Himatanthusdrasticus, a plant from the Apocynaceae family. After harvesting, H. drasticus leaves were macerated and a hydroalcoholic extract (HDHE) and fractions were prepared. Antimicrobial tests, such as agar-diffusion, Minimum Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were carried out against several bacterial species. Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes and Klebsiella pneumoniae were inhibited by at least one extract or fraction in the agar-diffusion assay . However, the lowest MIC value was found for HDHE against K. pneumoniae. In addition, HDHE and its fractions were able to inhibit biofilm formation at sub-inhibitory concentrations (780 µg/mL and 1.56 µg/mL). As the best activities were found for HDHE, we selected it for further assays. HDHE was able to increase ciprofloxacin (CIP) activity against K. pneumoniae, displaying synergistic  and additive effects (CIP + HDHE: 3 µg/mL + 400 µg/mL). This action seems to be associated with the alteration in bacterial membrane permeability induced by HDHE (as seen by propidium iodide labeling). This extract was non-toxic for red blood cell or human peripheral blood mononuclear cells (PBMCs). Additionally, it inhibited the lipopolysaccharide-induced proliferation of PBMCs. The following compounds were detected in HDHE using HPLC-ESI-MS analysis: plumieride, plumericin or isoplumericin, rutin, quercetin and derivatives, and chlorogenic acid. Based on these results we suggest that compounds from H. drasticus have antimicrobial and antibiofilm activities against K. pneumoniae and display low cytotoxicity and anti-proliferative action in PBMC stimulated with lipopolysaccharide.Plant-derived products have played a fundamental role in the development of new therapeutic agents. This study aimed to analyze antimicrobial, antibiofilm, cytotoxicity and antiproliferative potentials of the extract and fractions from leaves of  Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) + compatible with the molecular formula C15H10O7. This compound showed a fragmentation pattern with important peaks at m/z 285.26, which was attributed to the loss of H2O, and at m/z 199.00 and 149.88 [2) was identified based on a peak at m/z 471.22 [M + H]+, compatible with the molecular formula C21H26O12. Fragmentation pattern of this peak revealed a loss of water and a glucose unit at m/z = 453.28 and m/z 291.10, respectively [Quercetin (d 149.88 . Plumierectively .3) was determined based on the peak m/z 611.31 [M + H]+ compatible with the molecular formula C27H30O16. Fragmentation of this the precursor ion lead to the loss of a rhamnose unit (146 u) and yields an ion at m/z 465.20, while an aglycone ion at m/z 303.08 was obtained through loss of a hexose unit (162 u) [m/z 291.12 [M + H]+ was compatible with the molecular formula C15H14O6, and was attributed to plumericin or isoplumericin (4). Fragmentation provided important peaks at m/z 263.06  and m/z 230.98 , due to the neutral loss of ethylene unit and an acetyl unit, respectively +, compatible with the molecular formula C26H28O16. Fragmentation of this peak showed that a successive loss of a pentose and hexose unit, leading to the peaks at m/z 465.22 and m/z 303.06, respectively. This compound was identified as quercetin 3-apiosyl-(1->2)-galactoside (5) or quercetin 3-α-l-arabinopyranosyl-(1->6)-galactoside (5) or quercetin 3-lathyroside (5). However, the current study was not able to identify the isomer. The same situation happened with the peak at m/z 460.20 which was attributed to isoquercitrin or quercetin-3-O-glucoside or quercetin-3-β-O-glucoside (6). A loss a glucose unit (C6H10O5) yielded a fragment at m/z 303.04, which characterizes an aglycone ion [m/z 355.19, which corresponds to the molecular formula C16H18O9. A single fragment at m/z 162.84 was used for comparison with the Metlin database, allowing to identify this compound as chlorogenic acid (8) [Also, it was detected a peak at at cone ion . In addiacid (8) .H. drasticus. HDHE showed the best activity against K. pneumoniae  [K. pneumoniae has a double cell wall that constitutes an extra protection against the antimicrobial action by antibiotics, making the search of active compounds against this pathogen a hard task [This work aimed to analyze some biological activities of HDHE and its fractions obtained from leaves of tio < 2) . This eftio < 2) . Like otard task .H. drasticus extract on membrane permeability may explain its ability to increase ciprofloxacin activity, as result of an improvement in drug uptake. In summary, the combination of natural products and antibiotics is considered to have some benefits, such as improvement of therapy effectiveness and drug shelf life [In the present study, ciprofloxacin associated with the extract presented synergistic and additive actions. Ciprofloxacin is a synthetic quinolone with broad-spectrum antibacterial action. It induces structural damage in bacterial DNA resulting in the activation of the SOS response repair pathway associated to acquisition of virulence and drug resistance genes . The actelf life .K. pneumoniae biofilm formation, which, together with synergistic and additive to ciprofloxacin, may represent a potential for drug development.Another important strategy to fight microbial infections is the inhibition of biofilm formation . BiofilmH. drasticus, no toxicity was observed. These results suggest a selective action of this extract towards K. pneumoniae membrane.It was also analyzed whether the active extract had the ability to modulate LPS-induced PBMC proliferation. LPS  is a potent inducer of human T-lymphocyte proliferation as part of host pro-inflammatory response . In addiFinally, the presence of several bioactive compounds was detected by HPLC-MS, which may explain the pharmacological characteristics of the plant under study. For example, plumieride, plumericin, and isoplumericin have proven antimicrobial, anti-inflammatory and antiproliferative activities ,54,55. OH. drasticus were collected in the urban area of São Luís-MA (2°30′48″ S 44°17′20.7″ W), in November/2015. A sample of the plant  was sent to the Herbarium “Ático Seabra” of the Federal University of Maranhão (UFMA) for identification and was cataloged under registration number 1032/2015.Leaves of m/v. The macerate underwent a daily shaking process (three times per day) for 48 h. After maceration, the solution was filtrated and concentrated in a rotary evaporator under reduced pressure at 50 °C. After concentration, the final solution was termed H. drasticus hydroalcoholic extract (HDHE). HDHE was fractionated by liquid-liquid partition using 10 g of dry extract, which was resuspended in a hydromethanolic solution . This solution was submitted to successively liquid-liquid partitions using solvents with increasing polarity, yielding a hexane fraction (HDHEF), ethyl acetate fraction (HDEAF) and butanol fraction (HDBF).After harvesting, leaves (500 g) were cleaned with tap water and placed in an amber bottle containing a 70% ethanol solution, at a ratio of 1:3 Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), Salmonella enterica serovar typhimurium (ATCC 14028), Listeria monocytogenes (ATCC 15313), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 10031), Acinetobacter baumanni (ATCC 19606), Enterococcus faecalis (ATCC 19433).The microbial strains used in this work were obtained from the Culture Collection (Laboratory of Microbiology) of the CEUMA University, including 8 CFU/mL) were prepared. Each bacterial strain was seeded on Mueller-Hilton agar (MHA) plates . Next, wells (6 mm diameter) prepared in MHA plates were filled with 50 μL of 200,000 µg/mL extract/fractions solutions . Dimethylsufoxide  and ciprofloxacin  were used as negative and positive controls, respectively. The plates were incubated at 35 °C. After 24 h, the formation of inhibition diameter zone (IDZ) around the wells was recorded . The experiments were performed in triplicate.From recent overnight cultures, microbial suspensions with turbidity equivalent to 0.5 tube of the McFarland scale (1.5 × 108 CFU/mL) was added in each well. Ciprofloxacin (50 µg/mL suspension) and DMSO (1% suspension) were used as controls. The plates were incubated at 35 °C for 24 h, prior the addition of 30 μL of 0.03% resazurin sodium solution (Sigma-Aldrich®), an oxide-reducing indicator). After 40 min, changes from blue color to pink color were considered as microbial growth. Minimum Inhibitory Concentration (MIC) was defined as the lowest concentration capable of inhibiting bacterial growth. For determination of Minimum Bactericidal Concentration (MBC), samples (10 μL) of those wells where the inhibition was observed were transferred to MHA plates. After 24 h incubation, MBC was defined as the lowest concentration able of eliminate bacteria.HDHE and its fractions were serially diluted in 96-wells plates containing Mueller-Hinton broth . Concentrations ranging from 50,000 µg/mL to 48.82 µg/mL were tested. Subsequently, 1 μL of a microorganism suspension  were mixed to 140 μL of MHB and 50 μL of extract and fractions to reach subinhibitory concentrations (780 µg/mL and 1560 µg/mL). After 24 h of incubation at 37 °C, the formed biofilm was fixed with methanol (P.A.), stained with violet crystal (0.1%) and washed with ethanol (P.A.). As negative control, culture medium with bacterial solution was used. Biofilm biomass was measured using a spectrophotometer at 550 nm .K. pneumoniae ATCC 10031 by agar diffusion assay and microdilution assay. The diffusion agar assay was performed as described above, except for the addition of hydroalcoholic extract  to ciprofloxacin disks (5 μg). After 24 h, each IDZ was read and compared with those obtained using ciprofloxacin alone. In the microdilution assay, different combinations of HDHE and ciprofloxacin were tested as described in the MIC section. The results were used to calculate the fractional inhibitory concentrations (FIC), using the following equation [FIC ≤ 0.5: synergistic effect0.5 < FIC ≤ 1: additive effect1.0 < FIC ≤ 4: no interactionFIC > 4: antagonismThe effect of the active extract in combination with antibiotics was evaluated against equation :FIC:MICMMICA: MIC of extract when in combination with drug; K. pneumoniae suspension (1.5 × 108 CFU/mL) were treated with different concentrations of HDHE  for 4 h. Microorganisms killed by heat (100 °C for 10 min) were used as positive control, and bacteria grown without extract were used as negative control. Bacteria were centrifuged at 3000 RPM for 10 min, washed (2 times) and resuspended in 30 μM propidium iodide . The tubes were incubated for 10 min in the dark. The samples were collected on the flow cytometer  and data were analyzed using the FlowJo software . The results represent the percentage of incorporation of PI by bacteria (loss of membrane integrity).Aliquots of 1 mL of Universidade CEUMA (CEP-UNICEUMA) under registration number no. 44785215.6.0000.5084. The tests were performed in accordance with the Declaration of Helsinki 1975, revised in 2008.Blood samples were collected from three healthy volunteers with no recent history of taking either antibiotic or anti-inflammatory drugs, and/or infectious or inflammatory diseases in the last 3 weeks prior to sample collection; after a written informed consent was obtained. The study was reviewed and approved by the Human Research Ethics Committee of the After collection by venipuncture, whole human blood samples (5 mL) were centrifuged for 10 min at 1500 RPM, serum was discarded and pellet was washed 3 times and resuspended in PBS (pH = 7.4), yielding a 1% erythrocytes suspension. The erythrocytes suspensions were treated with HDHE , PBS (negative control), or hydrogen peroxide . After shaking for 60 min, the samples were centrifuged and the absorbance of the supernatant was read at 540 nm .