 min. The supernatant was mixed with three pre-chilled 95% (v/v) ethanol volumes to precipitate the crude EPS. The resultant crude EPS was treated with 8% (w/v) trichloroacetic acid to remove the precipitated proteins. After centrifugation, the obtained solution was dialyzed with ultrapure water using a dialysis bag  for 48 h and, finally, lyophilized.For EPS production, strain F2 was cultured in fresh MRS broth containing 40 g/L sucrose at 30°C for 48 h. Crude EPS were extracted from the strain and purified according to a previously reported method , with miThe lyophilized EPS was resuspended in ultrapure water and filtered through a 0.45 μm syringe filter. The filtrate was fractionated through a DEAE-52 anion-exchange chromatographic column  eluted with 0, 0.1, 0.3, and 0.5 M NaCl solutions at a flow rate of 2 mL/min. Each 4 mL of the elution was collected and then subjected to carbohydrates content measurement by the phenol–sulfuric acid method. The major peak named EPS-F2 were pooled, dialyzed, and then lyophilized. The EPS-F2 was further purified by gel filtration chromatography using a Sephacryl S-400 HR column . The elution was performed using ultrapure water at a flow rate of 1 mL/min .The total carbohydrate, protein, and sulfate contents were determined by the phenol-sulfuric acid colorimetric method, Bradford assay, and barium chloride-gelatin colorimetric assay. The UV spectrum of the EPS-F2 solution (5 mg/ml) was measured by a spectrophotometer at a wavelength of 200–800 .3 solutions at 25°C and then filtered through a 0.22 μm filter. Subsequently, 100 μL of EPS-F2 solution (5 mg/mL) was loaded onto the Shodex OHpak SB-803 separation system connected to a series of SB-804 and SB-805 separation systems at 60°C. NaNO3 solution (0.1 mol/L) was used as the mobile phase; at a flow rate of 0.4 mL/min.The homogeneity and average molecular weight of EPS-F2 were determined by high performance size-exclusion chromatography coupled with a differential refractive index and multi-angle laser light scattering detectors. The EPS-F2 sample was dissolved in 0.1 mol/L NaNOMonosaccharide composition of EPS-F2 was determined by high performance anion-exchange chromatography with pulsed amperometric detection. Briefly, the EPS-F2 (5 mg) was hydrolyzed by 1 mL of 2.0 M trifluoroacetic acid and then heated at 121°C for 2 h in a sealed glass ampoule. Excess acid was removed using methanol and then dried using a nitrogen blowing instrument. This process was repeated thrice, and the final hydrolyzed products were dissolved in distilled water. The hydrolyzate and standards were analyzed by a Dionex ICS-5000 ion chromatography system  equipped with a Dionex CarboPac PA-20 analytical column and a Dionex ED50A electrochemical detector.–1 at room temperature with a total 32 scans (Fourier transform infrared (FT-IR) spectra of EPS-F2 were recorded on an infrared spectrometer  at the wavenumber, ranging from 500 to 4000 cm32 scans .Glycosyl linkage analysis was performed according to a previously reported method , with mi2O, 99.9% D) and transferred to an nuclear magnetic resonance (NMR) tube for NMR analysis. 1D NMR (1H- and 13C NMR) and 2 D NMR  were determined by a Bruker 800 MHZ spectrometer  (Approximately 30 mg of lyophilized EPS-F2 sample was dissolved in 500 μL of deuterium oxide (Dzerland) .X-ray diffraction (XRD) data of EPS-F2 were recorded using an X-Ray diffractometer  equipped with a Cu Ka radiation source (λ = 1.5406 Å). The EPS-F2 was scanned at an angular angle ranging between 5° and 80° at a step size of 0.02°/min.Thermogravimetric analysis (TGA) and Differential thermal analysis (DTG) were performed between 50 to 800°C on TG 209F3 Tarsus equipment  . DiffereThe zeta potential and particle size of EPS-F2 (0.5%w/v) were determined at 25°C using a NanoPlus Zeta Potential and a particle size analyzer , respectively. The surface morphology and microstructure of EPS-F2 (5 mg) were visualized using a scanning electron microscope  at an accelerating voltage of 15 kV at 200× and 1000× magnifications .g for 10 min. The supernatant was lyophilized overnight. The WSI was calculated by the following equation:Water solubility index was determined according to a previously reported method , with mig for 30 min. Afterward, the supernatant was discarded, and the residues were weighed. The WHC was calculated by the following equation:According to the method reported by g for 10 min, the supernatant was discarded, and the residue was weighed. The OHC was calculated by the following equation:The OHC of EPS-F2 was determined according to the method reported by The EA of EPS-F2 was determined according to a previously reported method . Briefly–1) on the apparent viscosity of EPS at different concentrations (from 0.5 to 4%) were determined. The effects of temperatures , pH values , and salts (KCl and CaCl2) at different concentrations  on the apparent viscosity of 3% EPS-F2 were assessed.The rheological properties of EPS-F2 samples were determined by an air-bearing MCR 302 stress-controlled rheometer  equipped with a parallel plate . Additionally, the effects of shear rates  were determined using strain sweep measurements at logarithmically varying shear strains from 0.1 to 100%. The dynamic frequency sweep was performed at angle frequencies ranging from 0.1 to 100 rad/s to obtain the storage moduli (G’) and loss moduli (G”). The effects of KCl  on both G’ and G” of EPS-F2 (3%) were investigated. Subsequently, the temperature ramp oscillation analyses of EPS-F2 solutions (3 and 3.5%) were performed at a fixed strain (1%) and at an increasing temperature from 5 to 80°C.All the experiments were performed in triplicate, and the data were expressed as means ± SD. Origin Pro (version 8.5) software  was employed to summarize the data.MW563755) was closest to Enterococcus lactis BT159 (99.65% 16S rRNA gene). Thus, it was named Enterococcus sp. F2. This strain was observed in either pairs or short chains     and E. f5 g/mol) . Accordi5 g/mol) . In consE. faecium MS79  contained α-(1→6) glycosidic bonds , →6)-Glcp-(1→ (84.82%), and →3,6)-Glcp-(1→ (4.73%) and that the main chain of EPS-F2 consisted of glucopyranose units linked by α-(1–6) linkages and partially branched at the O-3 position with a single α-glucopyranose unit.The types of glycosidic bonds in EPS-F2 derived from the methylation analysis are summarized in 1H NMR  ppm were assigned to H–2, H–3, H–4, H–5, and H–6 of residue C, respectively. The corresponding chemical shifts of C–2, C–3, C–4, C–5, and C–6 were δ 73.05, δ 74.96, δ 71.13, δ 71.83, and δ 67.04 ppm, respectively. Compared with the previously reported data of glucose units  ppm of residue C were obtained from the se units , the sig13C chemical shifts obtained by HSQC spectrum were assigned to δ 100.51, δ 72.50, δ 82.18, δ 71.10, δ 71.74, and δ 66.80 ppm, respectively. The relative downward shifts of C-3 (δ 82.18 ppm) and C-6 (δ 66.80 ppm) indicated that residue B was →3, 6)-α-D-Glcp- ppm, respectively by combining the results in the TOCSY and COSY spectra. Based on these chemical shifts, the p- (Lactobacillus reuteri SK24.003 (292.6°C) (As illustrated in  (300°C)  and Lact292.6°C) . This diWeissella confusa MD1 has a melting point of 267.64°C and a melting enthalpy change (ΔH) of 337.7 J/g  is 120.09°C, and its ΔH is 404.6 J/g. These differences in the melting temperatures and ΔH values might be attributed to the different monosaccharide compositions. Leuconostoc