Weissella cibaria belongs to the Lactobacillaceae family and has been isolated from traditional fermented foods and saliva of children with good oral health. Previous investigations have shown that W. cibaria CMU  is expected to be safe based on results of in silico and in vitro analyses. However, there is a lack of studies assessing its safety in vivo. A toxicological safety evaluation of W. cibaria CMU was performed using an acute oral safety study in rats, a 14-day oral range finding study, a subsequent 13-week oral toxicity study in rats and a genetic toxicity battery . The results of the studies in rats showed that the acute lethal dose of W. cibaria CMU is > 5000 mg/kg body weight (bw)/day (1.8 × 109 CFU/kg bw/day) and the 14-day or 13-week no observed adverse effect level (NOAEL) is 5000 mg/kg bw/day (1.8 × 109 CFU/kg bw/day), the highest dose administered. W. cibaria CMU was non-mutagenic in the bacterial reverse mutation test and non-clastogenic or aneugenic in vitro and in vivo. In conclusion, the toxicological studies performed demonstrated W. cibaria CMU to be a safe strain to consume. This study is the first study examining the potential of a W. cibaria strain to cause genetic toxicity and subchronic toxicity in rats according to the Organization for Economic Cooperation and Development guidelines. Weissella genus includes a number of heterofermentative Leuconostoc-like lactic acid bacteria that are generally isolated from fermented foods [Weissella cibaria is described as a short, rod-shaped, Gram-positive, non-spore-forming, nonmotile, heterofermentative, and catalase-negative lactic acid bacterium [W. cibaria CMU  has been isolated from saliva samples of children who had little supragingival plaque and no oral diseases including dental caries [The ed foods , 2. Weisacterium . W. cibal caries .W. cibaria based on whole genome sequence analysis and phylogenetic homology [W. cibaria is registered as a safe raw material by the Korea Food and Drug Administration (KFDA) and is found in several commercialized oral care probiotics in Korea [W. cibaria CMU exhibits several properties for maintenance of oral health, including inhibition of biofilm formation by Streptococcus mutans, antibacterial activity against cariogens (S. mutans and Streptococcus sobrinus) and periodontopathogens  and inhibition of hydrogen sulfide and methyl mercaptan production by F. nucleatum and P. gingivalis [Strain CMU has been identified as homology –6. W. ciin Korea , 8. W. cngivalis .W. cibaria CMU group (0.8 × 108 colony forming units (CFU)/day) was compared with a placebo control group in healthy adults to compare the effects on halitosis and periodontal health and examine tolerability. The results showed no adverse effects of the strain at the level administrated. Available commercialized products provide a daily intake of W. cibaria CMU of 1 × 109 to 2.4 × 1010 CFU per day. Although W. cibaria is on the KFDA Food Ingredient List and is part of the International Dairy Federation List [In studies by Lee et al.  and Kangion List , but is W. cibaria CMU has been examined by phenotypic and genotypic analyses designed to assess the potential for antibiotic resistance, antibiotic resistance gene (ARGs) transferability, virulence, hemolysis, mucin degradation, toxic metabolite production, and platelet aggregation [W. cibaria CMU strain, both chromosomal and plasmid, was free of ARGs and revealed no ARGs transferability. In addition, the genomic sequence of this strain did not contain any virulence gene, β-hemolysis, mucin degradation, platelet aggregation, and toxic metabolites including d-lactic acid, bile salt deconjugation, ammonia production, β-glucuronidase activity, indole production, nitroreductase activity, phenylalanine degradation, and gelatin liquefaction. The safety of this strain was confirmed by negative reactions. However, animal toxicity or genotoxicity studies on W. cibaria strain have not been reported yet.The safety of regation . W. cibaW. cibaria CMU, by investigating the potential of the strain to cause toxicity to rats after acute or 14 days or 13 weeks of daily exposure. In addition, genotoxicity assessments including bacterial reverse mutations, chromosome aberrations, and micronuclei formation were also conducted.The purpose of the studies performed for the current evaluation is to add to the current database of safety information on W. cibaria CMU  in a vehicle of distilled water . The W. cibaria preparation contained 3.6 × 108 CFU/g. Preparations used for each study were prepared daily. For the in vivo studies, samples were collected from the dosage formulations to verify homogeneity and dose concentration on the day of administration.The test substance was Cell viability was determined using an EZ-Cytox cell viability assay kit . Briefly, EZ-Cytox kit reagent was added into each well for 2 h under standard culture conditions, after which 96-well plates were gently shaken thoroughly for 5 min on a rocker at room temperature. The absorbance of the treated and untreated samples at 450 nm was then measured on a multi-well microplate reader . DMEM medium supplemented with the same volume of kit reagent on an empty well was used as a blank. Cell viability was represented by percentage values compared to a control and then used to calculate the number of viable cells.All studies conducted in animals complied with the guidelines of animal ethics and were approved by the IACUC of Korea Conformity Laboratories (KCL). Approval numbers for each study are as follows: In vivo micronucleus test (IA20-02198); single oral dose toxicity study (IA20-01118); thirteen-week repeated oral Dose toxicity study (IA20-01554). All studies were performed at KCL in Incheon, Korea.The 14-day dose range finding and 13-week studies were performed according to OECD guidelines 407 and 408, respectively , 13. OECSpecific Pathogen Free (SPF) Sprague–Dawley (SD) rats from Orient Bio Inc.  were used in this study. Animals were acclimated for 5–8 days (depending on the study) before treatment and were approximately 8 weeks of age at the start of the acute study, and 6 weeks for the 14-day and 13-week studies. Animals were housed in wire mesh cages in groups no larger than three for the acute and 14-day studies and five for the 13-week study. The animal housing room was maintained on a 12-h light/dark cycle, with temperature and relative humidity of 24 ± 0.3 °C and 46.3 ± 1.5% in the acute study, 23.3 ± 0.5 °C and 51.1 ± 0.5% in the 14-day study and 22.7 ± 0.4 °C and 54.9 ± 1.5% in the 13-week study. Animals were supplied diet  and filtered water ad libitum. Animals were selected for use in the study only if their body weight fell within 20% of the mean. For the acute study, animals were fasted overnight prior to dosing and food was returned three to four hours after dosing; water was provided ad libitum during fasting. For the 14-day and 13-week studies, rats were randomly allocated into treatment groups to ensure adequate distribution of body weights.8, 9.0 × 108, and 1.8 × 109 CFU/kg, respectively. The test material was prepared just prior to dosing by dissolving the test material in distilled water at the appropriate concentration. Animals were dosed by gavage at a volume of 15 mL/kg bw. Following dosing, general clinical signs in all animals were observed more than once a day for 14 days before necropsy. On the day of administration, clinical signs were observed in all animals at 30 min and every hour until 6 h after administration. Body weights were taken at acquisition, grouping, immediately prior to dosing, and on days 1, 4, 7, and 14 after dosing. On day 14, animals were anesthetized with carbon dioxide (CO2) and humanely euthanized. Complete postmortem examinations were performed on all organs.Animals were divided into four groups (n = 5/sex/group), one of which was a vehicle control group (distilled water), and three test groups receiving the test material at 1250, 2500, or 5000 mg/kg body weight (bw). The viable cell counts in the low, medium, and high dose groups were 4.5 × 108 CFU/kg bw/day (Group 2), 9.0 × 108 CFU/kg bw/day (Group 3) or 1.8 × 109 CFU/kg bw/day (Group 4) for 14 days. Individual doses were calculated based on the most recent weekly body weights and were adjusted each week to maintain the targeted dose level for all rats . All doses were administered volumetrically at 15 mL/kg by oral gavage.In total, 40 rats (n = 20/sex) were assigned to four treatment groups (n = 5/sex/group)—vehicle control (Group 1) or the following amounts of the test substance: 4.5 × 10General clinical signs of all animals were observed and recorded daily. Body weights were recorded at acquisition, grouping, before administration, once a week during the study, and before necropsy. Food intake was measured right before the first administration and once a week during the study. Food consumption per animal was measured by the average intake (g/rat/day) of individual animals in a cage. Water consumption was measured in a similar manner to food consumption. The night before termination, the animals were subjected to an overnight fast in which water was still provided ad libitum. At study termination, animals were euthanized under isoflurane anesthesia and blood samples were drawn from abdominal aorta. Whole blood collected on EDTA-K2 was used for hematological analyses (white blood cell count (WBC), neutrophils (NEU), eosinophils (EOS), basophils (BAS), lymphocytes (LYM), monocytes (MON), large unstained cells (LUC), percent of neutrophils (NEP), percent of eosinophils (EOP), percent of basophils (BAP), percent of lymphocytes (LYP), percent of monocytes (MOP), percent of large unstained cells (LUP), red blood cell count (RBC), hemoglobin concentration (HGB), red cell distribution width (RDW), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocytes (RET), platelets (PLT), and mean platelet volume (MPV)). Serum was obtained from whole blood collected on 3.2% sodium citrate and analyzed for clinical chemistry (aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), total bilirubin (BIL), blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), glucose (GLU), total cholesterol (CHO), triglycerides (TG), total protein (PRO), albumin (ALB), ALB/GLOB ratio, lactate dehydrogenase (LDH), creatine phosphokinase (CPK), calcium (Ca), inorganic phosphorus (IP), magnesium (Mg), sodium (Na), potassium (K), and chloride (Cl)).At necropsy, postmortem examinations were completed on all organs. Weights of the following organs were obtained: testes, prostate, ovaries, uterus, spleen, liver, thymus, adrenals, kidneys, heart, lungs, brain, and pituitary gland. All collected organs were preserved in 10% phosphate-buffered formalin except for testes, which were fixed in Böuin solution. Histopathological examination was not performed.8 CFU/kg bw/day (Group 2), 9.0 × 108 CFU/kg bw/day (Group 3) or 1.8 × 109 CFU/kg bw/day (Group 4). Dose levels were adjusted based on body weights measured right before each administration, dosing volume was 15 mL/kg bw/day, and each dose was given by oral gavage for 13 weeks.Eighty animals (n = 40/sex) were assigned to four treatment groups (n = 10/sex/group)—vehicle control (Group 1), and three treatment groups receiving the following amounts of test substance: 4.5 × 10. On the day of termination, all animals were anesthetized with isoflurane and blood samples were drawn from the abdominal aorta. Blood samples collected on EDTA-K2 were analyzed for the same hematology parameters as described for the 14-day dose range finding study, but methemoglobin (metHGB) was added. Analyses for coagulation (prothrombin time (PT) and active partial thromboplastin time (APTT)) were added. Serum clinical chemistry was also the same as the 14-day study, with