The DPPH radical scavenging activity was determined for all samples applied in 6 concentrations . IC50 varied from 0.0700 to 0.9320 mg/mL (IC50 of ascorbic acid = 0.0757 mg/mL). The % of H2O2 inhibition in FOX assay was assessed for P1, P2, P3, P4 and P8 applied in 2 concentrations (5 and 0.5 mg/mL). A significant H2O2% inhibition was obtained for these samples for the lowest concentration. We firstly report the presence of resveratrol as bioactive compound in Western Romanian propolis. The principal component analysis revealed clustering of the propolis samples according to the polyphenolic profile similarity. The present study aimed to assess the phenolic content of eight ethanolic propolis samples (P1–P8) harvested from different regions of Western Romania and their antioxidant activity. The mean value of total phenolic content was 214 ± 48 mg gallic acid equivalents (GAE)/g propolis. All extracts contained kaempferol (514.02 ± 114.80 μg/mL), quercetin (124.64 ± 95.86 μg/mL), rosmarinic acid (58.03 ± 20.08 μg/mL), and resveratrol (48.59 ± 59.52 μg/mL) assessed by LC-MS. The antioxidant activity was evaluated using 2 methods: (i) DPPH  assay using ascorbic acid as standard antioxidant and (ii) FOX (Ferrous iron xylenol orange OXidation) assay using catalase as hydrogen peroxide (H This natural resinous mixture has been extensively used by ancient civilizations as folk medicine for its numerous beneficial properties that are synergistically related to its complex chemical composition [Populus nigra L.), comprises 50% resins, 30% wax, 10% volatile oils, 5% pollen, and 5% other organic constituents [Propolis is a sticky material harvested and processed by honey bees from buds, leaves, and the bark of trees  in order to secure and defend the hives  to assess the polyphenolic profile of eight ethanolic extracts of propolis samples harvested from different regions of Western Romania, including the presence of novel bioactive compounds, such as resveratrol, and (ii) to evaluate their antioxidant activity by 2 methods. Furthermore, in order to investigate the relationship between the phenolic profile of propolis samples and their geographical origin, principal component analysis (PCA) followed by clustering were performed.The main finding is the identification of resveratrol as an important bioactive compound in all propolis samples collected from Western Romania. Additionally, the dose-dependent characterization by two methods of the antioxidant capacity was performed.Total phenolic content (TPC) of the samples was determined spectrophotometrically (Folin-Ciocȃlteu method) and expressed as mg of gallic acid equivalents per g of sample (mg GAE/g)—The distribution of individual phenols in the eight samples is depicted in Kaempferol and quercetin were the major polyphenolic compounds identified in the Romanian propolis extracts, with the former representing 72.86 ± 11.33% of all individual polyphenols, and the latter 15.97 ± 9.68, respectively. The percentage of rosmarinic acid (ester of caffeic acid) from all polyphenols identified in propolis, varied between 4.34% and 14.04%; the mean concentration in the 8 extracts was 58.03 ± 20.08 μg/mL. Resveratrol, a powerful protective stilbene derivative, was also constantly present in variable concentrations in all eight samples  and reprChromatograms of individual samples (P1 to P8) are available as 50 (the extract concentration that determines the 50% DPPH inhibition) was further calculated for each sample and expressed in mg/mL   and was in mg/mL .As presented in The percentage of DPPH inhibition still remained high for the next two lower concentrations  for all samples but P6. The antioxidant activity showed an important decrease for all samples when applied in the lowest tested concentration (0.3 mg/mL), except for P2. Indeed, sample P2 at 0.3 mg/mL still preserved a radical scavenging activity of 78.16%.The concentration-dependency of the radical scavenging activity of the eight propolis samples is depicted in The percentage of DPPH inhibition for all samples (except P6) at 10 mg/mL, 5 mg/mL and 3 mg/mL was ~90% and thus similar to the one of ascorbic acid (at 0.105–0.13 mg/mL). Moreover, all samples still retained a high antioxidant capacity (88.60% ± 1.73%) when applied at 1.5 mg/mL, except, once again, for P6 (67.58%).Also, all samples (but P6) quickly reacted with the DPPH radical (~80 s) prior to reaching the equilibrium of reaction (that was inferior to the one of ascorbic acid) .An example of the time-dependency of the reaction is presented in 50 values . It is tempting to speculate that the low concentration of this powerful antioxidant may partly account for the low antioxidant activity of P6. Also, P7 had a rather high value of IC50 (0.5039 ± 0.0234 mg/mL) that might be explained by the fact that it contained the lowest amount of total polyphenols (170.24 mg/g). However, no statistical significance was reached when we calculated the Pearson coefficient as presented in The standing out value of IC2+) into ferric iron (Fe3+) which will react and form a colored complex with xylenol orange (XO) that can be measured at 560 nm. The % of H2O2 inhibition is presented for the samples that showed an important DPPH scavenging effect  applied in a high (5 mg/mL) and low concentration (0.5 mg/mL), respectively. In this assay catalase (CAT), the H2O2 scavenger, was used to compare the effect of propolis samples  is a rapid spectrophotometrical assay based on the ability of hydroperoxides to oxidize ferrous iron .As depicted in p-coumaric acid, rutin and quercetin, all being negatively correlated with this component. The second component had epicatechin as the main positive contributor, while the third component had total phenolic content as the major positive contributor. The similarity among polyphenolic profiles was examined when each sample was plotted using the first three principal components showing a clustering tendency, which allowed us to perform a cluster analysis , group 2—P6 and P8, which showed a similarity of 96.01%, group 3—P1 and P7 with a similarity of 94.28%. Samples