pseudomesenteroides XG5 dextran were 274.14°C and 101.80 J/g, respectively. This finding strongly supports the view that the thermal behavior of EPS from various lactic acid bacteria strains might be different.37.7 J/g . KanmaniBacillus paralicheniformis SR14 (293.73 ± 4.0 nm) (Lactococcus garvieae C47 (166.6 nm) (The X-ray diffractograms of EPS-F2 are presented in  4.0 nm)  and larg66.6 nm) . This reWeissella cibaria YB-1 (95.23 ± 4.45%) (Lactobacillus kefiranofaciens ZW3 (14.2%) isolated from Tibet kefir (Leuconostoc citreum B-2 (450%) (Leuconostoc lactis L2 (509.45 ± 28.59%)  , and higet kefir . Previou2 (450%)  and Leuc 28.59%) . Therefo 28.59%) . TherefoEnterococcus species. However, there is evidence of reporting dextran produced by other bacterial strains. For instance, dextran produced by Weissella cibaria YB-1 has high emulsification activity in sunflower oil (83.43 ± 3.65%)  . TherefoGenerally, the applicability of EPS as a gelling agent, stabilizer, and thickener in food products and film-forming substrates is dependent on their rheological properties in an aqueous solution. Therefore, determining the rheological properties of EPS has significant importance.–1). This result indicates that EPS-F2 has typical shear thinning or non-Newtonian fluid behavior, which is the typical behavior of EPS in aqueous solutions due to the orientations of their molecular components. Moreover, increasing the shear rate could alter the entanglement network structure between the molecular chains, leading to the random orientation of the molecular chains and decreased viscosity due to the decrease in the interaction of adjacent chains , the shear rate (s–1), and the consistency index parameter (mPa Sn), respectively. The flow behavior index (n) is the degree of non-Newtonian behavior: when 0 < n < 1, the fluid is non-Newtonian pseudoplastic. As summarized in The rheological properties of EPS-F2 at different concentrations (0.5–4%) were determined at 25°C in terms of the steady flow behavior. As illustrated in t chains . PseudopThe results of 3% EPS-F2 aqueous solutions exposed to 15, 25, 35, and 45°C temperatures are illustrated in Paenibacillus edaphicus NUST16 has higher viscosity in near neutral conditions.As depicted in + at different concentrations on the viscosity of EPS-F2 are illustrated in + at low shear rates, and the decrease became more prominent at higher K+ concentrations (0.3 and 0.4 M). It is apparent that the addition of K+ could weaken the electrostatic repulsion between molecules, causing them to expand and reducing the viscosity  were subjected to different shear strain rates ranging from 0.1 to 100% . At the The dynamic frequency sweeps results are illustrated in + on the viscoelastic properties of EPS-F2 at 25°C are illustrated in + concentration, the G’ value of EPS-F2 remained higher than the G” value over the frequency range tested, indicating that EPS-F2 predominantly has elastic behavior. Nevertheless, it was also found that the addition of K+ led to a slight decrease in G’ and G” values, which was consistent with the steady rheological properties test results. + at low concentrations results in a slight increase in the G’ value without affecting the G” value; however, the presence of Na+ at high concentrations led to a slight decrease in both values. The difference might be attributed to the different cationic interactions in EPS.The results of the effect of KThe effects of high temperature on the viscoelastic properties of EPS-F2 at two concentrations (3 and 3.5%) were determined by temperature ramp oscillation. As illustrated in Enterococcus. The functional properties and rheological properties of EPS-F2 were investigated after purification and characterization. The findings indicated that EPS-F2 consisted of only glucose with an average molecular weight of 1.108 × 108 g/mol. The FTIR, GC–MS, and NMR results confirmed that EPS-F2 contained →6)-α-D-Glcp--α-D-Glcp-(1→ linkage in the branch chain. The thermal properties determination, XRD, and SEM results revealed that it was an amorphous-crystalline biopolymer with excellent thermal stability. Additionally, EPS-F2 possessed significant functional properties, especially for WHC, OHC, and EA. Besides, the steady shear and dynamic oscillatory measurement results revealed the non-Newtonian behavior and liquid-like behavior of EPS-F2, respectively. However, further studies on this glucan are highly warranted to better understand its synthesis mechanism, and structural–functional relationship, and exploration of its potential applications in foods, pharmaceuticals, and other products.In conclusion, a novel EPS-producing strain F2 was isolated from Guizhou fermented soya beans and identified as The original contributions presented in the study are included in the article/GJ contributed to conceptualization, investigation, and writing – original draft preparation. LG contributed to methodology and validation. XL contributed to data curation. JH contributed to software. SZ and JC contributed to investigation and software. RZ and ZX contributed to formal analysis and software. YT contributed to conceptualization, funding acquisition, supervision, writing – reviewing and editing, and project administration. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Tph2−/−), the rate-limiting enzyme of 5-HT synthesis in the brain, were used to assess the role of central 5-HT in alcohol drinking behavior. Life-long 5-HT depletion in these mice led to an increased ethanol consumption in comparison to wild-type animals in a two-bottle choice test. Water consumption was increased in naïve 5-HT-depleted mice. However, exposure of Tph2−/− animals to ethanol resulted in the normalization of water intake to the level of wild-type mice. Tph2 deficiency in mice did not interfere with ethanol-evoked antidepressant response in the forced swim test. Gene expression analysis in wild-type animals revealed no change in Tph2 expression in the brain of mice consuming ethanol compared to control mice drinking water. However, within the alcohol-drinking group, inter-individual differences in chronic ethanol intake correlated with Tph2 transcript levels. Taken together, central 5-HT is an important modulator of drinking behavior in mice but is not required for the antidepressant effects of ethanol.Indirect evidence supports a link between disrupted serotonin  signaling in the brain and addictive behaviors. However, the effects of hyposerotonergia on ethanol drinking behavior are contradictory. In this study, mice deficient in tryptophan hydroxylase 2 ( Alcoholism is a chronic relapsing brain disorder defined by a loss of control over drinking despite known health consequences. Current first-line pharmacotherapies include naltrexone, the opioid receptor antagonist, and acamprosate, a drug that modulates glutamate and GABA-ergic signaling . DisulfiEpidemiological studies demonstrate the correlation between depression and alcohol dependence in humans ; howeverDysregulation of serotonin  neurotransmission has been linked to both depression  and ethaInterestingly, preclinical studies have shown that selectively bred alcohol-preferring rats displayed alterations in 5-HT function; however, there is inconsistency in the direction of observed changes ,25,26,27Tph2 mRNA is synthesized within the 5-HT neurons of the brainstem raphe nuclei and can be transported via axonal transport to other brain areas, such as the