® by Life Technology™, Grand Island, NY, USA) culture medium supplemented with 10% fetal bovine serum (FBS) and 1% of antibiotics  and 1% of antifungal .Samples of 5 mL of human peripheral blood were added to histopaque–containing tube (1:1 ratio). After centrifugation, peripheral blood mononuclear cells (PBMC) were collected from the formed ring. Cells were washed with PBS and resuspended in Dulbecco’s Modified Eagle Medium  were labeled with 10 μg/mL carboxyfluorescein succinimidyl ester  for 30 min at 37 °C, washed (2 times) and treated with the extract at the concentrations of 780 µg/mL and 1560 µg /mL in the presence or absence of LPS . PBMC treated with LPS or cultured with culture medium alone were used as controls. After 72 h, cells were harvested and proliferation was assessed by flow cytometry  and data were analyzed using the FlowJo software. The results represent the percentage of cells that underwent cell division.Samples of 1 mL of PBMC ; solvent B = MeCN. Elution profile = 0–5 min, isocratic 5%B; 5–6 min, linear gradient 5–20%B; 6–10 min, isocratic 20%B; 10–15 min, linear gradient 20–50%B; 15–20 min, linear gradient 50–72%B; 20–30 min, isocratic 72%B; 30–40 min, linear gradient 72–100%B; 40–45 min, linear gradient 100–5%B, 45–50 min, isocratic 5%B. The gradienty profile is shown in the m/z 100 and m/z 1500 in positive ion mode (ESI+). The following mass spectrometry parameters were maintained in the analysis: capillary 4500 V; drying gas flow 7.00 L·min−1; drying gas temperature, 300 °C; nebulizer gas pressure, 27.00 psi. After HPLC-ESI-MS run, the retention time and m/z for each chromatographic peak was used to determine MS/MS fragmentation parameters.The HPCL-ESI-MS analyses were performed at the Central Instrumentation Laboratory of the University of São Paulo using an LC-20A chromatography system  comprised of a CBM-20A controller, LC-20AD pump, SPD-20AC detector and a CTO-20A column oven and SIL 20AC—Shimadzu detector, coupled to an Amazon Speed ETD ion trap spectrometer . The HPLC analysis was performed using a Luna C18 (250 × 4.6 mm–5 µm) column  held at 40 °C. The following elution gradient was employed with a flow rate of 1 mL·minp-value of < 0.05 was considered as statistically significant. All tests were performed in at least two independent assays in quadruplicate. For flow cytometry assays, at least 20,000 cells were analyzed.Statistical analyses were performed using the software GraphPad Prism version 5.01 . Data were analyzed by two-way analysis of variance (ANOVA) followed by Tukey test. A K. penumoniae, since significantly diminished its viability and inhibited biofilm formation. The action mechanism is associated to the damage of bacterial membrane, which may be also related to HDHE ability to increase the activity of ciprofloxacin, as we demonstrated synergistic and additive actions. HDHE also reduces proliferation of human PBMCs stimulated with LPS, presenting no significant cytotoxicity. The bioactive compounds found in the extract by HPLC-MS analysis  have been reported as antimicrobial and anti-inflammatory agents. Particularly, the results regarding the inhibition of K. pneumoniae biofilm encourages further studies with H. drasticus derivative products to develop new compounds to be used in control of device-related infections.The results showed that HDHE can act as antimicrobial against


Gastrointestinal mucositis is a serious side effect of chemotherapy. Currently, no effective treatment exists for chemotherapy-induced mucositis, prompting the need to develop an anti-mucositis agent for use in clinics. The present study investigated whether azatyrosine-PBHA (AzP), a histone deacetylase inhibitor, has a therapeutic effect on intestinal mucosa. The results indicated that AzP did not affect the proliferation and viability of cancer cells, outcomes that are achieved by suberoylanilide hydroxamic acid (SAHA). However, AzP could decrease production of the inflammatory mediators interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and tumor-necrosis factor-α (TNF-α). In vivo histopathological assessment showed that AzP reduced cisplatin-induced injury to the jejunum villi and triggered weight loss in the C57BL/6 mice. Immunohistochemistry (IHC) results demonstrated that mice treated with AzP also recovered from cisplatin-induced injury to the intestinal mucosa. Mechanistic in vitro study using DAVID/KEGG enrichment analysis of microarray data and confirmation by a Western blot indicated the influence of AzP on the MEK/ERK and AKT-dependent pathway. In conclusion, the study demonstrated that AzP might regulate the MEK/ERK MAPK signaling pathway to attenuate MCP-1, TNF-α, and IL-6 production and provide opportunities for the development of new anti-inflammatory drugs targeting mucositis. Gastrointestinal mucositis is a frequent and severe side effect of radiotherapy and chemotherapy in cancer patients ,2. ApproHistone acetylation modulated by histone deacetylases (HDACs) serves as a key regulatory mechanism for gene expression and is involved in numerous developmental processes and diseases . Many hiThere are very limited therapeutic agents available for mucositis treatment , and an w/w) was selected for further research. Gavages of 50, 100, and 250 mg/kg of AzP were performed on days −1, 0 and 1, while cisplatin (11 mg/kg) was administered via intraperitoneal (IP) injection to C57BL/6 mice on day 0. The control mice were given PO HPβCD via sham-injection. No mortality was observed over the study period; however, cisplatin induced weakness in all animals within the cisplatin treatment group. This was followed by weight reduction throughout the experimental period. Relative mean body weight change (%) was recorded daily and expressed as mean ± standard deviation(SD) from baseline (day 0 = 100%). At day 5, 11 mg/kg cisplatin-only treatment reduced body weight to 76.0 ± 4.7%, while administering with AzP 50 mg/kg, 100 mg/kg, and 250 mg/kg at day 5 rescued the AzP-reduced weight loss to 78.7 ± 4.7, 80.5 ± 3.4 and 89.1 ± 8.1%, respectively  was used as an excipient with AzP, and the ratio 1:10 , the best characterized inhibitor, was used for comparison. 50 of pan-HDAC, HDAC1, HDAC 3, and HDAC 4 of AzP were 1650 ± 103 nM, 3440 ± 680 nM, 660 ± 99 nM and 1970 ± 110 nM, respectively. Results from Western blot also confirm that AzP elevated histone acetylation  while SAHA exhibited more potency in decreasing cell viability  was then performed twice in terms of the technical replicates, with a reproducibility of more than 0.975. Criteria of identification of differentially expressed genes were: fold change ≥2 or ≤ 2 and p-value < 0.05. Results showed that 2420 and 74 of 20,715 genes were significantly affected by AzP/DMSO and LPS/DMSO treatment, respectively. A total of 1159 and 25 descending genes and 1261 and 49 ascending genes were identified from AzP/DMSO and LPS/DMSO comparison, respectively. The heat maps of up-regulation (red), down-regulation (green), and mean gene expression (black) of 836 inverse correlation genes between two comparisons (AzP/LPS vs. LPS/Control) followed by hierarchical clustering analysis are presented in To confirm the therapeutic efficacy of AzP in C57BL/6 mice, duplications of the small intestinal epithelial cell line, IEC-6, cells were treated with 1% dimethyl sulfoxide (DMSO) , lipopolysaccharide  , or AzP (20 μM) co-treated with LPS (500 ng/mL)  for 8 h. Microarray using ROA v2.1 Rat OneArrayp < 0.5, To further investigate the possible mechanism underlying the anti-inflammatory effect of AzP, phosphorylation of MAPK proteins and Akt was tested in IEC-6 cells stimulated with 500 ng/mL LPS and/or 5, 10, and 20 μM AzP treatment. LPS significantly induced ERK phosphorylation up to 1.8 ± 0.3-fold compared to the control at 10 min p < 0.5, A. 5–20 μTo test whether AzP possesses potential genotoxicity, the Ames test, chromosome aberration test, and micronucleus test were performed. No mutagenicity or clastogenicity were found . In Ames tests, the number of revertants did not increase after treatment with various concentrations of AzP with or without S9 metabolic activation in all 5 test strains   B. The mon = 2) C. Mitogen = 2) A; p38 anTo conclude, AzP showed strong inhibitory effect on chemotherapy-induced mucositis, possibly through MEK/ERK and/or PI3K/Akt pathway. Combined with the negative results from genotoxicity evaluation, we believe AzP possesses high therapeutic potential as a safe and strong anti-inflammatory agent for mucositis treatment.Azatyrosine-PBHA AzP, A was synFetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F-12K medium, penicillin/streptomycin/glutamate and trypsin-EDTA were obtained from Gibco BRL . The horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) Ab and HRP-conjugated anti-rabbit IgG Ab were obtained from Amersham Biosciences . Except for acetyl-Histone H3 , other primary antibodies were purchased from Cell Signaling Technology . All of the other chemical reagents were purchased from Sigma-Aldrich .2 at 37 °C.Rat intestinal epithelial cell line, IEC-6, and Chinese hamster ovary epithelial cells (CHO-K1) were obtained from American Type Culture Collection . IEC-6 cells were cultured in DMEM high glucose medium (4.5 g/L) with 10% heat-inactivated fetal bovine serum (FBS) while CHO-K1 cells were maintained in F12K high glucose medium (4.5 g/L) supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere of 5% COPan HDAC enzyme , HDAC subtype 1, 3 and 4 activity were determined by using the HDAC fluorometric cellular activity assay  according to the manufacturer’s protocol. Different concentrations of AzP and SAHA were used, and the fluorescence intensity was measured on a fluorometric reader using excitation/emission wavelength of 360/460. Percent activity for each of the test compounds is calculated as follows: 50 corresponds to the drug concentration which achieves 50% activity of the untreated control. IC50 was then calculated and represented as mean ± SD.Activity = {(Sc − B)/(S° − B)} × 100. Wherein, S c represents the signal measured in the presence of AzP or SAHA, S° denotes signal measured in the absence of AzP or SAHA, B is measured in blank wells containing the medium alone. ICFollowing acclimatization, 25 male ICR mice  were randomly placed in 5 cages for micronucleus testing lasting 1 week. In addition, 45 female C57BL/6 mice  were used as mucositis models. All the animals were kept under a 12 h light/dark cycle at 23 ± 1 °C and 39–43% relative humidity. All animals had free access to water and food . All experimental procedures followed the “Guide for the Care and Use of Laboratory Animals” . The experimental designs were reviewed and approved by the Institutional Animal Care and Use Committee of Taipei Medical University .For the mucositis model experiments using chemotherapy, 45 female C57BL/6 mice were randomly placed in 5 groups for 3 days of treatment with AzP (50–250 mg/kg) P.O. on days −1, 0 and 1. Cisplatin (11 mg/kg) was intraperitoneally injected on day 0 as the chemotherapy. Control