P1, P7, P3 and P5 presented an important level of similarity (56.10%). Samples P2 and P4 were grouped together, but the similarity between them was low (13.40%) .50 ; indeed, a lower IC50 value of propolis samples corresponds to a greater capability to neutralize free radicals. It is worth mentioning that, for the poplar propolis extracts, correlations between total polyphenols/flavonoids and antioxidant capacity were not always reported in the literature [Since the antioxidant activity of propolis extracts has been classically ascribed to the polyphenolic content, the Pearson correlation coefficient (r) was computed as a measure of the linear association between quantitative variables. No statistically significant correlations between total phenolic content of Western Romanian propolis and either technique used to assess the antioxidant capacity were found in our study. As expected, the antioxidant activity evaluated by the DPPH method showed a significant, strong negative correlation with ICterature .The present paper was firstly aimed at characterizing the polyphenolic composition of ethanolic propolis samples collected from different regions of Western Romania.v/v) extracts [p-coumaric acid [The TPC of Western Romanian propolis varied from the lowest value of 170.24 mg GAE/g (sample P7) to the highest one, i.e., 333.83 mg GAE/g (sample P4) with a mean value of 214.30 ± 48.15 mg GAE/g of dry weight. Our results are in line with the data reported in the international literature for the TPC of propolis extracts, with values ranging from 30 to 200 mg GAE/g . Howeverextracts . Similarextracts . Two proextracts . Abubakeextracts . In 5 Etextracts . As for extracts .At variaric acid .Populus sp.  the major type of propolis in temperate zones [v/v), ethanol 60% extracted the highest amount of polyphenols. In their study, kaempferol varied between 1.33 and 3.54 mg/g and quercetin between 1.25–2.50 mg/g, respectively [50 = 0.01372 mg/mL), and anti-inflammatory activities for kaempferol [As for the individual polyphenols, four compounds were found in higher concentraions in all samples, namely kaempferol, quercetin, rosmarinic acid, and resveratrol, respectively . Kaempfete zones . Coneac ectively . In our empferol . Epidemiempferol . Similarempferol . Howeverempferol .Macaranga was the plant source used by Apis mellifera [The identification of resveratrol, as novel bioactive compound in Romanian propolis, is presented for the first time in this paper. Resveratrol, a powerful protective stilbene derivative, accounted for up to19.77% from all polyphenols in our samples. This is the most relevant finding of the present study and appears to be a characteristic of Western Romanian propolis since it was not reported in other types of Romanian propolis (reviewed in ref. ). As forellifera .Resveratrol is probably the most important polyphenol studied for the complex protective effects in ageing, cardiovascular pathology, malignancies and, more recently, the emerging field of cardio-oncology ,34,35. Ip-coumaric acid was detected only in sample P2, albeit in minute concentration—yet this sample had the highest antioxidant activity. Rutin was present in 5 out of the 8 samples, ranging from 1.03 ± 0.73 μg/mL to 10.11 ± 3.22 μg/mL of extract. Epicatechin was identified in low quantity in a couple of samples, representing between 0.24% and 0.56% from individual polyphenols. Gallic acid, protocatechuic acid, caffeic acid and ferulic acid were not detected in the analyzed samples.The phenolic acid As regarding the PCA analysis of the 8 propolis samples, we report a clusterization as follows: P3, P5; P6, P8; P1, P7 and P2, P4. Three clusters seemed very similar regarding the polyphenolic profile, the percentage of similarity being greater than 90% . Two samples, P2 and P4 represented the outliers, as their similarity with the rest of samples being very low. Analyzing their polyphenolic content, sample P2 is remarked for the highest content of individual polyphenols (1176.61 ± 161.59 µg/mL), whereas sample P4 presented the highest total phenolic content (333.83 ± 13.79 mg GAE/g). Samples P1, P7 were grouped with P3 and P5 as they showed a similarity of 56.10%.50 were determined. The IC50 varied from 0.0700 mg/mL for the P2 sample to 0.9945 mg/mL for P6 sample. Interestingly, sample P2 had a lower IC50 value when compared to ascorbic acid (IC50 = 0.0757 mg/mL), indicating the strongest anti-oxidant capacity. Whether this effect can be recapitulated in vivo, in experimental conditions associated with oxidative stress warrants further investigation. Mărghitaș et al. reported IC50 values between 0.3 mg/mL and 5.6 mg/mL for 13 propolis ethanolic extracts originating from Transilvania [50 varying from 0.007 to 0.066 mg/mL which was lower as compared to the value (0.184 mg/mL) for ascorbic acid used as control [50 varied between 0.33348 mg/mL and 0.60088 mg/mL, while for ascorbic acid was 0.28492 mg/mL [50 values from 0.043 to 0.269 mg/mL for 20 samples of Korean propolis [50 values for different propolis extracts varied between 0.633 mg/mL and 13.798 mg/mL [50= 16.55 ± 0.87 μg/mL) for Mexican propolis (ethyl-acetate extraction) [The percentage of DPPH free radical inhibition along with ICsilvania . Belfar  control . For 10 92 mg/mL . Wang etpropolis . Sun et raction) .Ahn et al. assessed the composition of several propolis samples harvested from 12 regions of China and concluded they were similar to poplar-type propolis. The authors used 3 techniques for the assessment of the antioxidant activity of the Chinese propolis: the inhibition of linoleic acid oxidation by means of beta-carotene bleaching, the DPPH radical-scavenging activity and thescavenging activity on 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation. All but one sample displayed a high antioxidant activity that was associated with the presence of caffeic acid, caffeic acid phethyl ester and ferulic acid . In a siIn a recent comprehensive study, Di Marco et al. used two antiradical assays (DPPH and FRAP) to assess the antioxidant activity of 460 Italia honeys and reported