the addition of bile acid (BA), high density lipoprotein (HDL), low density lipoprotein (LDL), cholinesterase (ChE), thyroxine (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH).General clinical signs were observed and recorded daily for all animals. Individual body weights were recorded at acquisition, grouping, right before administration, once a week during exposure, and right before necropsy. Food consumption was measured right before first administration and once a week during the rest of the study. Food intake per day and food intake per animal were calculated the same way as for the 14-day dose range finding study. Ophthalmic examinations were conducted on all animals before first administration and during the last week of the study. During the last week of test substance administration, urine was collected from five animals per sex per group using metabolic cages. Urine samples were analyzed for the following parameters: color, glucose, bilirubin, ketone bodies, specific gravity, occult blood, pH, protein, urobilinogen, nitrite, leukocytes, erythrocytes, epithelial cells, and casts. Animals were fasted overnight before termination, but water was provided ad libitumScheduled necropsy was completed on all surviving animals and complete postmortem examinations were performed on all organs. Vaginal smear examinations were completed in all female rats prior to necropsy to determine estrus cycle by the shape of epithelial cells using Giemsa staining . Weights of the 13 organs weighed in the 14-day study, plus the epididymides and thyroid, were taken at necropsy. Both sides of bilateral organs were weighed and preserved, and the thyroid was fixed prior to weighing. Organs and tissues from all animals were preserved in 10% neutral phosphate-buffered formalin . Preserved organs included the brain, pituitary, heart, lungs, liver, kidney, urinary bladder, mesenteric lymph node, thymus, spleen, adrenal gland, esophagus, aorta, spinal cord, sciatic nerve, skeletal muscle, skin, mammary gland, eye, stomach, colon, rectum, femur, sternum and bone marrow, trachea, tongue, prostate gland, testes, epididymis, seminal vesicle, pancreas, salivary gland, submandibular lymph node, thyroid and parathyroid glands, duodenum, jejunum, ileum, cecum, ovary, uterus, cervix, and vagina. Specimens from the vehicle control and high dose group were prepared and examined for all fixed organs and tissues.Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA. Experiments were conducted in the absence and presence of an S9 metabolizing system, using the preincubation method. The test product was formulated as a solution in distilled water to provide dose levels of up to 5000 μg/plate. The positive control chemicals sodium azide (NaN3), 9-aminoacridine (9-AA), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2), 2-aminoanthracene (2-AA), benzo(a)pyrene (BP), and 4-nitroquinoline 1-oxide (4-NQO) and their vehicle dimethyl sulfoxide (DMSO) were sourced from Sigma, with the exception of AF-2 and DMSO, which came from Wako  and Junsei  (respectively). The bacterial strains and the S9 tissue fraction  were sourced from Molecular Toxicology, Inc .Bacterial strains utilized in all experiments were S. typhimurium TA98 and TA100, sodium azide for TA1535, 9-AA for TA1537 and 4-NQO for E. coli WP2uvrA. The positive control for all bacterial strains in the presence of S9 mix was 2-AA, except for TA98, which used BP. Distilled water served as the negative control. Three replicate plates were prepared for each test condition. After exposure for 20 min at 37 °C, and incubation for 48 h at 37 °C, the number of colonies per plate was counted manually with an electronic register . The test result was recorded as experimental value, average value, and standard deviation for the number of revertant colonies per plate. The result was deemed “positive” if there was a dose-dependent increase and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation.For the experiments, the following substances were mixed in a test tube and poured over the surface of a minimal glucose agar plate : (1) the positive or negative control solutions or test formulations (0.05 mL each); (2) 0.5 mL of 0.1 M phosphate buffered saline or 0.5 mL of the S9 mix (for metabolic activation); (3) 0.1 mL of bacterial suspension; and (4) 2.0 mL of overlay agar supplemented with biotin and limited amounts of histidine and tryptophan . Concentrations of test article used were 62, 185, 556, 1667, and 5000 μg/plate. The positive controls in the absence of S9 mix were AF-2 for 2 and were subcultured every 3–4 days. The S9 preparation used for metabolic activation is described under the methods for the bacterial reverse mutation assay.Chinese Hamster Ovary cells (CHO-k1) were obtained from the Korean Cell Line Bank . The cells were cultured in F12 nutrient mixture  and 10% fetal bovine serum  at 37 °C and 5% COA preliminary dose range finding study was performed with eight concentrations up to 5000 µg/mL to determine the highest concentration used for each exposure scenario (24 h incubation without S9 and 6 h treatment and 18 h recovery with or without S9). For the 24-h incubation scenario, the relative increase in cell counts (RICC) was 46.40% and 60.39% in for 185.19 µg/mL and 61.73 µg/mL, respectively. For the 6 h exposure group without S9 mix, RICC were 46.77% and 61.55% for 555.56 µg/mL and 185.19 µg/mL, respectively. For the 6 h exposure group with S9 mix, RICC were 49.64% and 61.37% for 1666.67 µg/mL and 555.56 µg/mL, respectively. Based on these results, the maximum concentrations chosen for use in the chromosome aberration study were 185.19 µg/mL for 24 h, 555.56 µg/mL for 6 h-S9 and 1666.67 µg/mL for 6 h + S9. Two lower concentrations were also used in each assay, at threefold dilutions from the highest concentrations.For the assay, the positive control substance was mitomycin C without metabolic activation (S9 mix), and cyclophosphamide with S9 mix. The negative control was distilled water. Approximately 22 h after treatment, colcemid (GIBCO) was added to each culture plate for a final concentration of 0.2 µg/mL. The cultures were incubated for an additional 2 h, after which they were detached using 1X trypsin solution. The medium containing mitotic cells was centrifuged at 1000 rpm for 5 min. The cell pellets were resuspended in 75 mM potassium chloride solution and incubated at 37 °C for 20 min. The cells were fixed 3 times with Carnoy’s fixative solution (acetic acid:methanol = 1:3 v/v) for slide preparation. The slides were stained with 5% Giemsa solution for 5 min and observed microscopically. Two slide samples were prepared from each plate and 150 metaphase cells per plate were counted.Structural  and numerical aberrations were evaluated. Aneuploidy was not counted as numerical aberration since it occurs frequently in cell-lines such as CHO-k1. Endoreduplication was classified as polyploidy, and it was recorded if endoreduplication was frequently observed. The test material was considered to be clearly positive for clastogenicity (and/or aneugenicity) if at least one of the test concentrations exhibited a statistically significant increase compared with the corresponding negative control, the increase was dose-related and any of the results were outside of the distribution of historical negative control data.The study was conducted to evaluate the potential genotoxicity of the test material in an in vivo micronucleus assay. Male SPF CrlOri:CD1 (ICR) mice (n = 25) from Orient Bio Co., Ltd were used and divided into five equal groups: one negative control, one positive control and three treatment groups . The positive control used in the study was mitomycin C (2.0 mg/kg bw/day) and the negative/vehicle control was distilled water. Animals were dosed with the test material orally once a day for two days at 24-h intervals after being subjected to a 3–4 h fast, Mitomycin C was administered intraperitoneally rather than orally. Animals were observed on the day of administration from 30 min to 4 h after administration and at the time of autopsy for the occurrence of dead animals and abnormal signs. All animals were weighed before acquisition and grouping, administration of test substances, and before bone marrow collection. At 18 and 24 h after the last dose, all animals were euthanized, and bone marrow was collected from the thigh bone and suspended in fetal bovine serum. Bone marrow smears  were prepared on glass slides at room temperature and fixed in methanol for 5 min. To score the polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratio, slides were stained with 4% Giemsa solution. To score the micronucleated polychromatic erythrocytes (MNPCE) from PCEs, the slides were fixed, stained with acridine orange, and covered with cover glasses.The observation of slides was performed with the investigator blinded to test condition. The PCE to NCE ratio was determined by optical microscope at greater than 1000 × magnification. MNPCEs were observed by fluorescence microscope equipped with an FITC filter and more than 400 × magnification. The PCE/NCE ratio was determined by scoring the number of PCEs and NCEs in 500 erythrocytes per animal. Micronucleus frequency was determined by analyzing the number MNPCEs from 4000 PCEs per animal. Under the fluorescence microscope, PCEs appeared as a red-fluorescent and did not stain for nuclei using acridine orange. NCEs appeared only as shadow entities without fluorescence. With the optical microscope, PCEs stained with Giemsa appeared blue or purple, and NCEs appeared pink. In order to judge the presence of micronuclei, the largest size was defined as ½ the size of erythrocyte diameter and the smallest size was the limit of identification. Shapes included circle, donut, and semi-circle and color was defined as the same as the nucleus of the cell.The result was considered positive if all of the following acceptability criteria were met: (1) the frequency of MNPCE/4000 PCEs  is statistically reliable, increases dose-dependently, and/or shows a reproducible positive reaction at one or more concentrations, and (2) any of the results are outside the distribution of historical negative control data.p < 0.05. In the single dose oral toxicity study, body weight data were analyzed using the one-way analysis of variance (ANOVA) test to detect any differences between groups. In the 14-day dose range finding study, differences between groups were examined by the one-way ANOVA test. If a significant difference was detected, the Duncan test was used for data comparison if there was homogeneity of variance, and Dunnett’s T test was used if there was not homogeneity of variance. In the 13-week toxicity study, continuous data were compared using the one-way ANOVA test to determine if there was a difference between treated animals and controls. If there was a significant difference, the data were analyzed similarly to the 14-day study. Detailed clinical signs, urinalysis, urine sediments, and estrus cycle data were analyzed as non-continuous data and were converted by scale conversion and analyzed by the Chi-squared test. In the chromosome aberration assay, the number of aberrant metaphases, excluding gaps, and the incidence of polyploid plus endoreduplication were analyzed. Comparisons between groups were performed using the Chi-squared test. A linear logistic regression test was used to determine if the response was dose dependent. In the micronucleus assay, the negative control and treatment groups were compared by one-way ANOVA. If a significant difference was detected, the data were analyzed similarly to the 14-day study.The statistical package SPSS 12.0 K  was used for all analyses. The criterion for significance was 9 CFU/kg bw) (data not shown).There