cortex, hippocampus, striatum, hypothalamus, and cerebellum [TPH2 mRNA and protein in the dorsal and median raphe nucleus of alcoholic individuals [TPH2 gene have been associated with major depression . TPH2 is responsible for the conversion of tryptophan to 5-hydroxytryptophan (5-HTP), the rate-limiting step of 5-HT production. rebellum ,35,36. Sividuals ,38. A stividuals . In addi, R441H; ,41) and , R441H; ,43); howr review .Tph2 gene variants on the level of ethanol intake have been inconsistent. For example, mice with a hypofunctional R439H polymorphism in Tph2 gene , leading to a partial (60–80%) deficiency in 5-HT level in the brain [Tph2 knock-in mice drank amounts of ethanol comparable to wild-type animals; however, under aversive conditions, displayed enhanced motivation for ethanol [Tph2 gene, leading to reduction in Tph2 mRNA and protein levels and about a 15% decrease in 5-HT content in the brain [Preclinical studies examining the effects of he brain ,47,48,49he brain . In anot ethanol . Low prehe brain ,53,54,55Tph2-deficient (Tph2−/−) mice was observed in the forced swim test (FST); however, no difference in behavior compared to wild-type mice was detected in tail suspension and sucrose preference tests . To datTph2 transcript level in wild-type mice drinking ethanol. In this study, we employed Tph2−/− mice that lack central 5-HT throughout the life [Tph2−/− mice that had continuous access to ethanol solutions of increasing concentrations in a two-bottle choice procedure. We next evaluated whether the genetic ablation of Tph2 affected the antidepressant effects of ethanol in the FST. In the second part of the study, we assessed Tph2 expression in the brain of Tph2+/+ mice drinking ethanol by quantitative real-time PCR (qPCR). We demonstrate that Tph2 deficiency in mice enhanced ethanol consumption and that Tph2 transcript levels correlated with ethanol intake in mice.Taken together, the data discussed above prompted us to test the hypothesis that the life-long 5-HT deficiency affects the initiation of ethanol drinking and ethanol-induced antidepressant effects in mice. Furthermore, in search of a molecular mechanism linking 5-HT function and ethanol addiction, we analyzed the changes in the life . We firsAnimal experimental procedures were carried out in accordance with the European Communities Council Directive 2010/63/UE and had been approved by the local animal welfare and ethical review body , G 0343/09, date of approval: 24 March 2010). All the efforts were made to minimize animal suffering and to reduce the number of mice used.Animals were maintained at the animal facility of the MDC . Mice were housed in individually ventilated cages  in a colony room maintained at 21 ± 1 °C under a 12-h light–dark cycle (lights on at 7:00 am). Standard rodent chow  and water were available ad libitum. The behavioral experiments were conducted during the light phase (between 8:00 and 16:00) of the light–dark cycle.Tph2−/− mice on a C57BL/6 background  [Tph2+/+; n = 22) weighing 24.31 ± 0.64 g and 23.64 ± 0.33 g, respectively, at the beginning of the experiment (ca. 10 weeks old) were used. To generate C57BL/6 Tph2+/+ and Tph2−/− experimental groups, Tph2+/− females were bred with Tph2−/− or Tph2+/+ males. Genotyping was carried out using DNA isolated from the ear snips by multiplex PCR with primers Neo3: 5′-CTGCGCTGACAGCCGGAACAC-3′, TPH34: 5′-AGCTGAGGCAGACAGAAAGG-3′, and TPH54: 5′-CCAAAGAGCTACTCGA CCTACG-3′ to distinguish the Tph2-wild-type allele  from the Tph2-knockout allele .Male  n = 14) ,60 and wv/v diluted from 96% ethyl alcohol; Merck, Darmstadt, Germany) was dissolved in tap water.Ethanol  were single housed and habituated to two bottles (one empty and the second filled with water). The position of bottles was changed from the left to the right side to induce seeking behaviors in animals. After 6 days, randomly assigned mice were exposed to a two-bottle free-choice paradigm  in home cages for 29 days, using increasing concentrations of ethanol solution  [n = 10; Tph2+/+: n = 5, Tph2−/−: n = 5). Every two days, the bottles were refilled with ca. 14 mL of fresh solutions and the position of the bottles was alternated to control for side preference. Liquid  intake was measured by weighing the bottles in 2-day trials (after 48 h) for 28 days and on the final day of experiment (day 29) after 24 h. Ethanol intake was calculated as g of pure ethanol per kg of body weight per 1 or 2 days (g/kg). Preference was calculated as g of ethanol solution consumed within 1 or 2 days per total amount of liquid (%). Cumulative ethanol intake was calculated as g of pure ethanol per kg of body weight within 29 days (g/kg/29 d). We also calculated the average intake of ethanol (g/kg/d), preference (%), and average total liquid intake  consumed per kg of body weight per day (mL/kg/d)) during the initial 4 days of each ethanol concentration.One week before the experiment, animals (21 days) ,65,66,67Immediately after the end of the last ethanol exposure day, mice were introduced to the FST. As previously described ,68,69, mTph2 gene expression analysis, only the brain tissue from Tph2+/+ mice  was taken. First, the Tph2 expression level in the whole group of Tph2+/+ mice drinking ethanol (n = 17) was compared with water-drinking mice (n = 5).For the Tph2+/+ mice during the experiment, the level of Tph2 expression was then analyzed with respect to the animals’ ethanol intake levels in two drinking periods: during the initial days of ethanol drinking  or during the last week of access to ethanol (period of stabilized ethanol drinking). For each period , the average ethanol intake (g/kg/d) was calculated for each animal. In addition, an average preference (%) was calculated for each animal for each of the tested periods. The average ethanol intake scores were used to divide mice into “high ethanol-drinking” (n = 9) and “low ethanol-drinking” (n = 8) animals using a median split [Since there was considerable variability in the level of ethanol intake and preference for ethanol among an split , i.e., aTph2 expression was aimed at examining whether the inter-individual differences in Tph2 mRNA levels were notable prior to ethanol exposure (assuming that ethanol did not affect Tph2 expression) or were a result of the long-term exposure of animals to alcohol.Such analysis between ethanol intake levels in mice, classified as high- or low-ethanol drinkers at different drinking periods, and Immediately after the end of the FST, animals were sacrificed under non-stressful conditions by quick decapitation and the brains were rapidly dissected and chilled in ice-cold saline. Raphe nuclei of the brainstem, hippocampus , and the prefrontal cortex were dissected according to the Mouse Brain Atlas . Brain tTM spectrophotometer ND-1000 . A total of 1 µg of isolated RNA was reverse transcribed using random hexamer primers and Moloney Murine Leukemia Virus Reverse Transcriptase . The samples were then processed for qPCR to assess the expression of Tph2.Total RNA was isolated from the above brain areas using a phenol-based method . Further RNA cleanup and on-column DNase digestion were performed using RNeasy Mini Kit  according to the manufacturer’s instructions. RNA quantification and purity were assessed using NanoDropTph2 (NM_173391.3): SAB-RT_mTPH2_FW: 5′-GTCAATTACCCGTCC CTTCTC-3′; SAB-RT_mTPH2_REV: 5′-TTATTCAAGGCATCACACACTG-3′ (product length: 138 bp); a housekeeping gene, TATA box binding protein : forward 5′-CCCTATCACTCCTGCCACACC-3′; reverse 5′-CGAAGTGCAATGGTCTTT AGGTC-3′ (product length: 117 bp). For each tested brain region, standard curves with serial dilutions of cDNAs were performed to analyze the efficiency of the primers. Samples were run in triplicates in 384-well plate formats. A negative control lacking cDNA was included for each primer pair. All the data were normalized to the mRNA level of Tbp. To show differential expression of Tph2 in the brain of control mice, the abundance of Tph2 mRNA was calculated according to the following equation:Gene expression analysis was performed by qPCR using ABI Prism 7900 HT Fast RT-PCR System  with GoTaq qPCR Master Mix , 22.5 ng of cDNA, and the following primers: Tph2 transcript level, a comparative cycle threshold (CT) method  was uset-test for independent samples was used to study comparisons between means representing changes from the control values . In the case of the occurrence of unequal variances, Student’s t-test with Welch correction was used. In the case of the data in which the data distribution was not normal, a Mann–Whitney U test was used. A two-way repeated-measures analysis of variance (ANOVA) was used to analyze the liquid (or water) intake measurements , ethanol concentration). One-way repeated-measures ANOVA was used to process ethanol consumption and preference data  of the 2-day measurements performed over 28 days of the experiment. Two-way ANOVA was used to analyze the FST data ). One-way ANOVA was used to verify differences in Tph2 transcript level between different brain regions, and between “high ethanol-drinking” and “low ethanol-drinking” mice. ANOVA was followed by a post hoc Newman–Keuls test. All comparisons were made with an experiment type I error rate (α) set at p < 0.05.Data are expressed as the means (± SEM) or median and percentile range. The normality of data distribution was tested by the Shapiro–Wilk normality test. After checking all the assumptions , the appropriate statistical tests were applied. Student’s Tph2−/− and Tph2+/+ mice with continuous access to increasing ethanol concentrations in a two-bottle choice procedure.We first evaluated the ethanol drinking behavior in Tph2−/− mice was similar to Tph2+/+ animals at the beginning of the experiment .The body weight of Tph2 deficiency on ethanol consumption with a two-bottle choice paradigm did not change according to drinking days  = 1.40, p = 0.16, ηp2 = 0.055); p = 0.0049, ηp2 = 0.29) and day  = 6.03, p < 0.001, ηp2 = 0.20) on the level of ethanol consumption during the chronic exposure of Tph2−/− mice to ethanol , Tph2−/−: 139.45 ± 24.80 (g/kg/29 d); t = 2.34, df = 8.55, p = 0.046, η2 = 0.39).There was a significant increase in cumulative (during 29 days) ethanol intake in Tph2 deficiency in mice on preference for ethanol did not change depending on ethanol drinking days  = 0.92, p = 0.53, ηp2 = 0.037); p < 0.001, ηp2 = 0.39), but not genotype  = 3.79, p = 0.063, ηp2 = 0.14), on the preference for ethanol  of ethanol drinking changed according to different ethanol concentrations  = 4.76, p = 0.013, ηp2 = 0.17), genotype  = 9.68, p = 0.0048, ηp2 = 0.29), and ethanol concentration  = 29.94, p < 0.001, ηp2 = 0.56); Tph2+/+ mice was significantly increased compared to the 3% ethanol concentration by 76 and 86%, respectively  = 1.96, p = 0.15, ηp2 = 0.076; p = 0.00014, ηp2 = 0.31), but not genotype  = 2.87, p = 0.10, ηp2 = 0.11), on the preference for ethanol (p = 0.00014) or 6% (p = 0.00024) ethanol  intake according to treatment and ethanol concentration (a significant effect of genotype x treatment (ethanol) x ethanol dose interaction  = 3.81, p = 0.027, ηp2 = 0.11); p < 0.001, ηp2 = 0.55) and ethanol concentration  = 8.71, p = 0.00045, ηp2 = 0.21), but no effect of ethanol treatment  = 1.84, p = 0.19, ηp2 = 0.054) on total liquid consumption , 6% (p = 0.00038) and 10% (p = 0.00028) ethanol. Importantly, in Tph2−/− mice drinking ethanol and water, total liquid intake was enhanced only during the four initial days of exposure to 3% (p = 0.0035) and 6% (p = 0.0071), but not to 10% (p = 0.12), ethanol compared to appropriate Tph2+/+ water groups  x ethanol dose interaction  = 1.28, p = 0.28, ηp2 = 0.038); p = 0.0036, ηp2 = 0.24) and ethanol treatment  = 55.23, p < 0.001, ηp2 = 0.63), but no effect of ethanol concentration  = 0.92, p = 0.40, ηp2 = 0.028), on water consumption  ethanol concentrations. Continuous access to ethanol reduced average water intake in both genotypes.Overall, increasing the ethanol concentrations (6–10%) enhanced ethanol intake in both genotypes, with the highest (10%) concentration inducing a significantly higher ethanol consumption in Tph2 affected the antidepressant effects of ethanol. As measured in the FST, the effect of Tph2 deficiency in mice on the latency to the first immobility episode did not change according to ethanol exposure  = 0.069, p = 0.79, ηp2 = 0.0021); p = 0.14, ηp2 = 0.067) or treatment  = 0.057, p = 0.81, ηp2 = 0.0018) on latency parameter , but no effect of genotype  = 1.91, p = 0.18, ηp2 = 0.056) or treatment  = 0.99, p = 0.33, ηp2 = 0.030), on immobility. However, the post hoc Newman–Keuls showed no significant changes between the genotypes  measurement period revealed a significant effect of genotype x treatment interaction  = 4.17, Tph2 deficiency on swimming behavior in the FST during an initial 0–2 min did not alter according to ethanol treatment  = 1.87, p = 0.18, ηp2 = 0.055); p = 0.0019, ηp2 = 0.26), but not ethanol treatment  = 0.13, p = 0.72, ηp2 = 0.0041), on swimming during the 0–2-min measurement  = 0.12, p = 0.73, ηp2 = 0.0037); p = 0.042, ηp2 = 0.12), but not ethanol treatment  = 0.0001, p = 0.99, ηp2 = 0.000002), on climbing behavior during the 0–2-min measurement  = 1.20, p = 0.28, ηp2 = 0.036; p = 0.00086, ηp2 = 0.3), but not genotype  = 0.14, p = 0.71, ηp2 = 0.0044), on the immobility parameter was detected (Throughout the “test” (during a 2–6-min measurement), the effect of detected C, implyiTph2 deficiency on swimming time did not alter according to ethanol exposure  = 0.13, p = 0.72, ηp2 = 0.004; p = 0.000001, ηp2 = 0.54) and ethanol  = 10.90, p = 0.0024, ηp2 = 0.25) on swimming were found  = 0.82, p = 0.37, ηp2 = 0.025); p = 0.00012, ηp2 = 0.38), but not treatment  = 0.72, p = 0.40, ηp2 = 0.022), on climbing time was reported  = 199.99, p < 0.001, ηp2 = 0.97; Tph2 mRNA in the hippocampus and prefrontal cortex in control animals was 86- and 145-fold, respectively, lower than in the raphe nuclei , hippocampus , or prefrontal cortex  compared to mice drinking water  and preference  compared to “low ethanol-drinking” animals  = 0.58, p = 0.57, ηp2 = 0.058), hippocampus  = 0.065, p = 0.94, ηp2 = 0.0068), and prefrontal cortex  = 0.67, p = 0.52, ηp2 = 0.066) in these groups of mice  and preference  compared to “low ethanol-drinking” animals  = 3.83, p = 0.04, ηp2 = 0.29; Tph2 in the raphe nuclei of mice that exhibited high levels of ethanol intake was significantly increased compared to “low ethanol-drinking” mice  = 0.80, p = 0.46, ηp2 = 0.078; No change in the level of Tph2 expression in the prefrontal cortex of “high ethanol-drinking” and “low ethanol-drinking” mice during the last week of ethanol consumption  = 4.96, p = 0.019, ηp2 = 0.34; Tph2 in the prefrontal cortex of mice that exhibited high levels of ethanol intake was significantly decreased compared to mice drinking water (p = 0.022) or animals that consumed low levels