the highest antioxidant activity for the dark honeys .2O2 inhibition for the samples reported to have the highest and lowest DPPH scavenging activity, respectively. Interestingly, while both concentrations act as H2O2 scavengers  a superior antioxidant effect was found for the low dose as compared to the high one. A hypothesis was formulated in the literature regarding the hormetic effect of phytochemicals [We also thought to use a second technique, FOX assay for the assessment of the % of Hhemicals  as classhemicals ,46.Moreover, bee products, including propolis also contain fatty acids ; in partA limit of the present study is that we did not analyze other chemical components of propolis, in relation with the antioxidant activity. Indeed, it has been earlier suggested that flavonoids are responsible for the biological activities of European propolis . At variApis mellifera origin  were collected from the Western Romaniaduring 2015–2016: P1—Iteu, Bihor (BH) (47°21′7″N 22°25′7″E), 2015; P2—Bocşa, Caraș-Severin (CS) (45°22′29″N 21°42′38″E), 2015; P3—Șiria, Arad (AR) (46°16′2″N 21°38′18″E), 2015; P4—Ineu, Arad (46°26′N 21°50′E),2015; P5—Sărăuad, Satu Mare (SM) (47°28′45″N 22°37′32″E), 2015; P6—Folea, Timiș (TM) (45°29′54″N 21°18′0″E), 2015; P7—Iteu, Bihor (47°21′7″N 22°25′7″E), 2016; P8—Bocşa, Caraș-Severin (45°22′29″N 21°42′38″E), 2016 (Propolis samples of E), 2016 .The appearance of the 8 propolis extracts in relation to their sites of collection is presented in v/v) as solvent  at a ratio of 1:20 (g/mL). Samples were stirred for 60 minat ambient temperature using a platform shaker (Heidolph PROMAX 1020) and then filtered through a filter paper. Subsequent dilutions were prepared for the experiments.Raw propolis was kept in freezer and the cooled samples were grinded prior to the extracts’ preparation. Propolis extracts were prepared using ethanol 60% (v/v) with distilled water. The samples were kept at room temperature for 5 min and further treated with 1 mL Na2CO3 60 g/L . After incubation at 50 °C for 30 min, the absorbance was measured at 760 nm using a UV-VIS spectrophotometer . Calibration curve was obtained using gallic acid as standard (0–200 μg/mL) and the calibration equation was y = 0.0173x + 0.1224 (R2 = 0.9986), where x is the gallic acid concentration in μg/mL and y is the absorbance. Results were expressed as mg GAE/g propolis (mean ± SEM).The total phenolic content was determined according to the Folin-Ciocâlteu method referred to in . A volumThe separation and identification of polyphenols was performed by means of LC-MS  with electrospray ionization according to a technique described in ref.  and adapv/v) and to 0.5 mL DPPH  1 mM ethanol solution. The reaction was automatically monitored for 1200 s at 517 nm on a UV-VIS spectrophotometer . The absorbance was continuously measured from 5 to 5 s. Ascorbic acid 0.13 mg/mL in ethanol 60% (v/v) was used as positive control. Ascorbic acid was purchased from Lach-Ner Company (Czech Republic). Radical scavenging activity (RSA) was calculated with the formula: RSA (%) = 100 − (A517 (sample)/A517 (DPPH)) × 100), where RSA = radical scavenging activity of extract (%), A517 (sample) = sample absorbance measured at 517 nm at time t, A517 (DPPH) = the absorbance of DPPH solution measured at 517 nm at time t. The antioxidant capacity of the extracts was expressed as the IC50 value and compared to the one of ascorbic acid.The DPPH assay represents a classic method frequently used to assess the antioxidant capacity of plant extracts that was adapted from ref. . Moacă e2+ to Fe3+ ions which will then form a colored adduct with xylenol orange (XO) measurable at 560 nm. The reaction can be described as: Fe2+ + R-OOH → Fe3+ + RO·+ OH− and Fe3+ + XO →Fe3+-XO (blue colored adduct), where: XO = xylenol orange and R = H or a lipidic group.A standard solution of 100 μM hydrogen peroxide (H2O2) was firstly prepared, followed by the preparation of the working color reagent . The FOX assay was performed according to the method described in ref. . The pri2O2 scavenger, was used as positive control. A volume 100 μL of propolis sample plus 100 μL of standard hydrogen peroxide solution were mixed with 2 mL of working color reagent and incubated at room temperature (22–25 °C) for ~30 min. Samples (in duplicate) were read at 560 nm (spectrophotometer Jenway 6100). The results were expressed as % of hydrogen peroxide inhibition.Polyethylene-glycol (PEG)-catalase , a classic Ht test were used when appropriate. Inter-sample correlation and was conducted to investigate the underlying similarity of polyphenolic profiles. Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy and Bartlett’s test of sphericity were applied to verify the data suitability for PCA. The scree plots and the eigenvalues over 1 were considered as criteria for deciding the appropriate number of components to be extracted. Based on PCA extracted components, a hierarchical clustering was applied, using Ward’s minimum variance method and squared Euclidian distance. The Pearson linear correlation coefficients were supplementary determined and analyzed for certain variables describing the antioxidant activity. All reported probability values were two-tailed and a 0.05 level of significance was considered, while marking the highly significant values  as well. GraphPad Prism 7 and Minitab 18 were employed for data analysis.Results were expressed as mean ±standard error of the mean (SEM) as descriptive statistics. For the comparison of numerical values’ distribution across the samples, one-way ANOVA test followed by Bonferroni-adjusted multiple-comparisons between the pairs of samples and The present study firstly reports the presence of resveratrol as a novel and potent bioactive molecule in the composition of Western Romanian propolis; its contribution to the beneficial biological properties of individual propolis samples warrants further investigation. The polyphenolic profile of propolis samples from Western Romania was characterized and, based on PCA analysis, clusters with a high level of similarity were identified.