mice were injected with an appropriate vehicle. Mice were weighed daily and expressed as relative mean body weight change (%) from baseline (day 0 = 100%) and monitored closely for signs of morbidity over the course of the weeks to follow. On days 3 and 5, 3 of 9 mice were randomly chosen from each group and sacrificed for further intestinal analysis B. ControHistological and immunohistological observations of the intestine were performed on mice pretreated with AzP or HPβCD, followed by a course of cisplatin chemotherapy as described above. The mice were euthanized, intestines were removed, and a segment of the swiss-rolled trimming jejunum underwent formalin fixation and paraffin embedding. Hematoxylin and eosin stain were performed to observe the morphometry of villus length and crypt depth . Also, tg for 10 min at 4 °C and the supernatant was collected. Protein separation was performed using 10% reducing SDS-PAGE, electrotransferred onto polyvinylidene difluoride (PVDF), and immunoblotted with antibodies against inflammatory proteins as well as β-actin as control. Densitometry was performed using BioLight software V2000.01 . The optical density of specific protein was normalized to an internal control and expressed as a ratio to control.IEC-6 cells were incubated in different AzP concentrations with/without LPS stimulation for 24 h. The homogenate from cells or tissues was then centrifuged at 14,000× 260/280 values (>1.8) and OD260/230 values (>1.6), and the yield and quality were assessed using an Agilent 2100 Bioanalyzer . The ROA v2.1 Rat OneArray®  was used to assess gene expression with a total of 20,715 probes corresponding to the annotated genes in RefSeq v65 and Ensembl v76 databases, and 485 control probes used for the purposes of the study. The signal intensities were loaded into Rosetta Resolver® System  to do data preprocessing and 75 percentile centering normalization was applied. The errors of the sample were estimated by using the error-weighted approach at the same time. Both fold change and p-value for pair-wise sample comparison were calculated to evaluate differentially expressed genes, which was followed by further KEGG enrichment analysis . Using the DAVID v6.8 system , unsupervised hierarchical clustering by was performed by average linkage algorithm on selected differentially expressed gene lists after data transformation and mean centering.IEC-6 cells were treated with 1% DMSO, lipopolysaccharide , or AzP (20 μM) for 8 h, and samples from an independent duplication were collected for DNA microarray. Total RNA was extracted from cells using TRIzol reagent . The RNA concentration and purity were checked using ODSalmonella typhimurium, TA98, TA102, TA100, TA1537, and TA1535, were obtained from Moltox . Mutagenicity was evaluated by the incorporation method in the presence or absence of S9 metabolic activation [9 cells/mL), 2 mL soft agar , 0.5 mL S9 mixture (if necessary), and AzP were mixed well in a test tube. Immediately following mixing, the sample was poured into a minimal agar plate , and the plate was incubated for 48 h at 37 °C in the dark. The revertant colonies were counted, and a positive mutagenic response was identified when (i) the number of revertants was at least double the spontaneous yield; (ii) a statistical significance (p ≤ 0.05) was found; and (iii) a reproducible positive dose response was present.Five strains of tivation . A volum4) were dispensed in a 96-well plate for 24 h. Before being treated with varying concentrations of AzP (5–50 μM) or SAHA (5–50 μM) for 48 h, cells were fixed in 10% trichloroacetic acid for 10 min and washed with distilled water twice to remove excess trichloroacetic acid. Cells were stained with a 0.4% SRB solution dissolved in 1% acetic acid for 10 min and washed with 1% acetic acid twice to remove excess dye. The protein-bound dye was dissolved in 100 μL Tris-based solution (10 mM), and the absorbance was measured at 515 nm using a microplate reader. Cell Proliferation % = {(T48 − T0)/T0} × 100. Wherein, T48 represents the signal measured after 48-h treatment, T0 represent the signal measured before treatment at time 0. Relative cell proliferation (%) were expressed as mean ± SD compare with control. The cell proliferations of A549 and Met-5A cells were determined using the SRB assay. Cells , benzo(a)pyrene (5 μg/mL), or AzP  with or without S9 metabolic activation. Colemid (0.1 μg/mL) was administered 3 h before trypsinization, followed by treatment with 0.9% sodium citrate at 37 °C and Carnoy’s solution . The cells were stained with 3% Giemsa solution in 0.07 M phosphate buffer (pH 6.8) on glass slides. One hundred well-spread cells were observed for chromosomal alterations on each slide. The data were expressed as the total mean number of chromosomal aberrations per treatment ± standard deviation (SD) from three independent experiments.Chinese hamster ovary epithelial cells  were cultured in 6-cm dishes at a density of 2 × 10n = 5) were administered AzP orally by gavage, at doses of 5, 50, and 500 mg/kg body weight. Cyclophosphamide (200 mg/kg body weight) was administered as a positive control through intraperitoneal injection. At 24, 48, and 72 h after dosing, 2-μL blood samples were collected from the tail vein onto a slide stained with acridine orange (40 μg/mL). The ratio (%) of polychromatic erythrocytes (PCEs) to total erythrocytes was determined using fluorescence microscopy. In addition, the frequency of micronucleated polychromatic erythrocytes  was counted to a total of 1000 erythrocytes or PCEs per animal.Male ICR mice (n ≥ 3). Statistically significant differences between groups were determined using one-way analysis of variance (ANOVA) where a p value < 0.05 was considered statistically significant.All collected data were expressed as the means ± SD from at least 3 independent experiments  on chemotherapy-induced gastrointestinal mucositis. Results showed that AzP has promise in the prevention of the gastrointestinal mucositis that occurs during chemotherapy, and shows no genotoxicity and no clastogenicity.


Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and Listeria monocytogenes. The highest concentration with sensory acceptability was 6.25 g/L. At such concentration, the extract preserved fresh-cut fruits, increasing polyphenols (0.427 g GAE/kg fw (fresh weight)), flavonoid content (0.234 g QE/kg fw) and antioxidant activity (DPPH = 2.814 and ABTS = 0.551 mol TE/kg fw). It also reduced inoculated bacteria (range: 5.50 × 103 to 1.44 × 105 colony forming units (CFU)/g). These results showed the importance of considering consumer acceptability to determine the effective concentration of plant extracts as additives.Plant extracts have the potential to be used as food additives; however, their use have been limited by causing undesirable changes in the sensory attributes of foods. We characterized the mango seed extract as a preserving agent for fresh-cut mangoes. We established the maximum concentration of extract that, while increasing the antioxidant activity, and limiting microbial contamination of the fruit, did not negatively affect fruit sensory acceptability. The extract contained 277.4 g gallic acid equivalent (GAE)/kg dw (dry weight) of polyphenols and 143.7 g quercetin equivalent (QE)/kg dw of flavonoids. Antioxidant capacity values were 2034.1 and 4205.7 μmol Trolox equivalent (TE)/g against 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals, respectively. Chromatographic analysis revealed the presence of gallic and chlorogenic acids. The extract (16 g/L) inhibited the growth of  The consumption of healthy and ready-to-eat foods has been increasing in recent years due to its great convenience for the population. In this sense, the food industry has promoted the consumption of fresh cut fruits. However, owing to their nature, they are highly perishable and it is necessary to use preservatives to lengthen their shelf life. Currently, consumers demand products with great nutritional value and without synthetic preservatives, so it is necessary to look for antimicrobial and antioxidants natural agents. Plant extracts are among the most studied ingredients to achieve this goal. Taking this into account, the use of agro-industrial byproducts has been proposed as an alternative due to them being a rich and economic source of polyphenol compounds with bioactive properties ,2.The seed, one of the byproducts of mango processing, shows strong antioxidant capacity and it contains a concentration of bioactive polyphenols higher than, not only mango pulp and peel, but also seeds from other plant species ,4. MajorPhenolic compounds are the main constituents of the mango seed and such constituents can have predominantly sweet, bitter, pungent or astringent flavors . Some plMangoes “Haden” variety were obtained in a mature-green stage from a local market in Hermosillo, Sonora, Mexico. Fruits were selected according to homogenous color and free of physical damage. Mangoes were washed with chlorinated water (200 ppm) for 3 min and then, were allowed to dry at room temperature. The peel and seed were removed and the seed was stored at −20 °C for further use. The pulp was cut into small pieces and was physically and chemically characterized  15]. Co. Co15]. Mango seeds were chopped in small pieces (10 g) and put in a flask with 100 mL of ethanol: water (7:3). The sample was left to macerate in darkness for 10 days at 25 °C . SubsequTM system  linked to a PDA 2996 photodiode array detector (Waters). Ultraviolet-detection wavelength was set at 280 nm. Empower software (Waters) was used for controlling the instrument as well as for data acquisition and processing. The analysis was done at 30 °C using a reversed phase column . The mobile phase consisted of solvent A (7.5 mM acetic acid) and solvent B (acetonitrile) at a flow rate of 250 µL/min. Gradient elution was used starting at 5% solvent B for 0.8 min, 5–20% solvent B for 5.2 min, isocratic 20% solvent B for 0.5 min, 20–30% solvent B for 1 min, isocratic 30% solvent B for 0.2 min, 30–50% solvent B for 2.3 min, 50 –100% solvent B for 1 min, isocratic 100% solvent B for 1 min, and finally 100–5% solvent B for 0.5 min. At the end of this sequence, the column was equilibrated with initial conditions for 2.5 min. The pressure ranged from 6000 to 8000 psi during the chromatographic run and the effluent was introduced to a liquid chromatography detector  and the injection volume was 10 µL   . The ABTEscherichia coli O157:H7 ATCC 43890, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, Listeria monocytogenes ATCC 7644, and Staphylococcus aureus ATCC 6538. The minimum inhibitory concentration (MIC) was determined using the micro-well dilution assay as previously described [max (CFU) for each growth curve, were calculated using the Baranyi function and the R2 was calculated. All analyses were done in triplicates. Antibacterial activity of mango seed extract was tested against escribed . The ranescribed . The expTo determine the maximum acceptable concentration of mango seed extract added to fresh-cut mangoes, a preliminary sensory analysis was done with 100 consumers (20–60 years old) who were not aware of the treatment. For this, mangoes cv “Haden” were sanitized and cut in cubes of 2 cm. Subsequently, cubes were immersed for 2 min within aqueous solution of seed extract at different levels ; then, were allowed to dry and stored for 