were no mortalities, abnormal clinical signs, or macroscopic findings in the study. Normal weight gain was observed and there were no significant body weight changes between the test groups and the vehicle control group. Under the conditions of the study, the lethal dose was greater than 5000 mg/kg bw (1.8 × 109 CFU/kg bw/day) based on the results.There were no mortalities, abnormal clinical signs, or macroscopic findings in the study. There were no significant changes in body weight, body weight gain, food or water consumption, or organ weights between the test groups and the vehicle control group. Findings that were significantly different in treated compared to control animals are shown in Table There were no test substance-related mortalities or abnormal clinical observations. No abnormalities were found in the ophthalmological examination and there were no effects of the test substance on estrus cycle. Body weights of males in the 2500 and 5000 mg/kg bw/day groups were less than controls at week 4, all groups of treated males exhibited lower body weights than controls from weeks 5–8, and males in the 2500 mg/kg bw/day also had lower body weights than controls at week 12 . Cytotoxicity was not observed at any concentration and there was no mention of test material precipitation. The assay was valid, as the positive controls showed a distinct increase in revertants meeting the criteria for a positive response.As shown in Table Test concentrations were limited by cytotoxicity to 20.58, 61.73 and 185.19 µg/mL in the 24 h study, 61.73, 185.19 and 555.56 µg/mL in 6 h study, and 185.19, 555.56, 1666.67 µg/mL in the 6 h study + S9 (Table Result of the micronucleus study are shown in Table W. cibaria CMU (oraCMU®) is being developed for use in human food and dietary supplements. Results of previous studies indicate that the strain is susceptible to common antibiotics, with the possible exception of kanamycin and vancomycin, but does not transfer resistance of these antibiotics to other bacteria [W. cibaria and is not unique to W. cibaria CMU. Additional studies performed by Kang et al. [W. cibaria CMU does not harbor virulence genes associated with pathogenic bacteria, does not cause hemolysis, mucin or protein degradation, or platelet aggregation, and does not produce d-lactate or urease . However, animal toxicity or genotoxicity studies on W. cibaria strains have not been conducted previously. Results of the studies described herein add to the database of information that W. cibaria CMU will be safe for human consumption.bacteria . Geneticg et al.  showed t9 CFU/kg bw), the highest dose administered was well tolerated. This dose was chosen as the highest dose administered in a 14-day range finding and 13-week study in rats. In the 14-day study, changes in MPV of males receiving 2500 and 5000 mg/kg bw/day, NEP in females receiving 1250 and 2500 mg/kg bw/day, and BUN and TG in females receiving 1250 mg/kg bw/day were not considered to be toxicologically relevant due to lack of dose dependency and/or minor nature of the changes. Changes in these variables were not found in rats receiving up to 5000 mg/kg bw (1.8 × 109 CFU/kg bw) W. cibaria CMU for ninety days, supporting the conclusion that the findings in the 14-day study were not toxicologically relevant.When administered acutely to rats by gavage, up to 5000 mg/kg bw (1.8 × 109 CFU/kg bw/day), the highest dose administered. In the 13-week study, the most notable effect of administration of the test material was reduced body weight and food consumption of males in the 2500 and 5000 mg/kg bw/day groups. The study investigators did not consider the findings to be adverse because the body weights of the animals were within 10% of the vehicle control group. The 10% criterion is commonly used to determine whether an effect of a test material on body weight is adverse [The no observed adverse effect level (NOAEL) of the 13-week study is 5000 mg/kg bw/day  or clastogenic in an OECD Guideline 473 in vitro chromosome aberration assay in cultured CHO-k1 cells. Both of the assays met the criteria for validity that are required by the OECD [W. cibaria CMU is not clastogenic or aneugenic up to 5000 mg/kg bw. Toxicity was not noted in the micronucleus study—there was no effect of the test substance on body weight or PCE/PCE + NCE ratio and no abnormal clinical signs were noted. However, the study is valid and fit for purpose because the assay was sensitive enough to detect micronucleated PCE (as indicated by the positive control response), and the highest dose of W. cibaria CMU administered was the same dose evaluated for safety in rats. Further, the 5000 mg/kg bw dose is higher than the limit dose of 2000 mg/kg bw recommended by the OECD [the OECD , 15. Thethe OECD .9 CFU/kg bw/day) W. cibaria CMU is safe in rats and suggest that the strain could be safely consumed by humans. Furthermore, W. cibaria CMU is non-genotoxic as shown by the negative results of in vitro bacterial reverse mutation and CHO-k1 cell chromosome aberration studies and the in vivo micronucleus study in mice. This study is the first study examining the potential of a W. cibaria strain to cause genetic toxicity and subchronic toxicity in rats according to OECD guidelines.In conclusion, the results of the studies described in this manuscript show that oral administration of up to 5000 mg/kg bw/day (1.8 × 10


Despite over 30 years of clinical experience, high‐quality studies on the efficacy of bilateral versus unilateral deep brain stimulation (DBS) of the ventral intermediate (VIM) nucleus of the thalamus for medically refractory essential tremor (ET) remain limited.To compare benefits and risks of bilateral versus unilateral VIM DBS using the largest ET DBS clinical trial dataset available to date.Participants from the US St. Jude/Abbott pivotal ET DBS trial who underwent staged‐bilateral VIM implantation constituted the primary cohort in this sub‐analysis. Their assessments “on” DBS at six months after second‐side VIM DBS implantation were compared to the assessments six months after unilateral implantation. Two control cohorts of participants with unilateral implantation only were also used for between‐group comparisons.P < 0.001), with a 64.1% CRST improvement in the contralateral side (P < 0.001). An added improvement was observed in the axial tremor score , and CRST part B  score. Rate of adverse events was slightly higher after bilateral stimulation.The primary cohort consisted of n = 38 ET patients . The second side VIM‐DBS resulted in a 29.6% additional improvement in the total motor CRST score (In the largest ET DBS study to date, staged‐bilateral VIM DBS was a highly effective treatment for ET with bilateral implantation resulting in greater reduction in total motor tremor scores when compared to unilateral stimulation alone. Thalamic deep brain stimulation (DBS) is an effective treatment for medication‐refractory essential tremor (ET).Despite over 30 years of clinical experience with VIM DBS in ET patients, there are no randomized controlled studies comparing VIM DBS to the best medical therapy for ET.A large St. Jude/Abbott funded US prospective controlled study of VIM DBS for ET using omni‐directional leads showed that unilateral and bilateral VIM DBS implantation was safe and effective in patients with medically‐refractory ETDetailed methods of the original 12 center prospective, controlled trial were previously published.Participants were evaluated at baseline, and day 180 (±14 days) . If a second lead was implanted, additional evaluations were performed at day 180 (±14 days) following the second‐sided implant. Baseline and follow‐up assessments included the Fahn and Tolosa Clinical Rating Scale for Tremor (CRST) in both the “off” and “on” DBS states (using non ‐blinded rater data), DBS parameters , assessment for adverse events, patient questionnaires including the Short Form‐36 Health Survey Questionnaire, Beck's Depression Inventory (BDI‐II), and Mini Mental State Exam (MMSE). CRST part A is a systematic tremor severity assessment by a rater or clinician, part B assesses handwriting (dominant hand only), drawing and pouring (each hand separately), and part C assesses functional disabilities related to speech, eating, drinking, hygiene, dressing, writing, and working. The total CRST score is a summation of CRST parts A, B, and C. CRST part C and total CRST scores were not assessed at day 180 (±14 days) following the second sided implant.The primary target cohort included patients with unilateral VIM DBS who then went on to have a staged‐bilateral implant. This cohort was used for within group comparisons to identify differences in tremor scores following unilateral and staged‐bilateral implantation. Two other control groups were defined to allow for between‐group comparisons with the primary target cohort and to aid in identifying potential confounding factors. Control cohort 2 was composed of all subjects who received a unilateral implant only (excluding the ones who later went on to have a second‐side implant). Control cohort 1 was a “matched cohort” that was comprised of a subset of subjects from control cohort 2, with unilateral implant, that were selected based on demographic and tremor severity similarity to the primary target cohort.The primary outcome was difference in CRST part A scores while “on” DBS at day 180 after the second‐sided VIM DBS implantation compared to the scores 180 days after the unilateral implant for the primary target cohort.Secondary outcomes included differences in CRST sub‐scores  at day 180 after the second‐side implant when compared to 180 days after the unilateral implant for the primary target cohort. Of note, axial tremor subscores included CRST Part A Items 1–4 and 7, all postural/rest tremors only except voice (action tremor). The differences in CRST scores and sub‐scores in control cohort 1 and control cohort 2 at 180 days post unilateral implantation respectively, were compared to the outcome measures in the primary cohort at 180 days post unilateral implantation. Patient questionnaires and MMSE scores were not included in this sub‐analysis.Number of adverse events (device‐related and surgery‐related adverse events) were noted for the primary cohort, after unilateral and bilateral stimulation, and for the control groups. Device‐related adverse events included new or worsening cognitive and psychiatric symptoms, disequilibrium or gait impairment, dysarthria, and sensory or motor disturbances, as well as DBS system malfunction and other uncategorized side‐effects. Surgery‐related adverse events included post‐operative pain, headache, or abnormal sensations as well as surgical complications, wound healing issues, or infection.A centralized electronic database contained study data from each center with automated data checks for expected value ranges and audit trails from any manual changes to the data. To create control cohort 1, pairwise matches between subjects in the primary cohort and control cohort 2 were selected to minimize the following cost function:In this function, age is the subjects age in years at the time of implant, asymmetry is the absolute difference in the combined severity of the CRST for the upper and lower extremities, and cost is the total cost for a set of potential matches to the subjects in the primary cohort. Potential matches were prohibited if the subject's sex did not match or if the subject in control cohort 1 lacked data at day 180. Control cohort 2 included all subjects with unilateral‐only implants, and primary cohort included all subjects with staged bilateral implants.t tests and paired t tests were performed for comparisons of baseline and changes in outcome measures within and across cohorts.Baseline demographics