Preterm birth complications are one of the leading causes of death among children under 5 years of age. Despite advances in medical care, many survivors face a lifetime of disability, including mental and physical retardation, and chronic lung disease. More recently, both allogenic and autogenic cord blood cells have been applied in the treatment of neonatal conditions such as hypoxic-ischemic encephalopathy (HIE) and bronchopulmonary dysplasia (BPD).  To assess the safety of autologous, volume- and red blood cell- (RBC-) reduced, noncryopreserved umbilical cord blood (UCB) cell infusion to preterm infants. 7cells/kg) infusion for preterm infants <37 weeks gestational age. UCB cell characteristics, pre- and postinfusion vital signs, and laboratory investigations were recorded. Clinical data including mortality rates and preterm complications were recorded.  This study was a phase I, open-label, single-arm, single-center trial to evaluate the safety of autologous, volume- and RBC-reduced, noncryopreserved UCB cell  × 106 CD34+, (3.72 ± 3.25) × 105 colony forming cells (CFU-GM), and (99.7 ± 0.17%) vitality were infused to 15 preterm infants within 8 hours after birth. No adverse effects were noticed during treatment. All fifteen patients who received UCB infusion survived. The duration of hospitalization ranged from 4 to 65 (30 ± 23.6) days. Regarding preterm complications, no BPD, necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP) was observed. There were 1/15 (7%) infant with intraventricular hemorrhage (IVH), 5/15 (33.3%) infants with ventilation-associated pneumonia, and 10/15 (66.67%) with anemia, respectively.  After processing, (22.67 ± 4.05) ml UCB cells in volume, (2.67 ± 2.00) × 10 Collection, preparation, and infusion of fresh autologous UCB cells to preterm infants is feasible and safe. Adequately powered randomized controlled studies are needed. Preterm delivery is a global health problem. The rate of preterm birth ranges from 5% to 18% of babies born across 184 countries. An estimated 15 million babies are born preterm every year . InflammHuman umbilical cord blood cells (UCBC) are abundant in stem cells. These primitive cells can home into damaged tissues, produce anti-inflammatory and immune-modulatory factors by paracrine effects, and differentiate into tissue cells . PotentiRecently, delayed cord clamping in premature neonates have been reported to improve neonatal mortality and morbidity. The American College of Obstetricians and Gynecologists now recommends a delay in umbilical cord clamping in preterm infants for at least 30–60 seconds after birth . The potBased on these evidence, we hypothesized that autologous cord blood infusion was safe for preterm infants. We report the outcomes of the infusion of autologous, volume- and RBC-reduced, noncryopreserved cord blood cell to 15 premature neonates.7cells/kg) infusion for preterm infants <37 weeks gestational age.This study was a phase I, open-label, single-arm, single-center trial to evaluate the safety of autologous, volume- and red blood cell- (RBC-) reduced, noncryopreserved umbilical cord blood cells (UCBC)  of Guangdong Women and Children's Hospital were eligible if they were (1) preterm: <37 weeks gestation, (2) without congenital abnormalities, (3) without maternal chorioamnionitis, (4) had available UCB, and (5) the mother was negative for hepatitis B (HBsAg and/or HBeAg) and C virus (anti-HCV), syphilis, HIV (anti-HIV-1 and -2) and IgM against Cytomegalovirus, rubella, toxoplasma, and herpes simplex virus. The study protocol was approved by the ethics committee of Guangdong Women and Children's Hospital. All patients in the study were given an intensive care therapy in accordance with the departmental guidelines which included therapies including positive pressure mechanical ventilation, noninvasive respiratory support, oxygen therapy, and exogenous surfactant  replacement. Chest radiographs were performed at admission and 8 hours after CBT on the first day of life in all surviving patients. Blood gas was monitored every 24 hours until weaning from ventilation. All clinical diagnoses were defined according to a standard reference . Soon afg, 30 min, RT, Beckman, American), then was transferred to cryobags. Excessive nucleated cell-poor plasma was expelled. Meanwhile, MNC count, CD34 cell, CFU-GM, and sterility detection  were performed. Cell viability was measured via 7-aminoactinomycin D (7-AAD) detection kit through flow cytometry analysis . All infusions were administered in Guangdong Women and Children Hospital. Infusate and subject identities were double-checked by the research and clinical nursing staff. Infusions were also monitored by the research and clinical staff. Cells were infused over 15 minutes, followed by a 2 ml saline flush to clear the intravascular line.Guangdong Cord Blood and Stem Cell Bank is a public provincial blood bank affiliated to the Guangdong Women and Children's Hospital, which collects cord blood of every delivery in this hospital. Therefore, the cord blood of all the subjects had been routinely collected during the delivery. The procedure of cord blood collection and transfusion was performed in accordance with the cord blood bank guidance . The umbShortly, before, during, and until 24 hours after transfusion, heart rate, systolic, diastolic, and mean arterial blood pressure and arterial blood oxygen saturation level were monitored in peripheral blood continually and documented. Moreover, laboratory investigations in peripheral blood were monitored and kept stable during the whole treatment period, detailed in From January 1, 2009, till June 5, 2016, fifteen infants were enrolled for the treatment, gestational age ranged from 28 2/7 to 34 1/7 (31.2 ± 1.62) weeks and birth weight ranged from 1200 to 2220 (1582.7 ± 252.8) grams; 12/15 (80%) were delivered by cesarean section. All 15 patients who received the cord blood infusion survived. The duration of hospitalization ranged from 4 to 65 (30 ± 23.6) days. Details were shown in 8), mean (3.10 ± 2.17 × 108); cells postprocessing ranged from 0.86 to 7.83 (×108), mean (2.67 ± 2.00 × 108); cells concentration postprocessing ranged from 5.85 to 40.8 × 106/ml, mean (13.10 ± 10.35 × 106/ml); CFU-GM ranged from 0.72 to 11.27 (×105), mean (3.72 ± 3.25 × 105); amount of CD34+ cells in units varied widely, ranged from 0.1 to 16.22 × 106, mean (22.67 ± 4.05) ml; and