10 days at 5 °C. Sensory attributes were evaluated using a hedonic scale as ‘‘dislike very much”, “moderate dislike”, ‘‘neither like nor dislike”, “moderate like” and ‘‘like very much”  . Resultsg for 15 min at 4 °C  and filtered. This was repeated twice with 10 mL of methanol (80%) and the volume of the three supernatants were pooled and brought up to 40 mL [We assessed total phenol and flavonoid content and antioxidant activity of stored (10 days at 5 °C) fresh-cut mangoes treated or not with 6.25 g/L of mango seed extract. Sample tissue (10 g) was homogenized in 15 mL of methanol 80%), sonicated 30 min at 1 °C , centrifuged at 1200 0%, sonicE. coli, S. Typhimurium, L. monocytogenes and S. aureus with an inoculum of 1 × 106 CFU/mL, respectively) and were dried for 30 min in a biosafety cabinet  [Mangoes were sanitized and chopped in cubes of 2 cm. Samples (10 g) were inoculated by immersion during 2 min in different solutions of bacteria  . The inimax and Ymax). Also, the effect of mango seed extract on sensory acceptability, antioxidant activity and bacterial load reduction of fresh-cut mango was tested. Analysis of variance (ANOVA) was done (p < 0.05) to estimate significant differences between treatments and Tukey’s mean test was used for comparison (p < 0.05) using the software NCSS 2007 . A complete randomized design was done for all experiments. The effect of mango seed extract was tested on the kinetic parameters of bacteria growth , caffeic (0.770 g/kg dw), ferulic (0.104 g/kg dw) and cinnamic acids (0.112 g/kg dw). Mango seed extract showed a total phenolic and flavonoid content of 277.4 ± 0.75 g GAE/kg dw and 143.7 ± 1.86 g QE/kg dw, respectively. The main phenolic constituents identified in the seed extract were gallic (0.160 g/kg dw) and chlorogenic (0.100 g/kg dw) acids . AccordiE. coli, S. Typhimurium, L. monocytogenes and S. aureus at a MIC of 16 g/L and a MBC of 18 g/L for Gram-negative and 16 g/L for Gram-positive bacteria. Exposure to mango seed extract affected all bacterial growth parameters (E. coli and decreased both µmax (68.3%) and Ymax (39%) when compared to control. Kinetic parameters of S. Typhimurium were also affected at 16 g/L, with an extension of lag phase (10.5 h) and reduction of µmax (65.7%) and Ymax (39%). Similarly, the seed extract at 16 g/L extended the lag phase (12.5 h) and decreased µmax (56.3%) and Ymax (56.6%) of S. aureus. Regard of L. monocytogenes, the extract at 16 g/L was able to extent the lag phase to 24 h and no changes were observed in µmax nor Ymax. In addition, mango seed extract was able to inhibit rameters . An extrIt has been reported that Gram-negative bacteria are more resistant to mango seed extract  comparedPrevious studies showed that mango extracts contained compounds with higher molecular weight such as tannins, not detected here and that only simple compounds such as gallic acid were identified . This mip > 0.05) between treatments and consumers scored “liked very much” the odor of treated and control fruits. Color acceptance by consumers differed significantly among treatments. The panelists scored better on the color of the control fruits. Color acceptance of fruits treated with 1.5, 3.12 and 6.25 g/L showed moderate liking levels; while the fruits treated with the highest concentrations (10 and 12 g/L) were not pleasant for the panelists. Taste acceptability was positively affected using 1.5, 3.12 and 6.25 g/L of seed extract, and showed higher liking levels respect to the control (p < 0.05). However, all treatments were scored as “moderate like”. According to these results, 6.25 g/L of mango seed extract was selected as the maximum acceptable concentration that did not affect odor, color and taste of fresh-cut mangoes. The effect of mango seed extract on overall acceptance of fresh-cut mango storage for 10 days at 5 °C is showed in n = 100), and to evaluate the antioxidant and antibacterial activity of fresh-cut mangoes treated during storage. The effect of mango seed extract (6.25 g/L) on the total acceptability of the fresh-cut mangoes stored for 10 days at 5 °C is showed in p < 0.05) between the color of control and treated fresh-cut mangoes at 10 d of storage at 5 °C. Despite this significant difference, the level of color acceptance of treated fruits was scored as “moderate like” by consumers. However, no differences were found (p > 0.05) in odor and taste attributes between treated and control fruits, being both scored as “moderate like”. Mango seed extract at 6.25 g/L did not impact negatively on the taste of fresh-cut mango, and consumers expressed a better taste in treated fresh-cut mangoes indeed. This concentration was selected for further sensory evaluation . A similar result was observed with respect to the flavonoid content, which resulted higher in treated mango at day 0 than control fruits. At the end of storage, treated fruit retained its flavonoids level, in contrast to the control; this indicated that mango seed extract might have contributed to the preservation of flavonoid levels. Treated mangoes showed higher antioxidant activity than the control at day 0 and at the end of the storage, and an increment in antioxidant capacity during storage. Regarding the global effect, mango seed extract at 6.15 g/L increased the capacity to inhibit DPPH radical /kg fw), as compared to the control (2.10 mol TE/kg fw) (p < 0.05). Similar results were observed, using ABTS as compared to the control  (p < 0.05). Treatment with mango seed extract, increasing the total polyphenols and strengthening the antioxidant capacity of fresh-cut mango, places this as a potential candidate for the role of functional food with beneficial effects on human health.A significant effect of the treatment (6.25 g/L of seed extract) was observed on total phenolics, flavonoid content and on the antioxidant capacity of fresh-cut mangoes . OverallThe increase of antioxidant capacity exhibited by treated fruit could be justified simply by the addition of antioxidants taking place from the mango seed extract. A similar approach was followed in other studies: mango seed extract added to fresh-cut mango var. Haden increased polyphenols (7.4 times) and flavonoids (3.1 times) and the antioxidant capacity using DPPH, TEAC and oxygen radical absorbance capacity (ORAC)  . This hip < 0.05) inoculated bacteria in fresh-cut fruit. In fact, treated fruit showed 8.50 × 103 CFU/g of E. coli; on the contrary, control fruits exhibited 1.40 × 104 CFU/g. A similar effect was observed also when treated fruit was inoculated with S. Typhimurium (2.40 × 104 CFU/g) compared to the control, which contained higher bacterial load (6.30 × 104 CFU/g). Mango seed extract was also effective in reducing S. aureus compared to the control . Mango inoculated with L. monocytogenes showed a value of 1.68 × 105 CFU/g, treatment with mango seed extract determined a decrease of 2.40 × 104 CFU/g. Through the in vitro assays, mango seed extract proved to be more effective against Gram-positive than Gram-negative bacteria. Such results show the benefits of using mango seed extract to reduce the pathogenic bacterial load of fresh-cut mango without affecting its sensory acceptability. Mango seed extract at 6.25 g/L reduced (In other studies, mango seed extracts reduced 80% of aerobic mesophilic and 97% of total fungi in fresh-cut mangoes stored for 15 days at 5 °C [Sensory attributes of fresh-cut mangoes treated with mango seed extract at 6.25 g/L were rated acceptable by consumers. Moreover, at this concentration, mangoes showed a higher phenolic compound and antioxidant capacity and a lower bacterial load than untreated fruit. This study showed the benefits of mango seed extract as an antibacterial, antioxidant and sensory additive to use it in the formulation of functional foods.


Long-term oral warfarin is recommended in pediatric Kawasaki disease patients with large coronary artery aneurysms; however, heterogeneity is considerable. This study aimed to determine variables affecting warfarin dosage in Kawasaki disease. The enrolled individuals (194 children) were divided into four groups: (1) Cases with severe coronary artery lesions (CAL) of IV to V degrees or thrombogenesis treated with oral warfarin were assigned to Group A; (2) Group B, CAL of I degrees; (3) Group C, CAL of II and III degrees cases with small or medium-sized CAL not treated with warfarin; (4) Group D, normal children without Kawasaki disease. The relevant genotypes of CYP2C9, VKORC1 , and CYP4F2 were assessed. There were no statistically significant differences in CYP2C9, VKORC1, and CYP4F2 mutation frequencies among the 4 groups. In the 44 Group A patients, demographic features, clinical characteristics, and genotypes were recorded, and their associations with warfarin dose variability were assessed. Multivariate linear regression analysis revealed that height, VKORC1 1173, and CYP2C9 accounted for 61.2%, 7.9%, and 4.3% of dosing variability, respectively. Conclusions: Patient height is the main factor determining warfarin dosage, while genotype effects on warfarin dosage vary among studies. New formula should be defined using data obtained from children in cases with demonstrated efficacy. Kawasaki disease, an acute systemic vasculitis illness, causes CAA in about 25% of untreated patients; its occurrence rate could drop to 4% after treatment with intravenous immunoglobulins . MeanwhiAnticoagulant therapy has also been used in children for mechanical heart valve, Fontan operation, dilated cardiomyopathy, and all thrombosis types , 4. DespR)-warfarin and (S)-warfarin.; (S)-warfarin, metabolized by CYP2C9, is three to five times more active as anticoagulant than (R)-warfarin [This study aimed to determine non-genetic and genetic  factors affecting warfarin dose in Kawasaki disease. Warfarin is a racemic mixture of . To compare mutation frequencies, 3 control groups were set up: Group B, CAL of I degrees; Group C, CAL of II and III degrees cases with small or medium-sized CAL not treated with warfarin; Group D, no Kawasaki disease (blank control). CAL degrees and warfarin indications were based on Recommendations for clinical management of Kawasaki disease with coronary arterial lesions [Venous blood (1.5 ml) from every patient was collected for DNA extraction with TINAamp Blood DNA Kit (TIANGEN). PCR was performed in a 25 µl volume containing 0.7 µM primer, 120 ng DNA, 2× Power Taq PCR MasterMix, and ddH2O; 35 cycles were performed to obtain the final products, which were sent to Beijing genomics institute (BGI) for CYP2C9 , VKORC1 , VKORC1 , VKORC1 , and CYP4F2  genotyping.t test, 2-specimen Wilcoxon test, and ANOVA were used to measure associations of warfarin dose with age, height, weight, body surface area (BSA), sex, target international normalized rate (INR), and various genotypes in univariate analysis. A stepwise multivariate regression model was performed to determine the predicted formula for warfarin dose. Parameters with P < 0.20 in univariate analysis were entered into multiple linear regression analysis. Two-sided P < 0.05 was considered statistically significant.Data were presented as mean ± standard deviation (SD) and range for quantitative variables, and number and percentage for qualitative ones. Statistical analyses were performed with the SPSS Statistics Version 24.0 software. The Chi square test or Kruskal–Wallis test was used to compare gene mutation rates among the four groups. Pearson