of age, gender, race, disease duration, disease severity, dominant handedness, and hemisphere of initial implant for the primary cohort and two control cohorts were tabulated. Total CRST scores and subscores at baseline, 180 days after unilateral VIM DBS implantation, and 180 days after second‐side VIM DBS implantation (if applicable) were tabulated as means and standard deviations for the primary cohort and two control cohorts. For each time‐point, all available data were used when computing summary statistics. Outcome measures, including differences in the CRST subscores at each given timepoint were calculated as percentages. Two sample clinicaltrials.gov (NCT02087046). All sites received Institutional Review Board approval prior to consenting patients. Written informed consent was obtained prior to study procedures and device implantation. Abbott  sponsored the original trial.The study was approved by the United States Food and Drug Administration (FDA), and registered with In total, 38 patients were included in the primary cohort . Control cohort 1 included 38 matched unilaterally only implanted patients and the control cohort 2 included 80 patients with unilateral only implantation  are tabulated as means and standard deviations in Table Demographic data in the primary cohort group included mean age at implantation of 65.3 ± 9.5 years, mean duration of symptoms since diagnosis of 17.0 ± 11.9 years, mean duration since tremor onset of 28.6 ± 16.0 years, and baseline total Motor CRST of 17.9 ± 5.4 (Table P < 0.001) and 81.6% after bilateral VIM DBS (P < 0.001), with a 29.6% greater benefit after second side implantation (P < 0.001). (Table P < 0.001), 2.8% non‐significant improvement on the ipsilateral arm , and 21.4% improvement in axial tremor scores . There was a 21.1% and 25.0% improvement in head  and voice  tremor scores, respectively, following second‐sided implantation.The total motor CRST improved by 52.0% after unilateral (P < 0.001) and 68.1% after bilateral VIM DBS (P < 0.001), with a 24.8% greater benefit after second side implantation (P < 0.001).CRST Part B scores improved by 43.3% after unilateral stimulation (P < 0.001) and the total CRST scores improved by 53.8% post unilateral stimulation (P < 0.001). CRST Part C and total CRST scores were not assessed following bilateral implantation.CRST Part C scores improved by 70.3% after unilateral stimulation  improvement in their total motor CRST scores after unilateral implantation. After unilateral implantation, contralateral limb scores and ipsilateral limb scores improved by 82.4% (P < 0.01) and 21.1% (P = 0.03) respectively. Axial, voice, and head tremor scores improved by 61.1% (P < 0.001), 61.5% (P < 0.001), and 71.4% (P = 0.001) respectively. CRST part B, CRST part C, and total CRST scores improved by 44.3% (P < 0.001), 68.5% (P < 0.001) and 56.0% (P < 0.001) respectively.Outcomes were similar for both control cohorts and the primary cohort. Control cohort 1 had a 55.6% (P < 0.001) improvement in their total motor CRST scores post unilateral implantation. After unilateral implantation, patients in control cohort 2 had an 80.0% (P < 0.001) and 19.6% (P < 0.001) improvement in their contralateral target limb scores, and ipsilateral limb scores respectively. Axial, voice, and head tremor scores improved by 67.5% (P < 0.001), 58.3% (P < 0.001) and 66.7% (P < 0.001) respectively. CRST part B, CRST part C and Total CRST scores also improved by 45.3% (P < 0.001), 67.7% (P < 0.001) and 55.9% (P < 0.001) respectively.Control cohort 2 had a 57.2% . There was a difference in surgically induced AEs following bilateral when compared to unilateral surgeries, but not when comparing with control cohort 2, although they had two surgeries. Interestingly, the SAE rate was notably lower in the primary cohort when compared to either of the control cohorts, especially when considering only the SAEs reported as related to the system. This is despite the longer period of follow‐up for the patients in the primary cohort. We did not have a large enough bilaterally operated control group to compare the AE/SAE of one‐time versus staged surgery. In clinical practice, the number of surgically‐related AE may be smaller when bilateral procedures are done in a single‐ compared to a double‐step procedure.Other strengths of this sub‐study include low dropout rates and comparisons with two control groups. Weaknesses include this was a post‐hoc analysis in that statistical significances were not adjusted for multiple comparisons. Also, there was a lack of racial diversity (97.5% Caucasian), and the original clinical trial was not designed to assess the added benefit of a staged‐bilateral implantation when compared to unilateral implantation and unfortunately quality‐of‐life measures were not available. We also acknowledge with newer DBS systems now available with features including segmented leads and broader parameters, adverse events in clinical practice may be less that what we have reported in these patients treated with a legacy system.These data provide further evidence that second‐sided VIM DBS implantation significantly improves tremor severity when compared to a unilateral implant providing better bilateral hand tremor control in patients with ET. Patients should be educated about the benefits of having the second side implanted to provide greater overall tremor reduction, and that there is potential increased risk of adverse events.1. Research project: A. Conception, B. Organization, C. Execution; 2. Statistical Analysis: A. Design, B. Execution, C. Review and Critique; 3. Manuscript Preparation: A. Writing of the first draft, B. Review and Critique.PP: 1B, 1C, 2A, 2B, 2C, 3A, 3B; GD: 1A, 1B, 1C, 2A, 2C, 3B; SO: 2B, 2C, 3B; BC: 2B, 3B; PSL: 2B, 3B; AM: 2C, 3B; SG: 2C, 3B; KTM: 1A, 2C, 3B; TT: 2B, 2C, 3B; JLO: 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B.clinicaltrials.gov (NCT02087046). All sites received Institutional Review Board approval prior to consenting patients. Written informed consent was obtained prior to study procedures and device implantation. Abbott  was the sponsor of the original trial.The study was approved by the United States Food and Drug Administration (FDA), and registered with Abbott  was the sponsor of the original trial.PP: No additional disclosures to report.GD: Received personal fees from Boston Scientific, Jazz, Functional Neuromodulation, Thieme publishers. He receives funding for his research from the German Research Council .SO: Employee of Abbott.BC: Employee of Abbott.PSL: Non‐financial research support from Abbott laboratories, educational consulting for Boston Scientific and Medtronic, grants from Brain Neurotherapy Bio, Neurocrine, uniQure and Voyager, Advisory Boards for Biogen, BlueRock and Sio, consulting for Aspen, BrainXell, Corlieve, ClearPoint Neuro, Galvani, Huntington Study Group, Passage Bio, Sanofi.KTM: Received research support from Medtronic and Deep Brain Innovations, anticipates upcoming consulting with Boston Scientific and Medtronic.AM: Received speaker honoraria or consultancies fee from Abbott laboratories, Abbvie, and Lundbeck, and grant support from Abbvie and Lundbeck.SG: No additional disclosures to report.TT: Employee of Abbott.JLO: Received consulting from Abbvie, Abbott laboratories, educational and research grants from Medtronic, Boston Scientific, Abbvie, Supernus, Amneal, Merz, Biogen, Neuroderm.


Metallomics, Volume 14, Issue 8, August 2022, mfac051, https://doi.org/10.1093/mtomcs/mfac051This is a correction to: Wolfgang Maret, The quintessence of metallomics: a harbinger of a different life science based on the periodic table of the bioelements, In the originally published version of this manuscript, the credit line under Fig. 1 incorrectly stated that ‘Figure was adopted from W. Maret, Metallomics: The Science of Biometals and Biometalloids, chapter 1 in Metallomics, M. A. Z. Arruda, ed, Advances in Experimental Medicine and Biology 1055, 1–20, 2018 with permission of Springer International Publishing AG.’The correct version of the credit line under Fig. 1 is: ‘Figure was adapted from W. Maret, Metallomics: The Science of Biometals and Biometalloids, chapter 1 in Metallomics, M. A. Z. Arruda, ed, Advances in Experimental Medicine and Biology 1055, 1–20, 2018 with permission of Springer International Publishing AG.’There was an error in the sentence: “The amounts vary over at least three orders of magnitude from 18 mg iron to 2.4 µg vitamin B12 .”The correct version of this sentence is: “The amounts vary over at least three orders of magnitude from 18 mg iron for adult females to 2.4 µg vitamin B12  recommended per day.”Table 3 incorrectly referred to 1 : 1 : 1. It should refer to 1 : 1 instead.These errors have been corrected online.


Candida, the most common species of which is Candida albicans. The ergosterol pathway in yeast is a common target by many antifungal agents, as ergosterol is an essential component of the cell membrane. The current antifungal agent of choice for the treatment of candidiasis is fluconazole, which is classified under the azole antifungals. In recent years, the significant increase of fluconazole-resistant C. albicans in clinical samples has revealed the need for a search for other possible drug targets. In this study, we constructed a mathematical model of the ergosterol pathway of C. albicans using ordinary differential equations with mass action kinetics. From the model simulations, we found the following results: (i) a partial inhibition of the sterol-methyltransferase enzyme yields a fair amount of fluconazole resistance; (ii) the overexpression of the ERG6 gene, which leads to an increased sterol-methyltransferase enzyme, is a good target of antifungals as an adjunct to fluconazole; (iii) a partial inhibition of lanosterol yields a fair amount of fluconazole resistance; (iv) the C5-desaturase enzyme is not a good target of antifungals as an adjunct to fluconazole; (v) the C14α-demethylase enzyme is confirmed to be a good target of fluconazole; and (vi) the dose-dependent effect of fluconazole is confirmed. This study hopes to aid experimenters in narrowing down possible drug targets prior to costly and time-consuming experiments and serve as a cross-validation tool for experimental data.Candidiasis is reported to be the most common fungal infection in the critical care setting. The causative agent of this infection is a commensal pathogen belonging to the genus IMPORTANCE Candidiasis is reported to be the most common fungal infection in the critical care setting, and it is caused by a commensal pathogen belonging to the genus Candida, the most common species of which is Candida albicans. The current antifungal agent of choice for the treatment of candidiasis is fluconazole, which is classified under the azole antifungals. There has been a significant increase in fluconazole-resistant C. albicans in recent years, which has revealed the need for a search for other possible drug targets. We constructed a mathematical model of the ergosterol pathway in C. albicans using ordinary differential equations with mass action kinetics. In our simulations, we found that by increasing the amount of the sterol-methyltransferase enzyme, C. albicans becomes more susceptible to fluconazole. This study hopes to aid experimenters in narrowing down the possible drug targets prior to costly and time-consuming experiments and to serve as a cross-validation tool for experimental data. Candida. The following Candida species are all reported to cause cutaneous, mucosal, bloodstream, and deep-seated tissue infections: Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei. The most common of these is Candida albicans  noted several risk factors for invasive candidiasis that include