of ethanol (p = 0.049)  D. ImportTph2 mRNA in the raphe nuclei of control animals was almost two orders of magnitude higher than the level of Tph2 mRNA in the hippocampus and prefrontal cortex. Division of mice into “high ethanol-drinking” and “low ethanol-drinking” animals showed that, in high ethanol drinkers, the Tph2 expression level was increased in the raphe nuclei and reduced in the prefrontal cortex compared to low ethanol drinkers.Overall, the amount of Tph2 deficiency in mice induced an increase in ethanol consumption in a two-bottle choice test. Tph2−/− mice that were given continuous access to water only consumed more of that liquid compared to wild-types. However, following ethanol exposure, water consumption decreased to the level of Tph2+/+ animals with access to water and ethanol. Independent of ethanol treatment, Tph2 deficiency led to the reduction in swimming, but facilitated climbing behavior in the FST, without changes in immobility time. 5-HT depletion in Tph2−/− mice did not influence the development of an antidepressant phenotype (reflected by the reduction of immobility time and an increase in swimming) in the FST following 29 days of ethanol consumption. Evaluation of mRNA levels by qPCR in wild-type mice drinking water revealed, besides strong Tph2 expression in the raphe nuclei, very low levels of Tph2 transcripts in the 5-HT projection areas, hippocampus, and prefrontal cortex. Overall, chronic ethanol consumption in wild-type mice did not affect Tph2 expression in these brain regions. However, in mice drinking high levels of ethanol during the last week of ethanol exposure, a higher Tph2 expression in the raphe nuclei and lower level of Tph2 mRNA in the prefrontal cortex were noted in comparison to “low ethanol-drinking” mice.This study revealed that In our study, we employed a 29-day two-bottle choice procedure, in which animals had continuous access to water and increasing ethanol concentrations, a model often used by many research groups to evaluate the effect of genetic variations on drug response ,65,66. ITph2−/− mice is in line with previous studies showing that mice expressing the hypofunctional R439H allele for the Tph2 gene consumed more ethanol–sucrose solution, had increased preference for ethanol, and reduced sensitivity to ethanol compared to the wild-type animals [Tph2 knock-in mice exhibited no change in ethanol intake relative to the control animals, but, only under aversive conditions, motivation for ethanol consumption increased [Tph2 mutant mice, the variability in drinking-related outcomes between these animals and Tph2−/− mice is likely to be related to the extent of the brain 5-HT depletion. Indeed, R439H mutation in Tph2 gene resulted in partial (60–80%) reduction in 5-HT levels in the frontal cortex, amygdala, striatum, and hippocampus [Tph2 gene in mice used in the current study evoked severe (>98%) 5-HT depletion in all the tested brain regions, including those analyzed in R439H Tph2 mice [The finding of enhanced ethanol drinking in  animals . Howeverncreased . While tncreased ; or the ncreased ) may accpocampus ,47,48,49ph2 mice . Hence, Tph2−/− mice displayed a decrease in swimming, confirming previous data showing that swimming is a 5-HT-related parameter [Tph2−/− mice may suggest that these animals developed a modest depression-like state. These results partially support our previous study, in which Tph2−/− mice showed a reduced latency to immobility and increased immobility time in the FST, although, in this case, the swimming and climbing time were not assessed independently [Tph2−/− mice in the FST are heterogeneous: either a reduced latency to immobility or a mild antidepressant phenotype were reported [Tph2−/− mice in the FST may depend on specific protocols used to evaluate depression-like behavior , age of the animals, or genetic background on which the knockouts were created. In addition, the reduction in fat content or compensatory mechanisms resulting from a life-long depletion of 5-HT signaling in the brain in Tph2−/− mice may account for different outcomes in the FST .Tph2−/− mice. Interestingly, similar to Tph2−/− mice, in the Sardinian alcohol-preferring rat line, an animal model used to study alcohol dependence, an enhancement of ethanol drinking and reduction in the depression-like state were also accompanied by deficits in 5-HT function in the brain [Tph2−/− mice may serve as a model for the study of ethanol dependence in the future. Importantly, the increased ethanol consumption observed in mice depleted in brain 5-HT corresponds well to the characteristics of a subset of alcoholics with early-onset type II alcoholism (Cloninger type-2-like) that has also been associated with 5-HT hypofunction  allele for 5-HTT gene; [Tph2−/− mice will be instrumental in discovering novel non-serotoninergic medications for the treatment of type II alcoholics.Importantly, here we show for the first time that chronic ethanol consumption in 5-HT-depleted mice reduced immobility time and enhanced motor behavior, indicating that the 5-HT neurotransmission per se is not essential for the development of the antidepressant effect of ethanol. Additionally, it can be assumed that enhanced ethanol consumption in mice with low 5-HT function facilitated the antidepressant properties of ethanol and compensated for the negative state observed in drug-naive he brain ,76. Thusr review ,62, haveTph2−/− mice, including enhanced ethanol consumption could be due to the adaptation mechanisms compensating for the continuous depletion in 5-HT, rather than the lack of 5-HT per se. Previously, we detected no marked differences in gene expression in the whole brain of Tph2−/− mice compared to controls using Affymetrix [1A and 5-HT1B receptors in several brain regions, including the septum and frontal cortex of mice with constitutive inactivation of Tph2 [Tph2−/− mice, further functional and gene expression analyses of defined brain structures need to be performed.Behavioral phenotype in fymetrix . Importa of Tph2 . To confTph2−/− mice  10% ethanol consumption [7 receptor in the midbrain raphe nuclei of animals after chronic ethanol intake. Importantly, no change in 5-HT content and 5-HT1A receptor gene expression has been reported in the midbrain, frontal cortex, hippocampus, hypothalamus, and amygdala of mice after chronic ethanol [1A autoreceptor sensitivity in the dorsal raphe nucleus of mice, suggesting a reduction in central 5-HT tone after chronic ethanol [The effects of chronic voluntary ethanol consumption on neuroadaptations within the 5-HT neurotransmission, particularly in the raphe nuclei, have been studied to a limited extent. Consistent with our findings, a recent study has demonstrated no change in sumption . Additiosumption  have sho ethanol . In cont ethanol . These dTph2 mRNA in the whole brain resulting from the absence of one copy of the Tph2 gene in heterozygous Tph2+/− mice has led to about a 13% decrease in 5-HT level in the whole brain [Tph2 mutant mice revealed about a 22% reduction in 5-HT in the rostral raphe nuclei, but not in the hippocampus, frontal cortex, or thalamus [Tph2 gene [Tph2 mRNA level (by ca. 20%) and the number of TPH2-immunoreactive neurons (by 28%) in the dorsal raphe nucleus, and, consequently, a reduction (by 15%) in 5-HT content in the midbrain and cortex compared to high-ethanol-preferring C57B/6J mice [Tph2 [Tph2 transcript levels in the raphe nuclei observed in the