correlation analysis, unpaired A total of 59 children enrolled in Group A were hospitalized in the Children’s Hospital of Chongqing Medical University and assessed retrospectively; full data were obtained for 44 patients with a medium age of 3.6 years (3 months to 14.3 years), including 34 boys and 10 girls. Demographic and clinical characteristics, including target INR, cardiac thrombotic events, over-anticoagulation (defined as INR surpassing 4), warfarin dose, and others, are shown in Table Mean warfarin daily dosage was 0.101 ± 0.034 mg/kg (range 0.036–0.222 mg/kg), and was close to 0.1 mg/kg after grouping by age, weight, and target INR , fructose (29.5%), dipyridamole (15.9%), traditional Chinese medicine (11.4%), clopidogrel (6.8%), metoprolol (6.8%), levocarnitine (4.5%), benazepril (4.5%), tacrolimus (2.3%), vitamin E (2.3%), chalybeate (2.3%), calcium tablets (2.3%), and prednisone (2.3%). A total of 14/44 (31.8%) patients had special diets incorporating formula milk  and breastfeeding . In our study, only clopidogrel and prednisone effected warfarin intake dose, and the effect was uncertain and cases involved were small.Children enrolled in Groups A to D were 59, 50, 35, and 50, respectively. The 4 groups showed no statistically significant differences in sex and age. Genetic findings are presented in Table r = 0.742, P = 0.000). However, the prediction excessively estimated warfarin dose in 44 children , and included age, height, weight, race, CYP2C9 and VKORC1 − 1639, amiodarone, and enzyme inducers such as rifampin, phenytoin, and carbamazepine, for adult patients. Correction analysis showed a high relevance between the predicted warfarin daily dosage using the IWPC algorithm and clinical values (P = 0.056) and target INR (P = 0.968) were irrelevant to warfarin dose. The mean daily warfarin dose required in children with CYP2C9 *1/*1  was higher than that of individuals with CYP2C9 *1/*3 , with 0.105 ± 0.033 mg/kg and 0.078 ± 0.032 mg/kg, respectively. Children with homozygous wild-type CYP4F2 (1.632 ± 0.833 mg) needed lower amounts than those with CT  and TT . Patients carrying one VKORC1 1173 variant allele (2.479 ± 0.255 mg) required relatively higher doses than TT homozygotes ; children with VKORC1 − 1639GA (2.482 ± 0.233 mg) required a higher dose than those with AA , while VKORC1 3730 had the opposite result.Warfarin daily dose was closely associated with age, height, weight, and BSA. Figure P < 0.001), and explained the large majority of individual variability in warfarin dose requirement  = − 0.018 + 0.023 × height (cm) − 0.653 × VKORC1 1173TT − 0.46 × CYP2C9 *1/*3.Kawasaki disease, also called mucocutaneous lymph node syndrome, is an acute vasculitis disease in infants and young children. Inflammation in Kawasaki disease can cause wide polyangiitis, especially of the cardiovascular system, which can lead to coronary arteriectasis or even coronary aneurysm. Kawasaki disease with severe CAL or thrombotic formation requires a combination therapy of aspirin plus warfarin. Aspirin usage is fixed and valid, but warfarin dosage has large individual differences even in children with the same age or weight. Currently, a mature model for adults to predict warfarin dose is available, and studies increasingly focus on pediatric patients. Nevertheless, indications are different, and there might be distinct cardiovascular diseases, including the Fontan procedure, heart valve replacement, dilated cardiomyopathy, and coronary aneurysm, and other extra-cardiac diseases. No study has focused on only one disease such as Kawasaki disease. The primary reason might be fairly limited cases; we spent nearly 2 years of Southwest China, recruiting volunteers who matched inclusion criteria in the last 10 years, and only 44 patients were ultimately included in the experimental group. Another reason might be the viewpoint that indications possibly do not affect warfarin dose. In warfarin therapy for Kawasaki disease, clinicians focus more on dose-adjustment depending on clinical experience. Our research team comprises experts in the cardiovascular field, with great interest in warfarin treatment of Kawasaki disease, which is the most common acquired heart disease in developing countries. Besides, previous studies generally do not set control groups when assessing associations of warfarin dose and genotypes. Peng et al.  demonstrThis study assessed the associations of warfarin dose with genetic and non-genetic variables in children with Kawasaki disease, with appropriate control groups in order to compare genotype mutations of CYP2C9, VKORC1, and CYP4F2. Most children in the warfarin-treatment group were 6 months to 5 years, which is the common age of patients with Kawasaki disease. Height, VKORC1 1173, and CYP2C9 determined warfarin dose, and accounted for 73.4% of warfarin dose variability; patient height accounted for 61.2%, representing the main contributor. There were no statistically significant differences in CYP2C9, VKORC1, and CYP4F2 among the four groups, not supporting the implication of these genes in Kawasaki disease, CAL occurrence, or CAL severity.The first prospective cohort study with 59 patients the associations of VKORC1, CYP2C9, and VKA dosage in children demonstrated that age, VKORC1, and CYP2C9 explained 38% of dose variations, with age constituting the most important factor (28.3%) . HoweverIn the final model, CYP2C9 and VKORC1 1173 genotypes explained 4.3% and 7.9% interindividual variability of warfarin dose, respectively. These proportions were relatively lower than those reported in most adult and pediatric studies. CYP2C9 variant allele carriers (*2 or *3) require lower warfarin dosages than individuals with wild type allele (*1). In the present cohort, CYP2C9*1/*3 carriers required significantly lower warfarin doses than individuals with CYP2C9*1/*1; meanwhile, mutant genotypes were relatively rare, and the effects of other mutant CYP2C9 genotypes could not be evaluated. Adult patients with CYP2C9 mutations have high rates of serious or life-threatening bleeding and above-INR compared with wild-type counterparts . KaitlynIn Group A, no patient had the wild-type VKORC1 1173 or − 1639 gene, but showed homozygous mutations ; the mutation rate was higher than those reported for white and African American individuals, and similar to that of Asians. The patients with VKORC1 1173 CC required higher warfarin doses compared with the CT or TT groups. There was no wild type individuals, but the trend was consistent; compared with heterozygotes, cases with homozygous mutations required 36.1% less warfarin. Studies indicated that patients with VKORC1 − 1639 AA require significantly lower warfarin doses than the GG or GA groups. Avisa et al. [P = 0.004); however, the former cases were all infants. A study of 300 adults showed vegetarian or accidentally nonvegetarian patients intake lower warfarin doses than non-vegetarians [P = 0.002) or other diseases . This may be related to increased sensitivity, abnormal liver function, loss of appetite, reduced intake of vitamin K, and lack of exercise after surgery [Medicines and food could also affect warfarin dose. Children on phenobarbital/carbamazepine or receiving enteral nutrition required elevated doses . Drugs aetarians . Besidesetarians  found th surgery , 19.Inte surgery . The curA limitation of this study was its type, i.e., a single institution retrospective study, which failed to control for or/and analyze some factors, including test frequency of echocardiography and INR, the specific doses of other drugs, and diets. Another shortcoming was sample size in the experimental group, which resulted in reduced number of VKORC1 genotypes.This study demonstrated that height and VKORC1 1173 and CYP2C9 genotypes significantly determine warfarin dose in Kawasaki disease in Southwest Chinese. Warfarin dosage models based on pharmacogenetics have been generated for adults, but are not used in pediatric patients due to excessive estimation of warfarin dosage. Meanwhile, studies have explored the efficiency of warfarin dose based on genotyping , 20, 21,


Escherichia coli (EPEC) and hybrid E. coli (harboring genes of more than one DEC pathotypes) strains have complicated the issue of growing antibiotic resistance in diarrhoeagenic Escherichia coli (DEC). This ongoing evolution occurs in nature predominantly via horizontal gene transfers involving the mobile genetic elements like integrons notably class 1 integron. This study was undertaken to determine the virulence pattern and antibiotic resistance among the circulating DEC strains in a tertiary care center in south of India.Emergence of atypical enteropathogenic E. coli isolated.Diarrhoeal stool specimens were obtained from 120 children (< 5 years) and 100 adults (> 18 years), subjected to culture and isolation of diarrhoeal pathogens. Conventional PCR was performed to detect 10 virulence and 27 antimicrobial resistance (AMR) genes among the sulI, sulII, and sulIII were 83.3 and 21%, 60.41 and 42.1%, and 12.5 and 26.3%, respectively) and class 1 integron (int1) genes (41.6 and 26.31%) was higher in DEC strains isolated from children and adults, respectively. Other AMR genes detected were qnrS, qnrB, aac(6’)Ib-cr, dhfr1, aadB, aac(3)-IV, tetA, tetB, tetD, catI, blaCTX, blaSHV, and blaTEM. None harbored qnrA, qnrC, qepA, tetE, tetC, tetY, ermA, mcr1, int2, and int3 genes.DEC infection was observed in 45 (37.5%) children and 18 (18%) adults, among which  atypical EPEC was most commonly detected followed by  ETEC, [5 (11.1%) 2 (2%)] EAEC,  EIEC,  STEC, and no NTEC and CDEC was detected. DEC co-infection in 3 (6.6%) children, and 1(1%) adult and sole hybrid DEC infection in 3 (6.6%) children was detected. The distribution of sulphonamide resistance genes  contains supplementary material, which is available to authorized users. Escherichia coli. Few studies conducted in India have documented a high prevalence of diarrhoeagenic Escherichia coli (DEC) in children and adults , Aeromonas spp. [n = 1 (0.83%) and n = 1 (1%)], and Salmonella spp. [n = 2 (2%) detected only in adults].Other enteric bacterial pathogens detected in children and adults were n = 76) were males and 37% (n = 44) were females, and among the 100 adults, 57% (n = 57) were males and 43% (n = 43) were females  was 5.1 times  more than that of ETEC (n = 6). In adults, the likelihood of finding atypical EPEC (n = 10) was 2.7 times  more than that of ETEC (n = 4). However, the likelihood of finding atypical EPEC (n = 10) was 5.4 times  more than that the odds of finding EAEC (n = 2).Out of 120 children, 51.6% were less than 12 months of age  of the strains, while int2 and int3 were not detected in any of these strains (Tables All DEC strains isolated from children harbored one or more AMR gene. None of the isolates harbored .66% n = 0 of the n = 14) of the DEC strains isolated from adults harbored one or more AMR genes. No AMR gene was detected in remaining 26.31% of strains. None of the isolates harbored qnrA, qnrC, qepA, tetD, tetE, tetC, tetY, ermA, mcr1, int2, and int3 genes. Atypical EPEC strains harbored utmost classes of AMR genes than other DEC pathotypes ] followed by co-trimoxazole [n = 29 (43.2%)], tetracycline [n = 24 (35.8%)], levofloxacin [n = 22 (32.8%)], ciprofloxacin [n = 20 (29.8%)], ceftazidime [n = 18 (26.2%)], gentamicin [n = 13 (19.4%)], chloramphenicol [n = 9 (13.4%)], amikacin [n = 5 (7.4%)], and