viability of postprocessing units was high, ranged from 99.5 to 100%, mean (99.7 ± 0.17%). Details are shown in Cord blood volume collected ranged from 27 to 76 ml, mean (47.13 ± 19.10) ml; volume postprocessing ranged from 16 to 30 ml, mean (22.67 ± 4.05) ml; cells collected ranged from 0.97 to 8.11 ; time between collection (birth) and initiation of infusion ranged from 4.5 to 9 hours after birth, mean (6.77 ± 1.52 h); infused volume ranged from 2 to 28 ml, mean (10.27 ± 6.18 ml); pathogen detection  results were all negative.Infused NC ranged from 4.48 to 5.0 × 10The patient's vital signs and laboratory investigations were monitored during the whole treatment period, details were shown in The fifteen patients who received infusions all survived.Three patients had birth asphyxia, among them one suffered from IVH. None of the patients developed abnormal clinical features of central nervous system disorders such as convulsions, apnea, or dysphagia.12/15 (80%) presented with tachypnea and grunting soon after birth. The infants were diagnosed with RDS, 2/15 (13.3%) cases were grade I, 5/15 (33.3%) cases were grade II, 6/15 (40%) cases were grade III, and 2/15 (13.3%) cases were grade IV; and 12/15 (80%) received one dose PS replacement and 8/15 (53.3%) received intubation-surfactant replacement extubation–nasal continuous positive airway pressure (INSURE) therapy; however, one patient needed reintubation. 4/15 (26.7%) received mechanical ventilation; the median duration was 3.2 ± 1.8 days. The duration of oxygen therapy was (5.3 ± 3.0) days. No patient suffered from BPD, and chest radiographs showed improvement.Pseudomonas aeruginosa pneumonia, one was a case of Acinetobacter baumannii pneumonia, and 1 suffered from late onset sepsis, infected with Klebsiella pneumonia proved by blood culture.5/15 suffered from ventilation-associated pneumonia (VAP), of which two were cases of Klebsiella pneumonia, one was a case of No patients suffered from ROP.No patients suffered from NEC.10/15 (66.67%) suffered from anemia (≤140 g/l); 2/15 (13.33%) needed RBC transfusion.In our study, we treated 15 preterm infants with autologous, volume and RBC-reduced cord blood cells. The treatment was started within 8 hours after birth. No adverse effect of cell therapy was noticed. No patient died during treatment. No preterm complications such as BPD, NEC, or ROP were observed. Our study presents preliminary data on the safety of autologous cord blood cell therapy in preterm infants. We postulated that several factors contributed to the safety issue, among them, the most important one was the autologous cell source. Based on the autologous cell source, no GVHD-related complication was observed. Moreover, autologous cell source avoided ethical issues. A second factor that contributed to the safety issue was cord blood minimal-processing procedure. In our study, only density gradient centrifugation was employed to separate nucleated cells. Since our cell infusions were started within 8 hours after birth, no cryopreservation was needed; thus, no chemicals were added into the cord blood cells for cryopreservation. This minimal-processing procedure and immediate transfusion after processing helped to avoid contamination and possible chemical toxicity. It also alleviated decrease of viability which may happen during storage.8 mononuclear cells was collected before processing. After processing, cord blood volume and TNC were reduced to (22.67 ± 4.05) ml and (2.67 ± 2.00) × 108, respectively, including (22.67 ± 4.05) × 106 CD34+ and (3.72 ± 3.25) × 105colony forming cells (CFU-GM) in a vitality (99.7 ± 0.17%).In our study, mean (47.13 ± 19.10) ml cord blood with a total TNC of (3.10 ± 2.17) × 10Recently, delayed umbilical cord clamping 30–60 seconds after birth in preterms had been recommended by the American College of Obstetricians and Gynecologists and had been reported to reduce preterm-related complications. It is possible that delayed cord clamping increases supply of RBC and valuable stem and progenitor cells (SPC), thus may improve mortality and morbidity in premature neonates , 16. HowBPD is the main complication that contributes to morbidity and mortality in extremely premature newborns. It has been reported that among infants born with gestational ages of 22 to 28 weeks, 68% suffered from BPD . The patStudies on safety and feasibility of whole autologous cord blood transplant in preterm were also reported , 20. RudRegarding the administration route, there were reports on the advantage of damaged site administration when compared to intravenous infusion. However, on the one hand, the potential preterm complications were due to multiorgan damage. To achieve the multiorgan-targeted effect, we used intravenous infusion as the administration route, which may result in cells being trapped in organs such as lung and brain. On the other hand, in the report supported site administration, allogenic-MSC was used in intratracheal administration to treat hyperoxia-induced lung damage; it seemed to attenuate the side effects of rejection.In our study, we chose the infusion timing to be very soon after birth which is within the first 8 postnatal hours. Although some infants were delivered at midnight, we tried to process as early as within 8 hours after their birth. As it had been reported that it would take more than 1 week for progenitor cells to differentiate into damaged tissue cells, we administered CBC during the first hours after birth, so that it might provide enough time for these cells for differentiation.In conclusion, we demonstrated autologous, volume- and RBC-reduced, noncryopreserved cord blood cells transfusion soon after birth was safe and feasible in preterm infants. Autologous cord blood infusion avoid GVHD; meanwhile, the reduced volume would protect the fragile cardio-function of the preterm infants. This autologous, volume- and RBC-reduced, method guaranteed the safety of application. In addition, this was an autologous transfusion instead of “cell transplantation therapy”, and therefore it was not regulated by the FDA regulation licensing public cord blood banks distributing unrelated banked cord blood units for allogeneic transplantation in 2012. However, our study was the single-center descriptive study with limited number of preterm infants, further multicenter randomized controlled trials are needed to prove the effectiveness.