present study in “low ethanol-drinking” mice may potentially coincide with decreased central 5-HT levels and represent an ethanol-resilient phenotype. Further studies are necessary to verify whether reduction in Tph2 gene expression in mice consuming low levels of ethanol has any impact on TPH2 activity and/or 5-HT level.Previously, we have reported that a 50% reduction in le brain . Anotherthalamus . Importathalamus ,68, and thalamus ,54, harbph2 gene . Besides/6J mice ,55 that ce [Tph2 . Based oTph2 gene expression in the mouse raphe nuclei [Tph2 gene expression is sensitive to glucocorticoid signaling. Whether the high and low Tph2 gene expression observed in mice in the present study is related to the fluctuation in the glucocorticoid level in the brain and plasma needs to be further assessed.Rats consuming high levels of ethanol exhibited blunted hypothalamic–pituitary–adrenocortical axis responses compared to low-ethanol-consuming animals . On the e nuclei , indicatTph2 gene expression in the raphe nuclei, and possibly prefrontal cortex, may be specifically involved in the development of ethanol-drinking behavior in mice.In summary, we propose that the life-long 5-HT depletion in the brain predisposes to increased ethanol consumption. However, it is not clear whether the effect is due to the lack of 5-HT per se or, rather, a compensatory response to the long-term deficiency in 5-HT synthesis. Moreover, we conclude that the absence of central 5-HT does not interfere with the antidepressant properties of ethanol. Our results indicate that regulation of n = 17). Nevertheless, such conditions for selecting the “ethanol drinking phenotype” were sufficient to detect differences in the Tph2 transcript levels, suggesting specificity of the observed changes. Another limitation is that we did not measure the 5-HT level within these animals.Limitations of the study include the selection criteria of mice with high and low ethanol intake levels, which were due to the small group size (Tph2 expression in development of ethanol drinking behavior. Modulation of TPH2 expression and/or activity by appropriate tools in the future will verify whether such a strategy will induce resilience to ethanol.Further more detailed molecular analysis of a larger group size and rodent lines selectively bred for high and low ethanol consumption is urgently needed to confirm the present findings regarding the importance of


We investigated the effect of Korean mistletoe extract (KME) on inflammatory markers after high-intensity exercise by 20 university male rowers (KME group vs. CON group) consuming 110 mL KME/dose (2 times a day over 8 weeks). Blood samples were collected for measurement of serum cytokine levels at baseline, immediately after exercise, and following 30 minutes of recovery. Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and C-reactive protein (CRP) were used as markers for inflammation. After supplementation, IL-6 and TNF-α levels were significantly lowered in the KME group than in the CON group at baseline, immediately after exercise, and following 30 minutes of recovery. KME can reduce high-strength exercise-induced increases in the levels of serum inflammatory cytokines in active individuals and improve anti-inflammatory functions.Korean  Exercise promotes health, prevents disease, increases life expectancy, and is considered to some to be a medicine. However, elite athletes are a paradox that should be emphasized . For athβ), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) and amplify different aspects of inflammation through cytokine action [α is the major active cytokine during an acute inflammatory response and sends a stimulatory signal to leukocytes at sites of inflammation to eliminate microorganisms and decrease inflammation [κB), in combination with the TNF receptor 1, exerts a negative effect on induction of intracellular gene expression in response to oxidative stress [Strenuous exercise may serve as a physical stressor and can thus create a state similar to an inflammatory response . Inflamme action . These fe action , 6. TNF-ammation . Activate stress . IL-6 ise stress . IL-6 sie stress , 11. Infe stress .Nutrition planning may be useful to counteract inflammatory responses after strenuous exercise, and dietary supplements are able to inhibit inflammatory response and specific immune functions . Thus, mMistletoe  is an angiosperm that survives by absorbing moisture, minerals, and organic materials from several host trees. As it retains photosynthetic function, this makes it a hemiparasite that is not entirely dependent on its host. Mistletoe is chemically composed of viscotoxins, lectins, flavonoids, phenolic acids, terpenoids, sterols, phenylpropanoids, and alkaloids [Mistletoe also promotes antioxidant functions [mistletoe is used as a supplement to ameliorate symptoms of the above illnesses.lkaloids . It has lkaloids  and antilkaloids  but alsolkaloids  and trealkaloids . Mistletunctions , increasunctions , and hasunctions . As suchmistletoe extract supplementation on levels of inflammatory markers in university-aged male rowing athletes during an 8-week training period. The performance measure for high-intensity aerobic and anaerobic sport involved rowing a 2,000 m distance on an ergometer.Excessive long-term training and intense exertion during exercise can cause expression of inflammatory cytokines. Various measures have been explored to minimize this response, and dietary supplements with anti-inflammatory and antioxidant effects may help athletes to recover from repetitive intense exercise and thus prevent reductions in performance. To address this, we conducted this study to determine the effects of Study subjects included 20 university-aged male rowing athletes  at H University, Busan, Republic of Korea. Subjects individually had more than 5 years of rowing experience and were national-level athletes who won at national competitions. This study was conducted in accordance with protocols approved by the institutional review board at Pusan National University (PNU IRB/2016_24_HR). The subjects were fully informed about the study and understood its objective and intent prior to participation. Only those who gave voluntary written consent were included in this study. All subjects were athletes without a prior record of musculoskeletal or other injuries and had not been treated with medication in the 6 months prior to the study. The subjects were prohibited from taking other nutritional supplements or drugs for a month before commencement of the study and for its duration.mistletoe extract (KME) supplement (n = 10) and the control (CON) group (n = 10). All subjects undertook preseason training for 8 weeks (January to February), and the KME group were administered mistletoe extract during this period. All of the following parameters were measured using the same protocol and under the same conditions: 2,000 m rowing ergometer performance was assessed and serum IL-6, TNF-α, and CRP levels were measured before (0 week) and after (8 weeks) the training period. They were measured at baseline, immediately after the 2,000 m rowing exercise, and following 30 min of recovery. We set the recovery time of inflammation markers through mistletoe extract intake to 30 minutes based on previous recovery studies after 2,000 m rowing ergometer performance of rowers [mistletoe extract (110 mL) and had 30 min of recovery after the rowing test. The study