the use of broad-spectrum antimicrobials, immunosuppressive drugs, total parenteral nutrition, iatrogenic interventions, along with several challenges in the critical care setting, namely, the shift to more resistant  therapy . Among tstration . The mec species . The che species .Candida species, and some nonalbican Candida species have already been reported to have intrinsic resistance to azoles  noted a high level of resistance to azoles among o azoles . In fact noted a C. albicans, which can be costly and time-consuming. In this study, we propose a different approach to investigating fluconazole resistance in C. albicans, namely, using mathematical models to determine possible drug targets within the ergosterol pathway. The aim of this study is to narrow down possible drug targets prior to costly and time-consuming experiments and to serve as a cross-validation tool for experimental data. To our knowledge, this is the first study to investigate the fluconazole resistance in the ergosterol pathway of Candida albicans using mathematical modeling.There is an abundance of experimental investigations into azole resistance mechanisms in The rest of the paper is organized as follows: The results of the model simulations are shown in the Results section. These are followed by a discussion on how the results compare with previous literature, the conclusions drawn, and further recommendations that are proposed in the Discussion section. Then, in the Materials and Methods section, we discuss, in detail, the mathematical modeling process.C. albicans.The model was run to simulate several scenarios to investigate possible drug targets in the ergosterol pathway in C. albicans, as no ergosterol was produced, and ergosterol is essential for yeast survival. Cases 2 (0.01×) and 3 (0.1×) simulate the partial inhibition of lanosterol. They yield a higher percentage of the toxic sterol and ergosterol as the dominant metabolite but a lower percentage of ergosterol reduction, satisfying 2 out of 3 criteria which denote fair fluconazole resistance. Case 4 (1×) denotes the default case, which serves as our baseline. Cases 5 (10×) and 6 (100×) simulate the overproduction of lanosterol. They yield a lower percentage of the toxic sterol but a higher percentage of ergosterol reduction, satisfying 1 out of 3 criteria and denoting an equivocal result. The lanosterol concentration was varied as follows: 0×, 0.01×, 0.1×, 1×, 10×, and 100× the baseline value see , represe10.1128/msystems.00691-22.4FIG S2x axes denote the time in hours, and the y axes represent the concentrations in μM. The last two subplots, emphasized in dark borders, reiterate the pertinent plots of ergosterol and the toxic sterol  for easier comparison. Download FIG S2, PDF file, 0.1 MB.Plots generated by the simulation of variations in lanosterol concentration. The variations are as follows: 0× (red solid line), 0.01× (orange dashed line), 0.1× (yellow dotted line), 1× (green dashed dotted line), 10× (blue dense dashed dotted line), and 100× (purple dashed double dotted line). The Copyright © 2022 Yu et al.2022Yu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans, as no ergosterol was produced, and ergosterol is essential for yeast survival. Cases 2 (0.01×) and 3 (0.1×) simulate the partial inhibition of C14α-demethylase. They yield a higher percentage of toxic sterol and a higher percentage of ergosterol reduction, satisfying 0 out of 3 criteria, which denotes a good target for antifungals as an adjunct to fluconazole. Case 4 (1×) denotes the default case, which serves as our baseline. Cases 5 (10×) and 6 (100×) simulate the overexpression of the ERG11 gene. They yield a lower percentage of the toxic sterol and a lower percentage of ergosterol reduction, satisfying 2 out of 3 criteria and denoting fair fluconazole resistance. The C14α-demethylase enzyme concentration was varied as follows: 0×, 0.01×, 0.1×, 1×, 10×, and 100× the baseline value see , represe10.1128/msystems.00691-22.5FIG S3x axes denote the time in hours, and the y axes represent the concentrations in μM. The last two subplots, emphasized in dark borders, reiterate the pertinent plots of ergosterol and the toxic sterol  for easier comparison. Download FIG S3, PDF file, 0.1 MB.Plots generated by the simulation of variations in C14α-demethylase concentration. The variations are as follows: 0× (red solid line), 0.01× (orange dashed line), 0.1× (yellow dotted line), 1× (green dashed dotted line), 10× (blue dense dashed dotted line), and 100× (purple dashed double dotted line). The Copyright © 2022 Yu et al.2022Yu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans, as the dominant metabolite is ergosterol, and ergosterol is essential for yeast survival. Cases 2 (0.01×) and 3 (0.1×) simulate giving fluconazole with a dose lower than its minimum inhibitory concentration (MIC) . They yield a lower percentage of toxic sterol and a lower percentage of ergosterol reduction, satisfying 2 out of 3 criteria, which denotes fair fluconazole resistance. Case 4 (1×) denotes the default case, which serves as our baseline. Cases 5 (10×) and 6 (100×) simulate giving fluconazole with a dose higher than its MIC. They yield a higher percentage of toxic sterol and a higher percentage of ergosterol reduction, satisfying 0 out of 3 criteria and denoting a good target for antifungals as an adjunct to fluconazole. The fluconazole concentration was varied as follows: 0×, 0.01×, 0.1×, 1×, 10×, and 100× the baseline value see , represe10.1128/msystems.00691-22.6FIG S4x axes denote the time in hours, and the y axes represent the concentrations in μM. The last two subplots, emphasized in dark borders, reiterate the pertinent plots of ergosterol and the toxic sterol  for easier comparison. Download FIG S4, PDF file, 0.1 MB.Plots generated by the simulation of variations in fluconazole concentration. The variations are as follows: 0× (red solid line), 0.01× (orange dashed line), 0.1× (yellow dotted line), 1× (green dashed dotted line), 10× (blue dense dashed dotted line), and 100× (purple dashed double dotted line). The Copyright © 2022 Yu et al.2022Yu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans, as no ergosterol was produced, and ergosterol is essential for yeast survival. Cases 2 (0.01×) and 3 (0.1×) simulate the partial inhibition of sterol-methyltransferase. They yield a lower percentage of toxic sterol and a lower percentage of ergosterol reduction, satisfying 2 out of 3 criteria, which denotes fair fluconazole resistance. Case 4 (1×) denotes the default case, which serves as our baseline. Cases 5 (10×) and 6 (100×) simulate the overexpression of the ERG6 gene. They yield a higher percentage of toxic sterol and a higher percentage of ergosterol reduction, satisfying 0 out of 3 criteria and denoting a good target for antifungals as an adjunct to fluconazole. The sterol-methyltransferase enzyme concentration was varied as follows: 0×, 0.01×, 0.1×, 1×, 10×, and 100× the baseline value see , represe10.1128/msystems.00691-22.7FIG S5x axes denote the time in hours, and the y axes represent the concentrations in μM. The last two subplots, emphasized in dark borders, reiterate the pertinent plots of ergosterol and the toxic sterol  for easier comparison. Download FIG S5, PDF file, 0.1 MB.Plots generated by the simulation of variations in sterol-methyltransferase concentration. The variations are as follows: 0× (red solid line), 0.01× (orange dashed line), 0.1× (yellow dotted line), 1× (green dashed dotted line), 10× (blue dense dashed dotted line), and 100× (purple dashed double dotted line). The Copyright © 2022 Yu et al.2022Yu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans, as no ergosterol was produced, and ergosterol is essential for yeast survival. Cases 2 (0.01×) and 3 (0.1×) simulate the partial inhibition of C5-desaturase. They yield a lower percentage of the toxic sterol but a higher percentage of ergosterol reduction, satisfying 1 out of 3 criteria, which denotes an equivocal result. Case 4 (1×) denotes the default case, which serves as our baseline. Cases 5 (10×) and 6 (100×) simulate the overexpression of the ERG3 gene. They yield a higher percentage of the toxic sterol but a lower percentage of ergosterol reduction and ergosterol as the dominant metabolite, satisfying 2 out of 3 criteria and denoting fair fluconazole resistance. The C5-desaturase enzyme concentration was varied as follows: 0×, 0.01×, 0.1×, 1×, 10×, and 100× the baseline value see , represe10.1128/msystems.00691-22.8FIG S6x axes denote the time in hours, and the y axes represent the concentrations in μM. The last two subplots, emphasized in dark borders, reiterate the pertinent plots of ergosterol and the toxic sterol  for easier comparison. Download FIG S6, PDF file, 0.05 MB.Plots generated by the simulation of variations in C5-desaturase concentration. The variations are as follows: 0× (red solid line), 0.01× (orange dashed line), 0.1× (yellow dotted line), 1× (green dashed dotted line), 10× (blue dense dashed dotted line), and 100× (purple dashed double dotted line). The Copyright © 2022 Yu et al.2022Yu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The local one-at-a-time sensitivity analysis was plotted using a tornado chart for ergosterol, as shown in 10.1128/msystems.00691-22.2TABLE S1Table S1, PDF file, 0.05 MB.Model parameters. Download Copyright © 2022 Yu et al.2022Yu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ERG1 gene deletion results in the demise of C. albicans due to its lack of ergosterol. This agrees with the results of the study by Pasrija et al. (2005) that the erg1 mutant showed a total lack of ergosterol and led to increased azole susceptibility  a partial inhibition of lanosterol yields a fair amount of fluconazole resistance; (iv) C5-desaturase is not a good target of antifungals as an adjunct to fluconazole; (v) C14α-demethylase is confirmed to be a good target for fluconazole; and (vi) the dose-dependent effect of fluconazole is confirmed. This study hopes to aid experimenters in narrowing down the possible drug targets prior to costly and time-consuming experiments and to serve as a cross-validation tool for experimental data.From the model simulations, we found the following results: (i) a partial inhibition of sterol-methyltransferase yields a fair amount of fluconazole resistance; (ii) overexpression of the ERG1 gene that expresses squalene epoxidase, which is the target of antifungal allylamine. Moreover, the computational approach used in our study is also applicable to other, completely different pathways involved in the azole resistance mechanism, such as the CDR1, CDR2, and MDR1 genes, which are involved in the drug efflux transporter. We also recommend doing further experiments on the sterol-methyltransferase enzyme to confirm that its overexpression is a possible target for antifungals as an adjunct to fluconazole in order to validate our simulation result.We recommend extending the model to include other genes involved in the ergosterol biosynthesis pathway, such as the C. albicans is built by adopting the pathways of Sanglard et al. (2003) , and Maure 1 in ). We notERG11, ERG3, and ERG6. Various point mutations in the ERG11 gene were shown to alter the binding abilities of azoles to the C14α-demethylase enzyme  , which compared the sterol compositions of  (GC/MS) .The model uses ordinary differential equations (ODEs) with mass-action kinetics, as first described in , which hThe ODEs for the model with azole  are shown in The constraint equations are shown in C. albicans strain, it is mainly composed of ergosterol (98%), while the rest is classified as unknown (2%). We distribute the unknown 2% to the remaining metabolites in the pathway. In a fluconazole-resistant strain, the sterol