cefoperazone-sulbactam [n = 5 (7.4%)]  plasmid (absence of bfpA gene), whereas in typical EPEC both the genes eaeA and bfpA are present. In our study interestingly, among the atypical EPEC detected, most were found to harbor bfpA gene alone and very few strains were positive for the eaeA gene. Variations in the distribution of atypical EPEC have been documented in earlier studies   . Co-infen = 3) was relatively more compared to that of adults (n = 1) (Table n = 2), atypical EPEC-ETEC (n = 1)) of children were infected with hybrid DEC strains in Mexico [E. coli and are not restricted only to the DEC due to HGT. They emerge as potential outbreak agents in recent years leading to the high cause of morbidity and mortality. In addition, the increased severity of infection was noticed among the people who are infected with hybrid DEC strains and in DEC co-infections [The number of hybrid DEC strains observed in children  used in the agriculture as well as poultry farms [. showed that propagation of sul plasmid are majorly associated with incompatibility (IncF) replicons types (IncFI in sulI and IncFII in sulII) leading to resistance among the E. coli strains isolated from human stool specimen, and they also observed the co-transfer of other resistance genes [E. coli strains found that sul2 gene was transferred along with blaTEM in human [In children, all the DEC strains isolated were resistant to at least one class of antibiotic and 14 out of 19 DEC strains from adults harbored resistance genes for one or more class of antibiotics Table . The EPEry farms . These rce genes . Anotherin human . MajoritEnterobacteriaceae is plasmid-mediated (PMQR). The most common PMQR resistance genes were qnrB, qnrS, and aac(6′)-Ib. In children, all the pathotypes detected harbored either qnrS or qnrB genes or both, whereas, in adults, qnrS or qnrB was detected only in ETEC and atypical EPEC pathotypes (Table qnrA and qnrS genes in their DEC strains [aac(6′)-Ib-cr gene without qnr genes [qnr resistance genes among these DEC is variable.A major resistance mechanism of quinolone resistance in the es Table . A study strains . A studynr genes . These sn = 52) of DEC strains were found to harbor resistance genes to these antibiotics, blaTEM was positive in 83.33%, blaSHV in 31.48%, and blaCTX-M in 20.37% of adults [blaCTX-M gene (57.1%) while in children, the blaSHV gene (29.1%) was more common  was detected to be high in our study. In a study conducted by Ghorbani-Dalini et al., 96.3% . cat1 and cat2 genes were most commonly distributed in E. coli strains worldwide since plasmid-borne, irrespective of the source of isolation [The frequency of aminoglycoside and chloramphenicol resistance varied among the DEC strains . With resolation , 57. A ssolation . This potetA, tetB, and tetE genes without tetC, tetD, and tetY in the clinical isolates of ETEC in an outbreak [tetC, tetE, or tetY genes in our strains  was detected in DEC stains isolated from both children and adults in our study (Table int1 and int2 genes while none of the DEC strains harbored int3 gene [int1 and int2 genes in various DEC pathotypes [int2 gene) and very rarely class 3 integron (int 3 genes) have been reported so far among these strains. Class 1 integron was primarily linked with antibiotic resistance among these strains. Next to conjugative plasmid, the newly emerged sulIII gene is carried out in class 1 integron [Only class 1 integron Additional file 2:Tables S3. and S4. Correlation of DEC infection rate with age groups. (DOCX 16 kb)


The mechanistic study suggested that growth inhibition was related to ROS-mediated apoptosis, and ROS production was due to SOD inhibition initiated by DpdtbA rather than occurrence of ferritinophagy. In addition, DpdtbA also induced a downregulation of EGFR, p53, and AKT, which hinted that mutant p53 still played a role in the regulation of its downstream targets. Further study revealed that the downregulation of p53 was through stub1- (chip-) mediated autophagic degradation rather than MDM2-mediated ubiquitination. Taken together, the DpdtbA-induced growth inhibition in a mechanism was through inactivating the p53/EGFR/AKT signal pathway.Esophageal cancer (ESC) is one of the most deadly diseases for human. p53 in most cancers, including ESC cell, is mutated, and the mutated p53 losses its original function and acquires “gain of function” that allows for promoting the hallmarks of cancer, such as antiapoptosis, metastasis, invasion, angiogenesis, and resistance to chemotherapy. Targeting p53 through either introducing wild-type or degrading mutated p53 is an important strategy in cancer therapy. Di-2,2′-pyridine ketone dithiocarbamate s-butyric acid (DpdtbA) has significant growth inhibition against gastric cancer lines in previous study. Similar action in ESC cell lines but a novel molecular mechanism was observed in the present study. The results showed that DpdtbA exhibited an excellent antiproliferative effect for ESC cell lines (IC Cancer is one of most deadly diseases, while esophageal cancer (ESC) lies in the eighth position in cancer-caused deaths, and about half of all cases occur in China . DespiteThe epidermal growth factor receptor (EGFR), an ErbB family of receptor tyrosine kinases, plays an important role in cell proliferation and survival . Upon liAs mentioned above, different mutant p53 alleles play an important role in cancer development and progression; therefore, targeting mutant p53 through protein degradation, rather than its reactivation, might be another strategy for drug development. Dithiocarbamates as metal chelators own important biological activities in the treatment of bacterial and fungal infections, AIDS, and cancer . DpdtbA 50 ≤ 4.5 ± 0.4 μM for Kyse 450, 3.2 ± 0.6 μM for Kyse 510, and 10.0 ± 0.7 μM for Kyse 150) was observed compared to control (p < 0.05), but the cell line dependence was not evident. Next, the effect of DpdtbA on cell colony formation was further investigated. As shown in p < 0.05); the quantitative analysis is shown in Previous study demonstrated that di-2,2′-pyridine ketone dithiocarbamate s-butyric acid (DpdtbA) owned significant growth inhibition in gastric cell lines ; DpdtbA To test whether an induction of cell cycle arrest contributed to the antiproliferative capability of DpdtbA in ESC cells, cell cycle analysis was performed via flow cytometry. As shown in μM had a statistical significance for Kyse 450 (p < 0.05), but for Kyse 150 cells, 10 μM was required (p < 0.05). Moreover, the apoptotic portions in both cell lines were obviously different, which may be relative to IC50 value; as a whole, DpdtbA induced a limited apoptosis.Previous study revealed that DpdtbA-induced apoptosis contributed to the growth inhibition in gastric cancer lines ; similarTo seek additional evidence for the occurrence of apoptosis, the variations in nuclear morphology and fragmentation of chromosomal DNA were further investigated. As shown in Bcl-2 family members play key roles in the regulation of apoptotic progress. To understand how DpdtbA induced apoptosis, we further examined the alteration in the expression of apoptosis-related genes in ESC cells. As shown in p < 0.05, p < 0.01 at 10 μM, p < 0.01, In general apoptosis associated with ROS production, to determine the origin of ROS, the ESC cells treated by DpdtbA were stained by ROS dye, DCF, following flow cytometry analysis. The ROS production at different condition is shown in p < 0.01), hinting that depletion of EGFR might involve in the growth inhibition. Due to lower abundance of EGFR in Kyse 510, the cell line was further not included in the following investigation.It has shown that EGFR mediates cell proliferation , and genp < 0.01) in both cell lines, indicating that EGFR was involved in the regulation of AKT. In addition, it is well documented that p53 can regulate EGFR [p < 0.05, It has known that EGFR activation can trigger the alteration of others, including JAK/STAT and the PI3K/AKT signal pathway, thus promoting cell growth . The facate EGFR ; the p53α was used to confirm the action of p53. As shown in α impaired the DpdtbA-induced p53 degradation; PFT-α alone did not significantly modify p53 levels (p < 0.05), but the additional effect was not observed in the combination treatment, which might be due to the difference in interaction between PFT-α and mutant and wild-type p53 [α combination with DpdtbA on proliferation was further investigated; α attenuated DpdtbA-induced growth inhibition , in accordance with the result from western blotting. However, the addition of (NH4)2Fe(SO4)2 blocked the action of DpdtbA on both growth inhibition and downregulation of p53 .As mentioned above, DpdtbA induced p53 degradation; accordingly, its downstream target, EGFR and AKT, was also downregulated; it was suggested that the degradation of p53 was mainly a molecular event. Hence, the detail in p53 degradation required to be further determined. We then tested whether MDM2 played a role in such p53 degradation. To this end, the levels of p53 and MDM2 were assayed. As shown in Since stub1 chaperon-mediated autophagy involved the p53 degradation, accordingly, it may affect the downstream target of p53. To this end, autophagy-related genes, LC3, stub1, and along with the p53/EGFR/AKT axis were assessed. As shown in As mentioned above, DpdtbA induced growth inhibition, cell cycle arrest, and apoptosis, which involved ROS production due to SOD inhibition. Further mechanistic study revealed that DpdtbA led to stub1-mediated autophagic degradation of p53 that dominated the level of its downstream targets EGFR and AKT; thus, DpdtbA-induced growth inhibition could be through inactivating the p53/EGFR/AKT signal pathway.In vivo neoplastic cells can obtain iron from local environment, i.e., tumor microenvironment which is comprised of various cells, cytokines, and extracellular matrix; among those, tumor-associated macrophages (type II phenotype) are the main source of iron supply [Esophageal cancer (ESC) is one of the most deadly diseases, and the long-term survival of the patients is poor ; therefon supply . Obviousn supply , which pn supply . In addin supply . Similarn supply , 40, butn supply , while tOverexpression of epidermal growth factor receptor (EGFR) occurs in approximately 80% of patients with adenocarcinoma and squamous cell carcinoma , and manIn conclusion, DpdtbA-induced growth inhibition involved apoptosis and cell cycle arrest. Further study revealed that ROS production involved the apoptosis induction, and the rising ROS was stemmed from SOD inhibition initiated by DpdtbA rather than occurrence of ferritinophagy. Furthermore, DpdtbA could induce downregulation of EGFR, p53, and AKT, hinting that the mutant p53 still played a role in the proliferation of ESC cells. Additional study revealed that stub1- (chip-) mediated autophagy was responsible for the p53 degradation rather than MDM2-mediated ubiquitination, which inactivated the EGFR/AKT signal pathway. Taken together, DpdtbA-induced growth inhibition was through inactivating the p53/EGFR/AKT signal pathway.