design is shown in Subjects were randomly divided into two groups: the experimental group that was administered the Korean f rowers , 27. Themistletoe extract  daily during the 8-week training period, divided into two doses. A 110 mL dose was administered once every morning and again 1 h after the evening meal [mistletoe extract before and immediately following the 2,000 m rowing exercise, prior to blood collection. Mistletoe extract was prepared according to a method validated in previous studies on natural plants [Viscum album var. coloratum) grown at an altitude of 800 m or higher in Uljin (Mt. Tonggo and Mt. Baekam) in Gyeongsangbuk-do, Republic of Korea, was harvested and dried naturally for 1 month. After that, it was cut into pieces of 5 cm each and roasted, 1,650 g of mistletoe and 22,000 mL of water were added to the extractor, and the pressure was 0.15 kgf/cm2 at 110°C, and extraction was performed for 3 hours. After extraction, it was pasteurized for 40 minutes. The mistletoe extract was provided by sealing and refrigeration in a plastic pack of 110 mL .The KME group was administered 220 mL of ing meal . The KMEl plants , 29. Themistletoe extract were water (97%), crude ash (0.2%), crude fat (0.5%), crude protein (0.3%), carbohydrate (2.0%), sodium (3.9 mg/100 g), and flavonoids (32.0 mg/100 g).Analysis by the Korea Advanced Food Research Institute (2016) found that the main chemical components of An indoor rowing ergometer  that is typically used for physical or technical training of rowing athletes was used to assess exercise performance. Performance in a 2,000 m rowing test was measured. This was a physically demanding task that required maximum effort from the subjects. During the rowing performance test, heart rate was maintained at ≥80% heart rate reserve, and changes in heart rate were measured every 5 s using a wireless heart rate meter . Prior to the test, all subjects freely performed a warm-up on the rowing ergometer for 5 min.α using CymaxTM Human IL-6 and TNF-α ELISA Kits , respectively, and OD at 450 nm was measured using GENios . An immunoturbidimetric assay with CRPL3  and a Modular Analytics  analyzer was used to measure levels of CRP.Prior to blood collection, subjects were required to fast from 8:00 pm of the previous day. To measure cytokine levels at baseline, immediately after the 2,000 m rowing exercise, and following 30 min of recovery, 10 mL of blood was drawn from the forearm vein using a vacuum collection tube. The blood samples obtained were then centrifuged  for 10 min at 3,000 rpm. Serum was separated from blood cells, transferred to a microfuge tube, and was then stored at −80°C until analysis. Enzyme-linked immunosorbent assay (ELISA) was performed to determine serum concentration of IL-6 and TNF-p < 0.05, and 60% power [M) and standard deviation (SD) of the measured parameters were calculated using the Statistical Package for the Social Sciences (SPSS) version 23.0 for Windows . To analyze interaction of differences between the groups  and between periods , a two-way repeated measures analysis of variance analysis was performed. To assess changes within each group before and after supplementation, a paired t-test was performed. An independent t-test was also used to analyze the delta (Δ) change. The level of statistical significance was set at p < 0.05.Sample size was determined using a G-power version 3.1 Windows program , based on a 0.25-point effect size (default), an alpha level of 0% power . The meaThe physical characteristics of study subjects are summarized in t = −1.032, p = 0.329; CON group before: 174.39 ± 13.95 bpm, after: 178.91 ± 8.52 bpm, t = −1.514, p = 0.164). There was no significant difference in performance for the 2,000 m rowing test between the KME group (418.25 ± 14.03 s) and the CON group (420.98 ± 16.64 s) prior to the experiment . The 2,000 m rowing test times significantly decreased in both the KME group  and the CON group  at after 8 weeks. Total rowing stroke count also did not differ significantly for the 2,000 m rowing test in both groups prior to supplementation. The KME group had a rowing stroke count of 213.60 ± 11.5, and the CON group had a count of 222.80 ± 14.40 . Following supplementation, total rowing stroke count was significantly different between the groups. The KME group (207.10 ± 14.11) had a lower rowing stroke count than the CON group (221.60 ± 7.82), as shown in t = −2.843, p < 0.05).During the 2,000 m rowing ergometer trial test, there was no difference in heart rate between groups and periods  after the 8 weeks was different at baseline . Immediately following exercise, the concentration of serum IL-6 in the KME group was 9.95 ± 1.79 pg/mL before and 10.22 ± 1.49 pg/mL after supplementation. In the CON group, the concentration of serum IL-6 increased from 11.07 ± 2.38 pg/mL to 13.99 ± 3.21 pg/mL after the 8 weeks. An interaction effect  and the main effect of the groups  were observed immediately following exercise. Serum IL-6 significantly increased immediately after exercise in the CON group . There were significant differences between the groups after the 8 weeks . The concentration of serum IL-6 in the KME group after 30 min of recovery was 5.86 ± 1.98 pg/mL before supplementation and 5.45 ± 1.49 pg/mL after supplementation. In the CON group, the concentration of serum IL-6 was 7.59 ± 1.43 pg/mL before and 8.78 ± 2.70 pg/mL after the 8 weeks. The main effect of the groups  and comparison of differences between the two groups revealed that they were significantly different before  and after the 8-week period , as seen in After the 8 weeks, baseline levels of serum IL-6 decreased from 4.13 ± 1.16 pg/mL to 3.57 ± 0.55 pg/mL in the KME group and increased from 4.62 ± 1.41 pg/mL to 5.46 ± 0.54 pg/mL in the CON group. The main effect of the two groups . Immediately following exercise, the Δ-IL-6 was 0.27 ± 1.91 pg/mL in the KME group and 2.92 ± 3.79 pg/mL in the CON group . After 30 min of recovery, the Δ-IL-6 was −0.42 ± 0.88 pg/mL in the KME group and 1.19 ± 2.22 pg/mL in the CON group. This was again significantly different .Differences (Δ) between serum levels of IL-6 before and after the 8 weeks are represented in F = 8.678, p < 0.05) was observed for baseline levels of serum TNF-α; there was a significant main effect of group . Concentration of serum TNF-α decreased from 54.05 ± 16.75 pg/mL to 41.51 ± 3.68 pg/mL after supplementation in the KME group. In contrast, in the CON group, concentration of serum TNF-α was 56.57 ± 13.90 pg/mL prior to and 64.89 ± 10.06 after the 8 weeks. This demonstrated that the KME group had significantly lower TNF-α levels . Significant differences were observed between the two groups after the 8 weeks . Immediately following exercise, concentration of serum TNF-α in the KME group decreased from 140.04 ± 34.17 pg/mL to 93.27 ± 5.10 pg/mL after supplementation. In the CON group, concentration of serum TNF-α was 134.28 ± 23.27 pg/mL before and 138.46 ± 25.59 pg/mL after the 8 weeks. There was an interaction effect , a main effect of group , and main effect of time . The decrease observed in the KME group was statistically significant . After the 8-week period, the difference between the two groups was also statistically significant . After 30 min of recovery, serum concentration of TNF-α in the KME group was 65.31 ± 32.71 pg/mL prior to supplementation and 66.14 ± 19.05 pg/mL after supplementation. In the CON group, serum