composition is as follows: ergosterol (2%), eburicol (16.1%), obtusifoliol (34.5%), 14α-methyl-3,6-diol (45.2%), and unknown (2.2%). We again distribute the unknown 2.2% to the remaining metabolites in the pathway , they used a molar ratio of 1:2:50 for the enzyme, inhibitor, and substrate, respectively . We testble 8 in ). Follow10.1128/msystems.00691-22.3FIG S1x axes denote the time in hours, and the y axes represent the concentrations in μM. The last two subplots, emphasized in dark borders, reiterate the pertinent plots of ergosterol and the toxic sterol  for easier comparison. Download FIG S1, PDF file, 0.04 MB.Plots generated by the model calibrated from Kelly et al., (1997) . The norCopyright © 2022 Yu et al.2022Yu et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans are as follows: Susceptible (MIC ≤ 2 μg/mL), Dose-dependent (MIC = 4 μg/mL), and Resistant (MIC ≥ 8 μg/mL)  M27-A3  broth mi8 μg/mL) . We recaThe recalibrated model generated the plots shown in A local one-at-a-time sensitivity analysis was performed on the model parameters. We took −10% and +10% of the baseline values of the parameters, as shown in In experimental studies, antifungal susceptibility is quantified via a visual spotting assay on yeast extract peptone dextrose (YEPD) medium , the disC. albicans  proposed the use of percent ergosterol reduction as a quantitative method by which to evaluate antifungal susceptibility . Howeveralbicans . Anotheralbicans .C. albicans: (i) when the percent toxic sterol  is less than 17.73%; (ii) when the percent ergosterol reduction is less than 94.69%; and (iii) when ergosterol is the dominant fraction among the metabolites.Considering the above methods for antifungal susceptibility, we propose the following criteria, based on the data gathered in When all three criteria are satisfied, we designate it as good fluconazole resistance. When two out of three are satisfied, we designate it as fair fluconazole resistance. When only one out of three is satisfied, we designate it as an equivocal result such that no conclusion can be drawn. Conversely, when none of the criteria are satisfied, we designate it as a good target for antifungals as an adjunct to fluconazole.https://github.com/dlsu-scomb/candida-amr.The source code is available at


Meanwhile, the total phenolic content (TPC) increased by 21.0%, 10.6%, and 16.6%, respectively. Citric acid, sodium benzoate, and potassium sorbate all might reverse into the concentrated juice in amounts of 3.083 ± 0.477, 1.497 ± 0.008, and 0.869 ± 0.003 g/kg, respectively. These reversed food additives can make the TPC and TAC in juice steadier during its concentration and storage. Accordingly, food additives can be an excellent choice for DSs in the FO concentration process of juices, not only improving the concentration efficiency but also increasing the stability of blueberry juice.This study is to evaluate the athermal forward osmosis (FO) concentration process of blueberry juice using food additives as a draw solution (DS). The effects of food additives, including citric acid, sodium benzoate, and potassium sorbate, on the concentration processes are studied, and their effects on the products and membranes are compared. Results show that all these three food additives can be alternative DSs in concentration, among which citric acid shows the best performance. The total anthocyanin content (TAC) of blueberry juice concentrated by citric acid, sodium benzoate, and potassium sorbate were 752.56 ± 29.04, 716.10 ± 30.80, and 735.31 ± 24.92 mg·L The global market of concentrated juice, which is widely used to provide the ingredients for making the beverage, is estimated to be USD 81.8 billion in 2020. Concentration is a crucial step in concentrated juice production because concentrated juice may be stored for a long time and is portable ,4. It haAs a kind of athermal processing, membrane technologies, such as ultrafiltration (UF), nanofiltration (NF), and reverse osmosis (RO), have been applied to juice manufacture . However−2·h−1) exhibited a larger reverse salt penetration than glucose (2.5 g·m−2·h−1) in liquid egg white FO concentration, which could lessen the taste [In FO processes, the draw solution (DS), the most important factor of such technology, was commonly used with inorganic salts such as sodium chloride, potassium chloride, magnesium chloride, and others . These she taste . The shoEscherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes mixtures (>4 log CFU/g) on cherry tomatoes [Food additives, such as sodium benzoate, potassium sorbate, citric acid, sodium bisulfate, cyclamate, and sodium saccharin, are commonly used in fruit juices to reduce nutrient loss and prolong their shelf life ,18,19. Atomatoes . Similartomatoes . Meanwhitomatoes . HoweverIn this study, the effects of citric acid, potassium sorbate, and sodium benzoate on FO concentrations of blueberry juice were investigated. Differences in the water flux, membrane fouling, and reverse salt fluxes during FO were assessed. The effects of these food additives on the physicochemical properties and preservation of blueberry juice were also investigated. Meanwhile, the stabilities of blueberry juice concentrated by the FO process using different food additives were compared using a storage experiment.Blueberry pulp was made from blueberries by a Juicer . A total of 0.22% pectinase and 0.73% cellulase were added to the pulp and placed in a water bath  at 50 °C for 100 min. After filtration with gauze, the clarified blueberry juice was centrifuged  at 4000 rpm for 15 min and stored in a refrigerator at 4 °C for further use as FS. Citric acid, potassium sorbate, and sodium benzoate of analytical reagent were purchased from Yien Chemical Technology Co., Ltd. .2 produced from polyamide served as an FO membrane, and was previously purchased from Guochu Science and Technology .A thin Film Composite (TFC) membrane with an effective area of 20 cm−1), which was stable in preliminary experiments. FS tank was placed on a digital balance  Co., Ltd., Changzhou, China) connected to a computer for water flux acquisition. The concentration process was carried out for 8 h, and the water flux was calculated according to Equation (1) [W is the water flux (L·m−2·h−1), Δm is the mass change of feed liquid (g), ρ is the water density (1.0 g·cm−3), Δt is the concentration time (h), and A is the effective membrane area (m2).The FO processes were carried out using a bench-scale FO system, as shown in tion (1) .JW = Δm2 pieces, sprayed gold, and observed through a field emission scanning electron microscope  at an acceleration voltage of 15 kV.The original and fouled membranes were dried naturally after FO processes. Dried membranes were cut into 0.5 × 0.5 cm−1), ε is the molar extinction coefficient of cyanidin-3-O-glycoside  −1), and L is the optical path length (1 cm).TAC was determined according to the pH differential method with slight modification . To obtaAccording to the method described by Mondal et al. , the abs2 = 0.9996, 10–60 mg·L−1.TPC was analyzed using a modified Folin–Ciocalteu colorimetric method . A total2 = 0.9991, 10–60 mg·L−1.Total sugar content was determined by a phenol sulfuric acid method . A totalThe pH of blueberry juice was determined by a bench pH meter  Co., Ltd., Changzhou, China). TSS was determined by an Abbe refractometer . The particle size distribution of blueberry juice was determined by a laser particle size analyzer .0, a0, and b0 represent the color values of blueberry juice.The color difference analyzer  was used according to a reported method .(4)ΔE= equipped with UV detection was used to identify anthocyanins according to a developed method [−1. The column temperature was 25 °C, and the detection wavelength was 210 nm. Potassium sorbate in blueberry juice was separated using methanol and 0.02 mol·L−1 ammonium acetate solution (5:95) as a mobile phase [−1. The column temperature was 25 °C, and the detection was performed at 230 nm. Sodium benzoate was determined by the same method described for potassium sorbate. The standard equations of citric acid, potassium sorbate, and sodium benzoate were y = 2201x + 11,657 , y = 43,051,062x − 202,507 , and y = 25,568,511x − 118,381 , respectively.The citric acid in blueberry juice was separated by HPLC on a C18 column  using methanol−0.5% sodium dihydrogen phosphate (60:40) as the mobile phase . The injle phase . The injIn order to explore the effects of food additives on blueberry juice, the storage of concentrated blueberry juices with these three food additives was carried out. Concentrated and original blueberry juices were stored in a 50 mL sterile polypropylene tube at 25 °C. The TAC, TPC, TSS, pH, and anthocyanins profiles of blueberry juice were determined at weeks 0, 1, 2, and 3.All experiments and analytical measurements were performed in triplicate. Experimental results were expressed as the means ± standard deviation (SD). All experimental data were graphed by Origin 2021.−1 [+ is much smaller than Na+ and K+; thus, a slighter ICP of citric acid might result in larger water flux. Therefore, citric acid showed good potential as a DS since it exhibits a large water flux. Moreover, as citric acid could be an antioxidant and acidulant, there is no need to remove it from the product.−1 ,37. Due −1 . The sizAs shown in The variation trends of TPC, total sugar, and TSS during concentrations with different DSs were all similar. TPC, total sugar, and TSS were increased by about 20% with citric acid, while these with two others were about 12% and 17%. Binding between phenolic compounds and other solutes such as polysaccharides might occur in the solution, resulting in larger particles adsorbed on the membrane , which cTwelve anthocyanins were identified in blueberry juice, as reported in our previous study . As show−2·h−1, respectively. Correspondingly, the accumulations of food additives in the concentrated juice were 3.083 ± 0.477, 1.497 ± 0.008, and 0.869 ± 0.003 g/kg at the final measurement, respectively. FAO required that the contents of sodium benzoate and potassium sorbate in concentrated fruit and vegetable juice should not exceed 1.0 g/kg. Accordingly, in our study, the content of potassium sorbate in blueberry juice did not exceed the standard, but sodium benzoate exceeded. The citric acid in the juice was kept within the safety limit . Similar findings demonstrated that the reverse salt flux has no significant effect on the FS, and even some concentrated products do not need to remove the solute of reverse penetration [In the FO process, the solutes of DS could reverse into the feed solution. In this study, citric acid, sodium benzoate, and potassium sorbate all reversed into the concentrated juice, and their reverse fluxes were 18.14 ± 2.81, 9.43 ± 0.05, and 5.54 ± 0.02 g·metration . In geneIn In the study, the feasibility of using food additives as DSs for the FO process was demonstrated. Citric acid, sodium benzoate, and potassium sorbate were selected as DSs for blueberry juice concentration by FO. Citric acid exhibited higher water flux and more effective protection against anthocyanins than sodium benzoate and potassium sorbate. With citric acid as the DS, TAC in blueberry juice increased by about 26% and TPC by about 21%, which were higher than those with others. This indicated that citric acid had a particularly significant protective effect on anthocyanins. Citric acid could reverse into concentrated juice at the safety limit of FAO. These reversed food additives in concentrated juices could keep the juice more stable during storage. The protective effect of citric acid on the bioactive substances of juices demonstrated that food additives could be effectively applied in FO concentration processes of juices as DSs.