Oenanthe javanica (Blume) DC. in promoting nitrogen removal in free-water surface constructed wetlands (FWS-CWS) under low temperatures (<10 °C), pilot-scale FWS-CWS, planted with O. javanica, were set up and run for batch wastewater treatment in eastern China during winter. The presence of macrophytes observably improved the removal rates of ammonia nitrogen (65%–71%) and total nitrogen (41%–48%) (p < 0.05), with a sharp increase in chemical oxygen demand concentrations (about 3–4 times). Compared to the unplanted systems, the planted systems not only exhibited higher richness and diversity of microorganisms, but also significantly higher abundances of bacteria, ammonia monooxygenase gene (amoA), nitrous oxide reductase gene (nosZ), dissimilatory cd1-containing nitrite reductase gene (nirS), and dissimilatory copper-containing nitrite reductase gene (nirK) in the substrate. Meanwhile, the analysis of the microbial community composition further revealed significant differences. The results indicate that enhanced abundances of microorganisms, and the key functional genes involved with nitrogen metabolism in the planted systems played critical roles in nitrogen removal from wastewater in FWS-CWS. Furthermore, abundant carbon release from the wetland macrophytes could potentially aid nitrogen removal in FWS-CWS during winter.To investigate the role and microorganism-related mechanisms of macrophytes and assess the feasibility of  In rece−1 . Previou−1 ,4. The p−1 ,4. The m−1 ,7.It is generally assumed that wetland macrophytes are closely related to the abundance, activity, and diversity of the rhizospheric microorganisms in FWS-CWS ,5,8. TheTypha, Scirpus, Phragmites, Juncus, and Eleocharis [Oenanthe javanica (Blume) DC., a native aquatic macrophyte of China, has been proposed as an ideal candidate for nitrogen removal in CWs during the low-temperature season because of its advantages, such as fast growth in wastewater, tolerance to freezing temperatures, and capacity for repeated harvest [O. javanica for wastewater purification in FWS-CWS are still limited. In particular, the underlying microorganism-related mechanisms influencing nitrogen removal by O. javanica roots in FWS-CWS during low-temperature seasons are poorly investigated.The most common species used in FWS-CWS include those from the genera eocharis ,24,25. O harvest . HoweverOenanthe javanica (Bl.) DC. as well as other control systems, were set up and fed with effluents from a secondary wastewater treatment plant (WWTP) during the low-temperature season. The nutrient removal performances were measured and compared among the different treatment systems. Plant growth dynamics, root physiological characteristics, abundances of the key functional genes involved in the nitrogen removal process as well as the microbial abundances, diversity, and community composition in the substrate were investigated to establish the exact role and detailed mechanism of nutrient removal by macrophytes in FWS-CWS. This is the first study that systematically explains the effects of O. javanica on the nitrogen removal in WS-CWS under low-temperature conditions.In the present study, FWS-CWS planted with O. javanica and substrate sand (Tcw); (2) control systems without macrophytes but with substrate sand (Tcs) [O. javanica but without the substrate sands (Tcp); (4) control systems with blank incubators filled with wastewater only (Tck). Each system included four replicates. Cleansed sand  was used as the substrate in Tcw and Tcs. The water level of each system was 35 cm. Several concrete bricks wrapped in polyethylene bags were used in Tcp to maintain the same liquid height as the other two groups. Planted seedlings of O. javanica with similar size (65 ± 2.5 cm in length) were selected from a local nursery and cleaned to remove the rhizospheric soil. The initial density was 26 plants per m2 (72 plants per incubator). The secondary wastewater was obtained from a neighboring WWTP and their primary characteristics are listed in To investigate the precise role of macrophytes in facilitating nitrogen removal from wastewater in FWS-CWS during winter, different wastewater purification systems were built via several fiber-reinforced plastic incubators  in Huai’an, Jiangsu Province, Eastern China  on 20 November 2015. These systems were: (1) FWS-CWS planted with nd (Tcs) ; (3) con4+–N, NO2−–N, NO3−–N, total nitrogen (TN), and COD were determined through water quality analyzing systems  according to standard analytical procedures [The water in each system was sampled for water quality at the end of the 2nd, 4th, 6th, and 8th batches. The water temperature, pH, and dissolved oxygen (DO) were measured by a Temperature/Light Data Logger , a portable Multi-parameter Water Quality Meter  and a DO electrode , respectively. NHBiomass and nitrogen content of plant samples from the beginning and end of the experiment were determined according to . BrieflyThe microbial samples from the rhizoplane of wetland macrophytes and the substrates were obtained on 10 January 2016. For the rhizoplane samples, 5 g of root was obtained from five plants in each system and placed into clean phosphate buffer in a Falcon tube. After ultrasonic processing at 90 W for 30 min, the isolated biofilm from the rhizoplane was collected by vacuum filtration with 0.22 μm membranes . For subThe total genomic DNA from microbial samples was first extracted and purified using QIAamp Fast DNA Stool Mini