α (PFT-α), RPMI-1640, and other chemicals were purchased from Sigma-Aldrich. Gapdh, NCOA4, Bcl-2, and AKT antibody were obtained from EnoGene ; antibodies EGFR, stub-1, SOD, MDM2, Cyt-C, LC3, Bax, and p-AKT were purchased from Proteintech Group Inc. . Antibodies p53, p-p53, and ferritin were purchased from Cell Signaling Technology ; siRNA for MDM2 (stB0001232) and stub1 (stB0001238) were obtained from RiboBio .MTT, 3-methyladenin (3-MA), pifithrin-http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/), the status of TP53 is H179R in Kyse 450, R248Q and R155Q in Kyse 150, and nonmutation in Kyse 510. Briefly, the Kyse 450 cells (5 × 103/ml) were seeded equivalently into a 96-well plate, and the various concentrations of DpdtbA were added after the cells adhered. Following 48 h incubation at 37°C in a humidified atmosphere of 5% CO2, 10 μl MTT solution (5 mg/ml) was added to each well and additional incubation applied. Finally, 100 μl DMSO was added in each well to dissolve the formed formazan after removing the cell culture. The absorption of the solution that was related to the number of live cells was measured on a microplate reader  at 490 nm. Percent growth inhibition was defined as percent absorbance inhibition within appropriate absorbance in each cell line. The same assay was performed in triplet.A 10 mM DpdtbA  in 70% DMSO was diluted to the required concentration with DMSO. The MTT assay was conducted as previously described . The Kys50 or 1/10 IC50. Fourteen days later, colonies were fixed in 3.7% paraformaldehyde and stained with 0.1% crystal violet. Colonies containing 50 cells at least were counted under inverse microscope , and the clone numbers were analyzed subsequently.The cells in the exponential phase were trypsinized and seeded in 6-well plates at the density of 500 cells/well. The cells were kept in sustaining insulting of DpdtbA at a dose of 1/20 IC2DCF-DA was used to stain the cells.Cellular ROS determination was performed based on previously described . Apoptos5) were seeded in a 6-well plate and incubated for 24 h at 37°C (5% CO2). The medium was replaced with fresh medium supplemented or not (control) with DpdtbA (5 and 10 μM). Following 24 h of incubation, the cells were collected with centrifugation, washed with PBS, finally fixed in 70% ethanol, and stored at -20°C. After removing the 70% ethanol and washing with PBS, the cellular nuclear DNA was stained based on the company recommended protocol; the cells in staining buffer were directly subjected to flow cytometer analysis. For each sample, 10,000 events were collected, and fluorescent signal intensity was recorded and analyzed by CellQuest and ModiFit .The Kyse 450 cells (or Kyse 150 cells) .7 Kyse 450 (150 or 510) cells treated with or without the DpdtbA was scraped off in lysis buffer , and the suspension was incubated on ice for 30 min. and then collected the clear supernatant by centrifugation at 14,000 ×g. A colorimetric Bio-Rad DC protein assay was employed to determine the protein concentration on a microplate reader MK3 at 570 nm. Proteins (20-30 μg) were separated on a 13% sodium dodecyl sulfate-polyacrylamide gel at 200 V for 1 h. Then, the separated proteins were subsequently transferred onto a PVDF membrane at 60 V for 1 h. The membrane was washed three times with Tris-buffered saline (TBS) and was then blocked for 2 h in TBS containing 0.1% Tween-20 and 5% nonfat skimmed milk. The membrane was incubated at 4°C overnight with the primary monoantibody used at a dilution of 1 : 300 in TBS plus 0.1% Tween-20 (TBST). The membrane was washed several times with TBST and was subsequently incubated with HRP-conjugated secondary antibody  for 2 h at room temperature. After another wash of the membrane with TBST, the protein bands were detected using a super sensitive ECL solution  and visualized on an SYNGENE G:BOX Chemi XX9 .Briefly, 1 × 10t-test. Comparisons between multiple groups were performed by one way ANOVA with Dunnett's post hoc correction. A p value < 0.05 was considered statistically significant.Results are presented as the mean ± SEM. Comparisons between two groups were carried out using the two-tailed Student's


In contrast, 20% oxygen increased metabolite flux through the TCA cycle, activity of mitochondria, and ATP production. Acetylation of H3K9 and H3K27 was elevated at 5% oxygen while H3K27 trimethylation was decreased, conforming to a more open chromatin structure. RNA-seq analysis of 5% oxygen hPSC also indicated increases in glycolysis, lysine demethylases, and glucose-derived carbon metabolism, while increased methyltransferase and cell cycle activity was indicated at 20% oxygen. Our findings show that oxygen drives metabolite flux and specifies carbon fate in hPSC and, although the mechanism remains to be elucidated, oxygen was shown to alter methyltransferase and demethylase activity and the global epigenetic landscape.Metabolism has been shown to alter cell fate in human pluripotent stem cells (hPSC). However, current understanding is almost exclusively based on work performed at 20% oxygen (air), with very few studies reporting on hPSC at physiological oxygen (5%). In this study, we integrated metabolic, transcriptomic, and epigenetic data to elucidate the impact of oxygen on hPSC. Using  Oxygen is a critical, but often overlooked, metabolite within the stem cell niche. The mammalian reproductive tract, within which the preimplantation embryo develops, has been measured at 2-9% oxygen in the rat, rabbit, hamster, and rhesus monkey , 2. The αKG), NADH/NAD+, and TCA intermediates including citrate and succinate, which maintain the highly acetylated euchromatic landscape characteristic of pluripotency, while in contrast, differentiated cell types are generally heterochromatic [OCT4, SOX2, and NANOG pluripotency transcriptional promotors, leading to increased levels of H3K36me3, decreased H3K9me3, and a general increase in pluripotency markers, however none have integrated metabolic, epigenetic and transcriptomic data within a single culture system.PSC metabolism is characterized by a heavy dependenceon glycolysis, with ~50-70% of glucose being converted to lactate –11. In chromatic . Currenthromatic , in whic13C-glucose. Oxygen is therefore a key regulator of hPSC metabolism, directing the flux of glucose-derived carbon to lactate, alanine, glycogen, and glutathione at physiological oxygen and through the tricarboxylic acid (TCA) cycle to acetate at 20% oxygen. Coincident with altered intracellular metabolite flux, 5% oxygen increased acetylation and decreased methylation markers consistent with a more active gene regulatory network. RNA-seq analysis confirmed increases in transcription and glycolytic metabolism and confirmed the resulting epigenetic landscape at physiological oxygen levels. These findings show that 5% oxygen has a significant impact on intracellular metabolite fluxes in hPSC, metabolites which are known epigenetic cofactors, accompanied by increased H3K9/27ac and decreased H3K27me3. Although the mechanism remains to be elucidated, oxygen was shown to alter methyltransferase and demethylase activity influencing the global epigenetic landscape.Oxygen is a key factor, not just in PSC culture, but in all cell, tissue, and organ cultures. However, our understanding of how oxygen acts as a nutrient and signaling molecule in PSC is limited. Because metabolism is inextricably linked to epigenetics –21 and, Mitochondrial metabolism is often considered negligible in hPSC , 22–24, + leak , which has been related to mitochondrial function . Irrespe13C-glucose, followed by NMR-based fluxome analysis [13C flux analysis. hPSC adapted to either 5% or 20% oxygen culture were labeled with 13C-glucose over 0-4 h, and H,C-HSQC NMR spectroscopy was performed on polar and nonpolar cell fractions  and atmospheric (20%) oxygen results in distinct global metabolic profiles characterized by increased glycolytic and mitochondrial metabolism, respectively. To determine the underlying metabolite fluxes that contribute to these phenotypes, we performed metabolic labelling with uniformly labeled analysis . This meanalysis , 14, 30,ractions . This tilabeling , 29, or abolites . The anaabolites . Temporaabolites . This paabolites . Acetateabolites  where thHeatmaps of metabolite accumulation over time within oxygen treatments describe a rapid increase in all metabolic pathways at 5% oxygen, primarily between 2 and 4 h , indicat13C flux analyses  and SLC2A14 (GLUT14)  . Several(GLUT14)  and redu(GLUT14)  observedPCDH10, FBN1, VCAN, and POSTN), pro-apoptotic factors in response to mitochondrial damage (BNIP3), cell cycle (BTG2), early embryonic methylation patterning (BORIS), and signal transducers including hypoxia inducible factor- (HIF-) related genes . In response to culture at 20% oxygen, only 3 and 2 DEGs were upregulated >2-fold in the MEL2 and MEL1 hPSC lines, respectively. Of interest, follistatin (FST) which has a high affinity for activin A and leads to hPSC differentiation [OCT4, NANOG, MEIS1 OTX2, SOX11, GDF3, REX1, FGF4, DPPA2, DPPA4, and hTERT were unaltered due to oxygen (Supplementary OCT6 (POU3F1) [SOX2 and DNMT3B were decreased in the MEL1 and both hPSC lines, respectively (Supplementary 2(fc)=1 threshold.Further interrogation of RNA-seq data revealed the overall transcriptional response to oxygen conformed to a more open chromatin structure at 5% oxygen Figures  and S2. ntiation  was incrntiation , 34–36, (POU3F1)  was incrHIF-1α, HIF-1β, and HIF-2α were not oxygen regulated at the mRNA level; however, HIF-1 target genes ENO1, SERPINE1, ENO2, VEGFA, SLC2A1, EGLN1, PDK1, HK2, MAP2K1, and PFKFB3 were significantly increased at low oxygen , were also increased , KDM6A, KDM4B, and KDM3A were upregulated by more than 50%, while KDM6B, which specifically demethylates H3K27me3 [The results presented in the preceding paragraphs indicate that a change in oxygen is sufficient to alter hPSC histone methylation status. Next, we examined the transcript levels of methyl donors through the serine/glycine biosynthesis pathway and downstream methionine and folate cycles using RNA-seq data and qPCR . We have established that hPSC mitochondrial respiration is decreased at 5% oxygen, despite the absence of any apparent alterations to inner mitochondrial matrix organization. Beyond the production of ATP, mitochondrial respiration also generates ROS as Hrt chain , which art chain . Comparert chain  by direcrt chain  and indurt chain . Our stuMAT2A, but not MAT2B expression, which together generate EZH2 and SAM from methionine, was also downregulated at 5% oxygen. In support of this, an identical MAT2A/B expression pattern is observed when methionine is removed from hPSC culture [A euchromatic state was established at 5% oxygen, consisting of increased global H3K9/27 acetylation and reduced H3K27 trimethylation. Metabolite levels and pathway fluxes alter the epigenetic landscape, potentially regulating global or localized patterns of genes to elicit cell fate , 48, 49. culture , leading culture . Indeed, culture . While o culture , 34–36, A question arising from our work is why 5% oxygen resulted in the lower accumulation of intracellular acetate coincident with higher global H3K9/27 acetylation. Of all assessed metabolites, only acetate, an essential metabolite for most cancers , was les+ availability [MAT2A and EZH2 [Several key epigenetic cofactors can be derived from glucose metabolism and, thus, are potentially influenced by oxygen. The production of cytosolic acetyl-CoA requires glycolytic metabolism of citrate, while acetyl-CoA produced in the mitochondria from fatty acids is metabolised through the TCA cycle . The curlability  and incrlability . Conversand EZH2 . Furtherand EZH2 , suggestIn this report, we show that physiological oxygen (5%) promotes