Participants completed four experimental conditions on separate days: high-intensity interval exercise (HIIE) with intervals performed at 75% maximal oxygen uptake , HIIE with intervals performed at 90% V̇o2max (HIIE2), continuous moderate-intensity exercise (MIE) at 60% V̇o2max and a sedentary control condition (CON). All exercise conditions were completed on a cycle ergometer and matched for time (30 min) and average intensity (60% V̇o2max). Brachial artery flow-mediated dilation (FMD) and CVR of the middle cerebral artery were measured before exercise, and 1- and 3-h after exercise. CVR was assessed using transcranial Doppler ultrasonography to both hypercapnia (6% carbon dioxide breathing) and hypocapnia (hyperventilation). FMD was significantly elevated above baseline 1 and 3 h following both HIIE conditions (P < 0.05), but FMD was unchanged following the MIE and CON trials (P > 0.33). CVR to both hypercapnia and hypocapnia, and when expressed across the end-tidal CO2 range, was unchanged in all conditions, at all time points . In conclusion, these novel findings show that the acute increases in peripheral vascular function following HIIE, compared with MIE, were not mirrored by changes in cerebrovascular reactivity, which was unaltered following all exercise conditions in healthy young adults.The acute effect of exercise intensity on cerebrovascular reactivity and whether this mirrors changes in peripheral vascular function have not been investigated. The aim of this study was to explore the acute effect of exercise intensity on cerebrovascular reactivity (CVR) and peripheral vascular function in healthy young adults (NEW & NOTEWORTHY This is the first study to identify that acute improvements in peripheral vascular function following high-intensity interval exercise are not mirrored by improvements in cerebrovascular reactivity in healthy young adults. High-intensity interval exercise completed at both 75% and 90% V̇o2max increased brachial artery flow-mediated dilation 1 and 3 h following exercise, compared with continuous moderate-intensity exercise and a sedentary control condition. By contrast, cerebrovascular reactivity was unchanged following all four conditions. Atherosclerosis is a precursor to overt cardiovascular disease, and endothelial dysfunction is the first detectable manifestation of the atherosclerotic process . AerobicCerebrovascular function plays an important role in the risk of cerebrovascular diseases such as stroke, dementia, and cognitive decline , 11. RecA recent systematic review explored the effect of HIIE on cerebrovascular function . In totao2max)   but theno2max) , 26. Preo2max), HIIE performed at 75% V̇o2max, and HIIE performed at 90% V̇o2max, which were all matched for time (30 min) and average intensity (target: 60% V̇o2max). It was hypothesized that 1) both HIIE protocols would increase brachial FMD compared with MIE and a resting control; 2) increases in middle cerebral artery blood velocity (MCAv) during exercise would be higher during HIIE completed at 75% V̇o2max compared with MIE and HIIE completed at 90% V̇o2max; and 3) CVR would be unchanged following MIE, increased following HIIE completed at 75% V̇o2max, and decreased following HIIE completed at 90% V̇o2max.This study aimed to investigate the acute effect of exercise intensity on peripheral and cerebral vascular function in healthy adults. Specifically, this study compared continuous moderate-intensity exercise  changes in FMD, to detect an effect size of ∼1.2 . This ren = 10  throughout. Participant characteristics are described in Following ethical approval from the University of Exeter ethics committee (180613/A/07), 12 healthy adults volunteered to take part in this study. Exclusion criteria included smoking, contraindications to exercise, cardiometabolic disease, and the use of any medication or supplement known to influence vascular function. One participant did not complete the study due to an unrelated injury, and one participant was removed due to inadequate acquisition of vascular data. Consequently, data are presented as o2max was determined as the highest 10 s average . The mean V̇o2 response time from the incremental ramp test was accounted for when prescribing the power outputs for each exercise trial  ramp test to exhaustion on an electronically braked cycle-ergometer . V̇se trial .The four subsequent experimental visits were completed in a different order for each participant to control for any potential order effect. At least 48 h separated experimental visits, and the means ± SD time to complete the four visits was 25 ± 7 days. Participants were instructed to avoid vigorous exercise and alcohol consumption in the 24 h preceding each visit. Following an overnight fast (including abstaining from caffeine), participants reported to the laboratory at 0800 and were provided with a standardized cereal breakfast consisting of 50 g cornflakes and 150-mL semiskimmed milk. The macronutrient content is unlikely to have influenced vascular function , 30. Pero2max , or two different 30 min HIIE protocols, with the work rate of the active intervals corresponding to either 75% (HIIE1) or 90% (HIIE2) V̇o2max. Specifically, HIIE1 included a 2.5-min warm-up and 2.5-min cooldown at 45% V̇o2max and five 3-min intervals at 75% V̇o2max, interspersed with four 2.5-min recovery intervals at 45% V̇o2max. HIIE2 included the same 2-min warm-up and cooldown, and five 2-min intervals at 90% V̇o2max, separated by four 4-min recovery intervals at 45% V̇o2max (o2max).Immediately following the assessment of baseline resting vascular measures, participants completed either seated rested in the laboratory , or 30 min of cycling at 60% V̇ V̇o2max . Each exo2 and end-tidal carbon dioxide (Breath-by-breath V̇AUC)  with a 13 MHz linear array transducer, in accordance with current guidelines . BrieflyAUC) . FMD wasexercise . The FMDexercise . The wit2   and end-tidal COween day . The witocapnia) . To addrocapnia) , 39, CVR : small <0.5, moderate 0.5–0.8, and large ≥0.8 (Data are presented as means ± standard deviation (SD). All analyses were performed using SPSS version 26 (IBM). Differences in the physiological responses during each of the experimental trials were explored using a one-way ANOVA. All vascular responses were analyzed using a mixed model ANOVA, with trial  and time  as the main effects. Statistical significance was accepted when 4, large . Follow-rge ≥0.8 .o2max 72 ± 19 W, 60% V̇o2max 118 ± 25 W, 75% V̇o2max 161 ± 29 W, 90% V̇o2max 211 ± 39 W. By design, mean V̇o2 was not different between exercise trials  and MIE , whereas HIIE1 was greater than MIE . Mean P = 0.007, d = 0.75) and HIIE1 . P < 0.001, d = 1.16) and in HIIE1 compared with MIE . Average MCAv mean , MCAv peak , and area under the MCAv mean curve versus time  were greater in HIIE1 compared with CON.The group responses to each experimental trial are presented in P = 0.002, η2 = 0.305, P = 0.029, d = 0.20) and HIIE2 .There was a time by trial interaction effect for brachial baseline diameter  after HIIE1 compared with pre. In the HIIE2 trial, the change in brachial artery diameter was greater 1 h  and 3 h  after HIIE2 compared with pre. Between trials, there were no differences between trials at the pre time point . One hour after exercise, the change in brachial artery diameter after occlusion was greater in the HIIE1 trial than CON  and MIIE  but not different compared with HIIE2 . The change in brachial diameter after occlusion was only different between HIIE2 and CON at the 3 h time point .There was a time by trial interaction for the absolute change in brachial artery diameter after occlusion , time , or trial by time interaction  for the time taken to achieve peak dilation after occlusion.There was no main effect of trial  or MIE (P > 0.521) trials. In the HIIE1 trial, FMD was elevated 1 h  and 3 h  after HIIE1 compared with pre. In the HIIE2 trial, FMD was elevated 1 h  and 3 h  after HIIE2 compared with pre.There was a time by trial interaction for allometrically adjusted FMD . One hour after exercise, FMD in the HIIE1 trial was greater than in CON  and greater than in MIIE  but not different compared with HIIE2 . FMD was also greater 1 h after HIIE2 compared with the same time point in CON  and greater than MIE . No differences between trials were observed at the 3 h time point .Between trials, there were no differences in FMD between trials at the pre time point . There was no effect of trial , time , or trial by time interaction effect  for SRAUC.Mean SRP = 0.713, η2 = 0.064, P = 0.02, η2 = 0.379). Resting MCAv was higher at the pre time point compared with 1 h post  and 3 h post  across the experimental trials.There was no trial by time interaction for resting MCAv , time , or trial by time interaction  for resting mean arterial pressure. There was no main effect of trial , time , or trial by time interaction for resting CVC .There was no main effect of trial . There was no time by trial interaction for the increase in MCAv during the hypercapnic challenge . There was no main effect of trial , time , or trial by time interaction  for CVR hypercapnia.The responses to the hypercapnic stimulus are presented in P = 0.594, η2 = 0.060). There was no time by trial interaction for the fall in MCAv during hyperventilation . There was no main effect of trial , time , or trial by time interaction  for CVR hypocapnia.o2max) acutely improved brachial artery FMD in healthy young adults, compared with MIE and a sedentary control condition. However, the present study found no effect of exercise intensity on CVR, with CVR to both hyper- and hypocapnia remaining unchanged following the exercise and sedentary control conditions. Finally, the present study found no significant differences in the overall MCAv response to exercise between exercise conditions (expressed as MCAv AUC), although the profile of MCAv during exercise differed between protocols.This study found that HIIE . It is possible this may have resulted in a similar shear stress stimulus between the two HIIE trials, where the same number of intervals were also performed. However, it was not possible to measure brachial artery shear stress during the exercise conditions, and technical challenges limit the ability to measure limb shear rates at such high intensities. Furthermore, other factors have previously been shown to influence postexercise changes in FMD  . It rema2 breathing, whereas Burma et al. used a rebreathing protocol, which elevates The data from the present study are in contrast to the only previous study investigating CVR following acute HIIE, where Burma et al.  observedsess CVR . As highsess CVR  and are o2max would elicit the greatest increases in MCAv during exercise, compared with HIIE performed at 90% V̇o2max and MIE at 60% V̇o2max, as this is the intensity that is thought to elicit the greatest increases in MCAv during incremental exercise  are almost double during HIIE compared with work-matched MIE .No conflicts of interest, financial or otherwise, are declared by the authors.A.R.B. and B.B. conceived and designed research; M.E.W., J.L.K., and A.B.L. performed experiments; M.E.W., J.L.K., A.B.L., and B.B. analyzed data; M.E.W., J.L.K., A.B.L., A.R.B., and B.B. interpreted results of experiments; M.E.W. and B.B. prepared figures; M.E.W. drafted manuscript; M.E.W., J.L.K., A.B.L., A.R.B., and B.B. edited and revised manuscript; M.E.W., J.L.K., A.B.L., A.R.B., and B.B. approved final version of manuscript.