Kit  and the yield was evaluated with SpectraMax 190 . Subsequently, the integrity was detected with 1% agarose gel electrophoresis and stored at −20 °C until further use.amoA), nitrous oxide reductase gene (nosZ), dissimilatory cd1-containing nitrite reductase gene (nirS), and dissimilatory copper-containing nitrite reductase gene (nirK) was conducted using the Illumina-Eco real-time PCR system . The primers were synthesized by Genergy Biotechnology Limited Corporation  and the details are listed in Quantitative PCR analysis of seven target functional gene fragments, , archaea , anaerobic ammonia oxidation (anammox), bacteria (ANO 16S rRNA gene), ammonia monooxygenase gene  and 785R (5′-GACTACHVGGGTATCTAATCC-3′). Raw fastq files were demultiplexed, quality-filtered, and merged by FASTX-Toolkit (version 0.0.14) and Mothur program  . All reaThe Illumina sequencing raw data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive database .p > 0.05) between the mean values of the treatments were determined using Duncan’s test. Nonparametric tests were used for non-normal distribution data.Statistical Package for Social Sciences (SPSS) 17.0  was used for statistical analysis. The data were analyzed using a one-way analysis of variance to compare the performance of each mesocosm, and statistically significant differences (4+–N (64.58%–70.68%). However, no significant differences in the average removal rates of NO3−–N and NO2−–N were observed among the three systems in the four detected batches. The best removal efficiency with regard to TN was observed in Tcp (40.57%–46.41%) in the 2nd, 4th, and 6th batches and in Tcw (48.37%) in the 8th batch. Additionally, over time, an increase in the effluent COD concentrations was observed in Tcw and Tcp. It increased from 9.2 mg·L−1 to 26.9 mg·L−1 and from 9.1 mg·L−1 to 38.2 mg·L−1 in Tcw and Tcp, respectively. Accordingly, the ratio of COD/N in the effluent sharply increased from 0.5 to 2.1 and from 0.7 to 2.5 in Tcw and Tcp, respectively.The performance of nutrient removal varied greatly across the different systems . Tcw and−2 while the plant root biomass was enhanced by 11.8–13.0 g·m−2 during the operation period. Accordingly, the nitrogen content also increased by 0.110–0.143 g·m−2 and 0.108–0.117 g·m−2 in the shoot and root, respectively. Unlike the plant growth dynamics indicators, the physiological characteristics of plant root presented a mild fluctuation in Tcw and Tcp during the operation period. The plant root vitality showed a slight decline at the end of 2nd, 4th, and 6th batches in comparison to the beginning, but increased to 58.7–63.6 μg TTC·g−1 root·h−1 at the end of the 8th batch. Further, the ROL rates increased by 0.32–0.52 μmol O2·g−1 root·h−1 in Tcw and Tcp at the end of 8th batch, though they presented a slight decline at the end of the 2nd and 4th batches when compared to the beginning.amoA, nosZ, nirS, and nirK) in the samples from plant roots or sand in the three systems are shown in nirK, the abundances of bacteria, archaea, amoA, nosZ, and nirS in samples from rhizoplane in Tcw (Ps) were higher when compared to samples from rhizoplane in Tcp (Pw). The copy numbers in Ps were recorded as 8.947 × 1010 copies·g−1 root (bacteria), 1.954 × 1011 copies·g−1 root (archaea), 7.335 × 107 copies·g−1 root (amoA), 1.491 × 107 copies·g−1 root (nosZ), and 2.517 × 108 copies·g−1 root (nirS), while the numbers were 7.017 × 1010 copies·g−1 root (bacteria), 7.871 × 1010 copies·g−1 root (archaea), 4.828 × 107 copies·g−1 root (amoA), 6.812 × 106 copies·g−1 root (nosZ), and 2.017 × 108 copies·g−1 root (nirS) in Pw. On the other hand, significantly high abundances of all seven target functional gene fragments were observed in samples from sand in Tcw (Sp) when compared to samples from sand in Tcs (Su). The copy numbers of bacterial 16S rRNA, archaeal 16S rRNA, anammox bacterial 16S rRNA, amoA, nosZ, nirS, and nirK in Sp were recorded as 7.987 × 108 copies·g−1 sand, 1.049 × 1010 copies·g−1 sand, 2.392 × 107 copies·g−1 sand, 5. 413 × 107 copies·g−1 sand, 1.716 × 106 copies·g−1 sand, 2.842 × 107 copies·g−1 sand, and 7.865 × 106 copies·g−1 sand, respectively, while they were 3.220 × 108 copies·g−1 sand, 7.930 × 109 copies·g−1 sand, 1.241 × 107 copies·g−1 sand, 3.658 × 107 copies·g−1 sand, 3.897 × 105 copies·g−1 sand, 2.170 × 107 copies·g−1 sand, and 4.343 × 106 copies·g−1 sand, respectively, in Su.The absolute abundances of bacteria, archaea, and anammox as well as the four functional genes, , comprised a total of 48 genera , Aeromonas (2.44%), Bacillus (1.72%), and Rhizobium (0.60%). In Pw, Pseudomonas accounted for 65.76%, followed by Aeromonas (7.23%), Exiguobacterium (5.14%), Paenibacillus (2.93%), Rhizobium (1.01%), Acidovorax (0.94%), Flavobacterium (0.67%), and unclassified-Rhodobacteraceae (0.61%). The order for Sp was Halomonas (14.49%), Rhodoferax (7.73%), Pseudomonas (5.19%), Arenimonas (5.00%), unclassified-Bacteroidetes (4.18%), Flavobacterium (2.79%), Perlucidibaca (2.46%), unclassified-Rhodobacteraceae (2.20%), Simplicispira (1.93%), Hydrogenophaga (1.82%), and Thiobacillus (1.35%). The order for Su was Halomonas (18.68%), Pseudomonas (9.64%), Arenimonas (5.01%), Rhodoferax (3.19%), unclassified-Xanthomonadaceae (3.16%), Perlucidibaca (2.93%), Hydrogenophaga (2.79%), unclassified-Bacteroidetes (1.88%), unclassified-Rhodobacteraceae (1.84%), and Thiobacillus (1.71%). In addition, more abundant nitrifying bacteria were observed in Sp in comparison