concentration of TNF-α was 91.68 ± 20.32 pg/mL prior to and 94.22 ± 20.64 pg/mL after the 8 weeks. There was no statistically significant interaction. We observed significant differences between the two groups prior to  and after supplementation , as shown in An interaction effect . Immediately following exercise, Δ-TNF-α was −46.77 ± 36.14 in the KME group and 4.18 ± 33.38 pg/mL in the CON group . After 30 min of recovery, Δ-TNF-α was 0.83 ± 21.26 pg/mL in the KME group and 2.54 ± 18.43 in the CON group .Differences (Δ) in levels of TNF-Baseline concentration of serum CRP in the KME group was 0.21 ± 0.16 mg/L prior to and 0.22 ± 0.15 mg/L after supplementation. In the CON group, it was 0.33 ± 0.20 mg/L before and 0.35 ± 0.31 mg/L after the 8-week period. Immediately following exercise, concentration of serum CRP in the KME group was 0.27 ± 0.23 mg/L prior to and 0.24 ± 0.15 mg/L after supplementation. In the CON group, it was 0.36 ± 0.24 mg/L before and 0.32 ± 0.28 after the 8-week period. After 30 min of recovery, the concentration of serum CRP in the KME group was 0.21 ± 0.17 mg/L prior to and 0.22 ± 0.19 mg/L after supplementation. In the CON group, the concentration was 0.30 ± 0.18 mg/L before and 0.34 ± 0.26 mg/L after the 8-week period. No interaction effect was observed, and there were no significant differences between the two groups .t = −0.107, p = 0.916). Immediately following exercise, Δ-CRP was −0.03 ± 30.24 mg/L in the KME group and −0.04 ± 0.13 mg/L in the CON group . After 30 min of recovery, Δ-CRP was 0.01 ± 0.22 mg/L in the KME group and 0.04 ± 0.21 mg/L in the CON group .Differences (Δ) in concentration of serum CRP before and after the 8-week period are represented in mistletoe supplementation on the release of proinflammatory cytokines induced by strenuous exercise. Immediately following a bout of strenuous exercise , there was a rapid increase in serum inflammatory cytokines such as IL-6 and TNF-α. Supplementation with mistletoe extract after exercise for the purposes of quick recovery dampened the increase in IL-6 and TNF-α from baseline levels. Daily administration of the mistletoe extract supplement for 8 weeks prevented possible increases in the levels of IL-6 and TNF-α induced by long training periods. Immediately following a break after strenuous exercise, levels of serum IL-6 and TNF-α decreased. Our data suggest that mistletoe extract decreases levels of inflammation in elite athletes. This is the first study to investigate the effects of such an antioxidant treatment on exercise-induced oxidative stress.This study demonstrated the effect of mistletoe extract. Our results demonstrated that the time taken to row 2,000 m on the rowing ergometer significantly decreased in each group. As both groups had undertaken 8 weeks of training, the time for the trial decreased compared to the baseline level in both groups. In particular, the KME group reduced the record of 2,000 m rowing performance. Furthermore, the total number of strokes was significantly lower in the KME group compared to the CON group.Training protocols include long training times and high-intensity training for the purposes of reinforcing skills and improving performance. In this study, the subjects performed high-intensity training for 8 weeks. During the off-season period, performance of rowing athletes in a 2,000 m rowing ergometer trial was compared pre- and postsupplementation with Mistletoe extract stimulates the release of peroxisome proliferator-activated receptor gamma coactivator 1-alpha and sirtuin 1 in myoblasts, resulting in improved endurance by upregulating mRNA expression of genes associated with mitochondrial creation and function [mistletoe extract supplement enhancement of endurance reported that administration of mistletoe extract for 12 weeks extended time to exhaustion on the treadmill by 2.5 times and swimming time by 212% and reduced postexercise lactate levels in the experimental group compared to the control group [mistletoe extract during treadmill exercise also increased distance and duration of exercise till exhaustion compared to control [mistletoe extract may be due to increased capacity in the electron transport chain system through increased oxygen consumption by mitochondria [function . It has function . Anotherol group . Adminis control . This suchondria . Alternachondria .via binding to the cell surface receptor, (2) revitalizing inflammatory channels, (3) release of inflammatory molecules, and (4) recruitment of inflammatory cells [α and IL-1β and is therefore classified as both a pro- and anti-inflammatory cytokine [Several mechanisms are involved in inflammatory responses: (1) detection of harmful stimuli ry cells . Cytokinry cells , 34. An ry cells . IL-6 plcytokine . Howevercytokine , and thicytokine . Inflammcytokine .mistletoe has higher inhibition of lipid peroxidation than an equivalent concentration (10 mg) of vitamin C and demonstrates high antioxidative properties [mistletoe plays a role as a natural antioxidant as it contains polyphenols, has a superior ability to donate electrons, and increases activity of superoxide dismutase [mistletoe, shows potential in preventing and treating diseases associated with the generation of reactive oxygen species [Mistletoe's anti-inflammatory effects also include inhibition of cytokines induced by prostaglandin E2 biosynthesis through selective inhibition of cyclooxygenase-2 (COX-2) [mistletoe may exert anti-inflammatory effects via activating transcription factor 3 and mitogen-activated protein kinase (MAPK) and inhibiting NF-κB signal transduction, decreasing inflammatory effectors such as inducible nitric oxide synthases (iNOS), COX-2, TNF-α, and interleukin-1 beta (IL-1β) [Korean operties . Oak treismutase . In part species . Mistlet (COX-2) . It has  (IL-1β) .α was elevated after the 2,000 m rowing trial and induced IL-6 expression. The KME group had a lower concentration of IL-6 and TNF-α compared to the CON group after 30 min of recovery, both before and after postexercise supplementation. This indicated that supplementation with mistletoe extract was effective in treating acute exercise-induced inflammation when coupled with 30 min of recovery. The KME group also had a lower concentration of serum inflammatory cytokines compared to the CON group at baseline, immediately after exercise, and after 30 min of recovery postsupplementation. This finding implies that supplementation with mistletoe ameliorates inflammatory responses induced by long-term intensive training.Serum TNF-α. Because CRP increases dramatically during the inflammatory process and remains elevated for long periods of time, it is thought to be considered in future studies of chronic inflammation levels in athletes.However, despite the rapid increase in serum inflammatory cytokines after the 2,000 m rowing trial, there was no significant change in the level of CRP and no interaction effect. Acute phase proteins including CRP also increased after excessive exercise, but it was confirmed that the increase was delayed compared to IL-6 and TNF-mistletoe extract had anti-inflammatory and positive effects on performance during long-term high-intensity training and extreme exertion . Thus, we propose that mistletoe extract may be a valuable anti-inflammatory supplement that protects against exercise-induced oxidative damage in athletes who have increased inflammation due to high-intensity training.Supplementation with