Action processing is crucial for sports activities. Using event-related potentials (ERPs), the present study investigated whether semantics were activated in action processing and, if so, whether semantic activation was modulated by action duration. Badminton athletes were recruited to finish a lexical decision task following an action-semantic priming paradigm, in which short (400 ms) or long (1000 ms) action videos served as primes, and semantically congruent or incongruent action words served as targets. The ERP results showed a P300 effect, that is, larger P300 amplitudes were observed for targets primed by semantically incongruent action videos than for targets primed by semantically congruent action videos, only when the action videos were long and not when the action videos were short. Moreover, a late positive component (LPC) was only sensitive to action duration, showing that the targets primed by long action videos elicited larger LPC amplitudes compared to the targets primed by short action videos. These results suggested that semantics could be activated in action processing and that semantic activation was modulated by action duration, supporting a link between the language system and action processing. Increasing evidence has claimed that there are strong links between language and the motor system ,2. It haAction processing is a key component of human cognition and is especially important for excellent performance in sports activities . An obseRecently, several studies have focused on action processing at the abstract level and found that the language system might also contribute to action processing ,22. For Semantic priming has served as an important tool in studies exploring semantic processing in nonlinguistic stimuli, such as sounds , music , and odoFollowing previous studies ,24,25, aOverall, the present study aimed to investigate whether action semantics were predicted/activated during badminton action processing and, if so, whether and how semantic activation was modulated by action duration. Badminton athletes were instructed to finish a cross-domain semantic priming procedure followed by a lexical decision task, in which a long (1000 ms) or short (400 ms) action video served as the prime and a conceptual congruent or incongruent action word served as the target, and participants were asked to decide whether the target stimulus was a real word. We hypothesized that if the action concept could be activated during action processing, then the semantic priming effect would be observed, as indicated by an N400, P300, and/or LPC effect. In contrast, if the semantic concept could not be activated during action processing, then the semantic priming effect would not be observed. In addition, given that a long action video might provide more information than a short action video, we hypothesized that the semantic activation might be stronger when the action video is long than when the action video is short, as reflected by a stronger semantic priming effect for videos with long durations than for those with short durations.Twenty badminton players  participated in this study. They were qualified as the second level or above the level of the national standard . They had a mean of 9.6 years of training experience  and practised badminton at least twice a week, 2 h each time, in the last 2 years. All participants were right-handed with normal or correct-to-normal vision. They were native speakers of Mandarin Chinese and did not report any neurological disorders. Informed consent was obtained prior to the experiment. The data of three male participants were excluded from the statistical analysis, two due to partly missing data and another due to excessive artefacts.We used 120 video–word pairs as experimental materials in a lexical decision task. In each pair, a video served as a prime stimulus, and an action word served as a target stimulus. The videos were silent colour clips , selected from the 2018 World Badminton Championship and the last eight of the finals at the end of that year. Kinovea software was used to capture 1000 ms and 400 ms of the video up to the point of 40 ms before the contact between the shuttlecock and the racket . Each dups ≥ 0.844; M ± SD: long–congruent, 85.71 ± 11.26%; long–incongruent, 85.71 ± 11.26%; short–congruent: 85.24 ± 15.23%; short–incongruent: 85.24 ± 15.23%) or reaction time . These results indicated that the processing difficulty of the action videos was well matched among the four conditions.To match the processing difficulty of the videos among the four conditions, seven badminton players  who were qualified as the second level or above the level of the national standard were recruited to watch the videos and judge their action concepts by pressing “J”, “K”, or “L” to represent clear, drop, or smash, respectively, as quickly and accurately as possible. These players did not participate in the electroencephalogram (EEG) experiment. Two-way repeated measures analysis of variances (ANOVA) was performed with action duration (long or short) and semantical congruence (congruent or incongruent) as two factors, and the results did not reveal any significant effect of either accuracy  were presented in a pseudorandom order with no more than three consecutive trials under the same conditions. The 120 stimulus pairs were presented in two blocks, and each block consisted of 60 trials. In each trial, a white fixation cross was presented in the centre of a black background for 1000 ms. Then, a 1000 ms or 400 ms prime video was presented. Following a 100 ms black screen, the target stimuli were presented in white on a black background for 300 ms. Participants were instructed to watch the video and to judge whether the target stimulus was a word or not as quickly and accurately as possible  was recorded from electrodes placed above and below the left eye, and horizontal EOG was recorded from electrodes placed at the left and right outer canthi. The sampling rate was 1000 Hz. The impedance was kept below 5 kΩ for all electrodes.ps ≥ 0.10; M ± SD: long–congruent, 27.82 ± 1.94; long–incongruent, 27.47 ± 2.15; short–congruent, 28.12 ± 1.90; short–incongruent, 28.59 ± 1.91).EEG data were preprocessed using EEGLAB  and ERPLBased on visual inspection, larger P300 amplitudes were elicited by target words primed by incongruent action videos compared to those elicited by target words primed by congruent action videos in long duration conditions, and not in short duration conditions, and larger LPC amplitudes were elicited by target words primed by long action videos compared to those elicited by target words primed by short action videos. For statistical analysis, the latency windows of P300 and LPC were selected as 200–400 ms and 500–700 ms, respectively, based on previous studies ,35 and vp = 0.024, η2p = 0.28). However, follow-up simple effect analysis revealed neither a significant main effect of congruence for long or short durations , nor a significant main effect of duration for congruent or incongruent conditions .Mean accuracy and reaction time in the lexical decision task were computed, and the results are shown in p = 0.943, η2p ≤ 0.001) nor a significant main effect of congruence  = 0.65, p = 0.434, η2p = 0.04). There was also no significant interaction between duration and congruence  = 0.43, p = 0.521, η2p = 0.03).To compute the mean reaction time, the data with erroneous responses (4.90% of the data) and with reaction times shorter than 300 ms or longer than 1000 ms (5.44% of the data) were removed first. Then, two-way repeated-measures ANOVA was conducted for the remaining data. The results revealed neither a significant main effect of duration  = 0.01, Overall, the behavioural results did not reveal any significant effect involving duration and congruence factors.p = 0.007, η2p = 0.38), showing that incongruent words elicited larger P300 amplitudes than congruent words , 0.93 ± 0.30 μV). For the central regions, the ANOVA results revealed a significant main effect of congruence  = 4.99, p = 0.040, η2p = 0.24) and a significant interaction between congruence and hemisphere  = 4.43, p = 0.020, η2p = 0.22). Follow-up simple effect analysis revealed that incongruent words elicited larger P300 amplitudes over the right hemisphere than congruent words  = 7.15, p = 0.017, η2p = 0.31; mean ± SE: incongruent, 6.07 ± 0.87 μV; congruent, 5.37 ± 0.83 μV; difference (incongruent–congruent), 0.70 ± 0.26 μV) but not over the left and medial electrodes . For the parietal regions, the ANOVA results revealed neither a significant main effect of congruence nor a significant interaction between congruence and hemisphere . Overall, a congruence effect was observed only over the frontal and right central regions.In the 200–400 ms time window, there was a significant main effect of congruence and a significant three-way interaction of congruence × hemisphere × anteriority see . This thp = 0.045, η2p = 0.23; mean ± SE: long, 5.27 ± 0.88 μV; short, 3.70 ± 0.59 μV; difference [long-short], 1.57 ± 0.72 μV) but not over the left and medial electrodes .Moreover, repeated-measures ANOVA revealed a significant interaction between duration and hemisphere see . The folp = 0.003, η2p = 0.43) and a significant interaction between congruence and anteriority  = 6.57, p = 0.017, η2p = 0.29). The follow-up simple effect analysis revealed that, compared to congruent words, incongruent words elicited larger P300 amplitudes over all three anteriority levels  = 14.34, p = 0.002, η2p = 0.47; mean ± SE: incongruent, 6.10 ± 1.15 μV; congruent, 4.24 ± 1.10 μV; difference (incongruent–congruent), 1.87 ± 0.49 μV; central: F  = 8.20, p = 0.011, η2p = 0.34; mean ± SE: incongruent, 5.94 ± 0.89 μV; congruent, 4.96 ± 0.81 μV; difference (incongruent–congruent), 0.96 ± 0.34 μV; parietal: F  = 7.53, p = 0.014, η2p = 0.32; mean ± SE: incongruent, 4.20 ± 0.71 μV; congruent, 3.22 ± 0.66 μV; difference (incongruent–congruent), 0.98 ± 0.36 μV). For the short duration, the ANOVA revealed neither a significant main effect of congruence nor a significant interaction between congruence and anteriority , suggesting that no P300 effect was present in the short condition. Overall, a P300 effect in response to congruence was observed only in the long condition, not in the short condition.Most importantly, repeated-measures ANOVA revealed a significant three-way interaction of duration × congruence × anteriority see . This thIn the 500–700 ms time window, repeated-measures ANOVA revealed a significant main effect of duration see , showingOverall, the ERP results revealed a congruence effect for the long duration condition but not for the short duration condition in the 200–400 ms time window and showed a duration effect for both the congruent and incongruent conditions in the 500–700 ms time window.The human conceptual system encapsulates people’s knowledge about the world and supports a wide variety of cognitive operations, including attention, memory, language, thought, and sociocultural cognition . The preFollowing previous studies investigating conceptual processing in nonlinguistic systems ,24,25, wUnlike the behavioural data, the ERP data revealed a semantic priming effect with a modulation of the P300 component; that is, larger P300 amplitudes were elicited by targets primed by incongruent action videos compared to targets primed by congruent action videos over a long duration. The P300 component has often been observed in the “oddball” paradigm, with low-probability target items eliciting higher amplitudes than high-probability nontarget items, and has been linked to attention and subsequent memory processing . In the One might argue that there was a reverse N400 rather than a P300 effect in the early time window, that is, more negative-going waveforms were elicited by congruent targets compared to incongruent targets. Actually, a reverse N400 effect has been found for the priming effect, with more negative-going waveforms elicited by related targets than by unrelated targets when the prime was masked  or when To our knowledge, this is the first study to directly investigate whether action semantics can be predicted and activated in action processing. Our results indicated that individuals can predict the upcoming actions and activate the action semantics at the abstract representation level, providing neurophysiological evidence for semantic prediction and activation in action processing. The neural correlates of this semantic prediction and activation need to be explored in future studies: specifically, how the brain utilises the visually presented action information to infer the goal and intention at the abstract representation level in action processing. Moreover, contrasting ERPs elicited by words and pseudowords matched in visual properties would reveal getting access to lexico-semantic information, which will provide an opportunity to explore the potential difference between accessing to lexico-semantic and action-semantic information.The P300 effect was observed only in the long condition but not in the short condition, indicating that action duration modulates semantic activation in action processing. Action duration has been found to influence decision making in sports activities; that is, a long action video provides rich information to support athletes in comprehensively processing and making analytical decisions, whereas a short action video provides little information, and athletes can only make intuitive decisions . SimilarIt is worth noting that a block design was used in the present study to prevent the participants feeling the action videos disordered or guessing the presentation rules of the materials. However, the blocks with long duration videos would take more time that the participants may need to concentrate more, or they may be more engaged and paying more attention, resulting in semantic activation in long action videos condition rather than in short action videos condition. To exclude this possibility, a mixed design could be used to verify the results in the future.Moreover, one might argue that 400 ms video clips might be quite brief and ambiguous, and in this case the actions between clear/drop/smash were not easily distinguished. This might result in the absence of the P300 effect in the short condition. However, this possibility could be ruled out given that the action videos had been pretested offline and the mean accuracy of judging the corresponding action concepts was above 85% in the short duration condition (with no difference with the long duration condition).Significant effects of action duration on the LPC component were found with enhanced amplitudes observed for long durations than for short durations. However, no effects of action-semantic congruence were found on the LPC component. In previous studies, the larger LPC in response to semantically related targets compared to unrelated targets has been linked to the semantic relationship detection process ,41. IncoMoreover, given that the LPC has been proposed to reflect the retrieval of episodic information ,45, we tThe present study showed that the P300 and the LPC, two ERP components that index different stages of information processing, are differentially sensitive to action-semantic congruence and action duration in the action-semantic priming paradigm. Our data indicated that semantic concepts could be predicted and activated in badminton action processing and that semantic activation was modulated by action duration. These results support the strong link between linguistic system and action processing.