a euchromatic state, increasing acetylation, activating demethylases, and deactivating methyltransferases, providing a basis for explaining how oxygen could influence hPSC by providing a more flexible transcriptional state. Moreover, this euchromatic state at 5% oxygen coincides with a wave of transcriptional activity and a time-dependent increase in the flux of glucose-derived carbon through multiple metabolic pathways. The requirement for pluripotent cells to maintain specific metabolic, epigenetic, and transcriptional profiles implicates oxygen as a key developmental regulator through its ability to influence metabolism. These findings have broad implications because the role of oxygen in metabolism, transcription, and epigenetics has obvious implications for all aspects of pluripotent cell culture, including differentiation assays, and disease modelling.2 in air (20% oxygen), or 5% CO2 and 5% O2, and balanced with N2. Cell passaging took place every 5 days using Dispase (Stem Cell Technologies), and the medium was refreshed every 24 h in a biosafety cabinet. To minimize exposure to 20% oxygen conditions during handling, the medium was preequilibrated under each respective oxygen condition. All cell cultures were acclimated to 5% or 20% oxygen conditions for a minimum of 2 passages before use.hPSC MEL1 and MEL2  were cultured in mTeSR1™ medium (Stem Cell Technologies) on PSC qualified Matrigel™- (BD Biosciences) coated tissue culture plates (BD Biosciences). Cells were cultured in humidified CB 150 incubators (Binder Inc.) at 37°C with 5% COμM; AdipoGen) for 1 h prior to dissociation with TrypLE Select (Invitrogen) and seeded as single cells at a density of 40,000 cells/well in Seahorse XF24 assay microplates (In Vitro Technologies) and cultured at 5% or 20% oxygen. For cell number determination, a parallel seeding of 40,000 cells/well of a 96-well clear bottom plate  in mTeSR1™ + Y-27632 was performed. After 24 h, the medium was refreshed with mTeSR1™, and after a further 24 h, cells in the assay plate were prepared for analysis. Cells were rinsed with 500 μL of warmed (37°C) Seahorse XF base medium (In Vitro Technologies) containing 13.7 mM glucose, 0.392 mM sodium pyruvate, and 2.94 mM L-glutamine (the levels found in mTeSR1™) at pH 7.4. A final volume of 630 μL was added to the assay plate before it was placed in a 1% CO2 incubator for 1 h to stabilize the pH of the assay medium and the plate. A standard mitochondrial stress test assay (Seahorse Bioscience/In Vitro Technologies) was performed with an initial basal recording followed by sequential injections of oligomycin , carbonyl cyanide-p-trifluoromethoxyphenylhydrazone  and rotenone/antimycin A  every 24 minutes. Cells parallel seeded on clear 96-well plates were fixed in 70% ethanol and stained with DAPI. To determine the cell number, 5 representative images were taken of the cell monolayer from each of 4 separate wells and analyzed using ImageJ, and the average cell number quantitated after adjusting for the well surface area. Assay results were normalized to the cell number, and metabolic parameters were determined using the publicly available Seahorse XF Cell Mito Stress Test Report Generator (Agilent Technologies).hPSC were treated with Y-27632 (10 μL resin (50% absolute acetone: 50% resin) in an open tube under agitation for 2 h. Resin was refreshed and infiltrated for 4 h under vacuum, then set in 100% resin at 80°C. Samples were sliced with a microtome, fixed to TEM grids, and imaged using a Jeol 2100F transmission electron microscope with an Oxford X-MaxN 80 T EDXS detector (Oxford Instruments), an EELS Spectrometer imaging filter (Gattan), and an Orius SC1000 CCD camera (Gattan). The microscope was operated at an accelerating voltage of 80 kV.Approximately 4 million hPSC per treatment were processed for TEM using a protocol adapted from Underwood et al. . hPSC wev/v) KOSR (Thermo Fisher), 0.1 mM NEAA (Thermo Fisher), and 10,000 U PenStrep (Thermo Fisher). hPSC cultured at 20% oxygen for over 5 passages were treated with Y-27632 (10 μM) for 1 h prior to dissociation with TrypLE Select. A 3 mm coat of methylcellulose was added to wells of a 24-well ultralow attachment plate (Corning) and incubated at 37°C for approximately 40 min until solidified. hPSC were harvested with 5 mL KOSR+20% FBS, then 6.25 million cells were centrifuged at 300 g and resuspended in 5 mL of KOSR+20% FBS+0.25 mL of undiluted Matrigel (Corning)+10 μM Y-27632. Approximately 5,000,000 cells (400 μL) were loaded into each well on top of the methylcellulose layer and the plates were centrifuged at 300g for 5 min until tight spheres of cells had formed; then, 200 μL of KOSR medium+10 μM Y-27632 was added and incubated overnight at 20% oxygen. After 24 h, following attachment of the spheres to the methylcellulose layer, the medium was aspirated and replaced with 400 μL/well of KOSR medium+20% FBS. After a further 24 h, the medium was completely aspirated and a second 3 mm layer of methylcellulose was added on top of the cell aggregate and placed in the incubator for ~40 min. Once solidified, 400 μL of KOSR medium+20% FBS was added on top of the methylcellulose layers. The medium was replaced every 48 h for 8 weeks. After 8 weeks of differentiation, in vitro teratomas were extracted, fixed, and stained with H&E for morphological analysis.Methylcellulose (Sigma) was resuspended to make a 10% solution in knockout serum replacement (KOSR) medium consisting of DMEM/F12+L-glutamine (Thermo Fisher) with 20% (13C6 D-glucose (Sigma) was supplemented into glucose-free mTeSR1™ medium. Long-term 5% and 20% oxygen-adapted hPSC were treated with Y-27632 (10 μM) for 1 h, dissociated with TrypLE Select, and 4 × 106 cells were seeded as single cells in 100 mm dishes (Becton Dickinson) in glucose-free mTeSR1™ + 13C6 D-glucose+Y-27632 and cultured at 5% or 20% oxygen. Upon reaching confluency, the medium was changed 4 h prior to labeling, with in-house glucose-free mTeSR1™ supplemented with D-glucose. After 4 h, custom mTeSR1™+13C6 D-glucose was added to timepoint dishes 1, 2, and 4 h, which were returned to their respective incubators for 1, 2, or 4 h, respectively. Timepoint 0 h was harvested immediately. For each collection, 10 mL of spent 13C6 D-glucose medium was collected and stored at -80°C. Ice cold PBS was added to the cells prior to scraping, cell suspensions were centrifuged at 300g for 3 min, the supernatant was aspirated, and the pellets were snap frozen in liquid nitrogen before being stored at -80°C. A 1 cm2 area of cells was left on each plate for cell number determination.To determine metabolite kinetics, uniformly labeled 2O; Cambridge Isotopes) with 1 mM DSS (Sigma). The organic concentrate was reconstituted in deuterated chloroform  and analyzed immediately. Samples were loaded into 3 mm glass tubes (Wilmad-LabGlass) and analyzed on a 63 mm bore 800 MHz spectrometer (Varian/Agilent Technologies) using VnmrJ software (Agilent Technologies) by collecting gradient-enhanced 1D-1H and 2D-1H,13C-heteronuclear single-quantum coherence (cCHSQC) spectra. Acquisition and recycling time for aqueous, organic, and media samples were 6 h and 1.5 h, 4.5 h and 1.25 h, and 0.5 h and 0.125 h, respectively. Spectra were processed using Mnova (Mestrelab Research), by integrating peaks relative to DSS or CDCl3. Chemical shift data from the Human Metabolome Database and Biological Magnetic Resonance Data Bank [Sample preparation and NMR spectra acquisition were performed as previously described . In detaata Bank , 57 wereFor the quantification of histone acetylation and methylation markers, hPSC were fixed in PFA for 15 min, permeabilized using 1 M HCl for 20 min, and blocked with 0.1% Triton X-100 and 5% species-specific serum in PBS for 1 h. Primary antibodies were incubated at 4°C for 24 h at the following concentrations: H3K9ac (Abcam) at 1 : 500, H3K27ac (Abcam) at 1 : 1000, and H3K27me3 (Abcam) at 1 : 250. Secondary antibodies Alexa Fluor 488 or 568 (Invitrogen) were used at 1 : 1000 and incubated at RT for 45 min. Nuclei were counterstained with 300 nM DAPI for 5 min at RT. For each marker of interest, exposure times for DAPI and primary were kept constant. Five representative colonies were imaged per treatment, and 10 cells were randomly selected per colony for fluorescence intensity analysis using ImageJ. Corrected total cell fluorescence (CTCF) was calculated using the following formula : CTCF = μM, Invitrogen) for 20 min at 37°C, counterstained with 300 nM DAPI, and washed twice with PBS. Cells were then run on a Beckman Coulter CyAn analyzer (Beckman Coulter), where a minimum of ten thousand live cell events were acquired using DAPI gating for each sample. Flow density plots were generated using Summit 4.3 software (Beckman Coulter). Mean log10 fluorescence data values were recorded and normalized for the statistical analysis.To determine mitochondrial ROS production, hPSC were dissociated using TrypLE Select, centrifuged at 300g, incubated in mTeSR1™ with the MitoSOX Red superoxide indicator (1 μg of RNA using M-MLV Reverse Transcriptase (Promega). Triplicate reactions of each sample were run using EvaGreen Master Mix (Solis BioDyne) on a ViiA7™ thermocycler (Invitrogen). No-template control samples with nuclease-free water in place of cDNA were included for every primer set, and a –RT control was included to confirm appropriate cDNA synthesis. The relative abundance of each gene was normalized to that of RPLP0 and analyzed using Q-Gene which is based on the ΔΔCt method. Standard curves were generated for each gene to control for primer-specific efficiencies. All PCR products were verified by sequencing. See Supplementary Total RNA was isolated from hPSC using TRIzol (Invitrogen). RNA was isolated using a chloroform-induced triphasic solution before DNase treatment using DNase-I (Ambion). cDNA was synthesized from 1 μg of total RNA as input. Sequencing was performed with 12 samples per lane, using 50 bp single-end reads. The quality of resultant raw FASTQ data files was assessed with FastQC. Data files were uploaded to the Galaxy platform [https://usegalaxy.org/ and processed using the following pipeline: FASTQ Groomer to convert raw Illumina FASTQ files to Sanger Quality score format, Trim Galore! to trim the 13 bp of Illumina standard adapters, RNA STAR to align reads to the UCSC GRCh37/hg19 human reference genome, featureCounts to measure gene expression, and DESeq2 to identify DEGs. GO analysis of Biological Function terms and KEGG pathways were determined using DAVID. DEGs were submitted to NetworkAnalyst [RNA from MEL1 and MEL2 hPSC cultured at 5% and 20% oxygen was isolated using the RNeasy Plus Mini Kit (Qiagen). The RNA integrity numbers (RIN) of 10 for all samples were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was analyzed on the Illumina HiSeq 2500 Sequencing System (Illumina) at the Australian Genome Research Facility (AGRF) using 1 platform  at httpskAnalyst  to visuap < 0.05). Statistical analyses were performed using Student's t-test to calculate differences between two groups, or following a two-factor ANOVA, significant main effects and interactions were further analyzed using simple main effects calculated using the MS residual from the initial ANOVA. All analyses were performed in Prism 7 (GraphPad) and unless otherwise stated n = 3, where n is the number of biological replicates.All data are presented as the mean ± SEM (