to Su. The relative abundance of unclassified-Nitrosomonadaceae was recorded as 0.0889% and 0.0333% in Sp and Su, respectively.The primary genera (relative abundance >1.00%) in the four systems with the addition of two nitrifying bacteria . Actinomycetes, Aquifaceae, Bacteroides, Firmicutes, Proteobacteria, and even archaea as well as fungus [Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria [Halomonas, Rhodoferax, Pseudomonas, and Arenimonas constituted the four primary genera of denitrifying bacteria in both the planted and unplanted systems. Hence, it is likely that the difference in community composition of denitrifying bacteria was not the material cause of the difference in nitrogen removal efficiency between the planted and unplanted systems. In contrast, significant differences between the planted and unplanted systems were obviously revealed by the quantitative analysis of the critical functional genes, including nosZ, nirS, and nirK.Denitrifying microorganisms exist across a wide range of microbial groups, which involve s fungus . A totalbacteria . In thisnirK gene and dissimilatory cd1-containing nitrite reductase encoded by the nirS gene. The third step is the conversion from nitric oxide to nitrous oxide. The last step is catalyzed by the product of the nosZ gene which converts nitrous oxide into nitrogen gas. Additionally, the nosZ gene is often regarded as the marker of complete denitrification while the nirS and nirK genes usually act as the markers for the second denitrification step [nosZ gene, presented significant increases in the planted systems in contrast to the unplanted ones. This result indicates the remarkable facilitation of the quantity of denitrifying bacteria by wetland macrophytes which could be ascribed to the increased supply of biodegradable carbon. It is widely believed that the denitrification in CWs depends on organic carbon levels. Ye and Li [Denitrification, which converts nitrate to nitrogen gas, consists of four steps and commonly occurs under anaerobic conditions ,55. The ion step ,55. In te and Li  concludee and Li ,25,57. Oe and Li , on the e and Li ,58. In te and Li , the eleBrocadiales of the phylum Planctomycetes [Brocadiales were detected by the analysis based on V3–V4 regions of the 16S rRNA gene in the current research. Nevertheless, the quantitative analysis of the anammox bacterial 16S rRNA gene, which is usually regarded as the marker of anammox process, clearly indicated the presence of anammox bacteria [amoA gene, which indicated that the ammonia process was not the dominant pathway of the ammonia nitrogen removal. This result was consistent with the opinion that nitrification–denitrification primarily contributes to the removal of ammonia nitrogen when the C/N ratio is less than or equal to 6:1 in the CWs [Besides the nitrification–denitrification process, anammox has been widely indicated as a new ammonium oxidation process which occurs under anaerobic conditions. Compared with the nitrification–denitrification pathway, the anammox pathway has the advantage of a lower demand for carbon sources ,10,55. Tomycetes ,61. HoweFlavobacterium in the class Flavobacteria, in the planted systems may also relate to the nitrogen removal via the denitrification process [Analysis of bacterial community composition revealed that Proteobacteria was the dominant phylum followed by Bacteroidetes in not only the plant rhizoplane, but also the rhizosphere in the current system. Proteobacteria has been widely considered an active participant in nitrogen removal in the CWs for its high diversity of metabolism which involves global carbon, nitrogen, and sulfur cycling . The res process . In summO. javanica had a significant facilitating effect on the nitrogen, especially TN, removal from wastewater in the FWS-CWS during the low-temperature season with mean water temperature lower than 10 °C. Because the macrophyte could provide extra organic carbon by root exudation and plant residues, it enhanced the microbial abundance, diversity, and evenness, as well as the abundances of amoA, nosZ, nirS, and nirK, and those closely related to nitrification–denitrification, on the rhizoplane and in the substrate. This suggests that FWS-CWS planted with O. javanica is a reliable option for a higher removal rate of nitrogen during low-temperature seasons. However, the increased COD concentration in planted systems may also cause secondary pollution and pose a new challenge for the wastewater purification, especially, when the wetland macrophytes begin to shrivel and die under low temperatures. Therefore, further studies on the selection of plant species, the control of plant density, as well as feasible improvements to the approach, for instance, adopting the FWS-CWS as part of an integrated wastewater treatment train, merit more attention in the future.In conclusion, the presence of


Penicillium brasilianum (strain LaBioMMi 136) has been reported to be a great producer of secondary metabolites and a source of enzymes of biotechnological interest. Here, we report the draft genome sequence of P. brasilianum (strain LaBioMMi 136), isolated as an endophyte from the plant Melia azedarach (family Meliaceae). Penicillium brasilianum (strain LaBioMMi 136) has been reported to be a great producer of secondary metabolites and a source of enzymes of biotechnological interest. Here, we report the draft genome sequence of P. brasilianum (strain LaBioMMi 136), isolated as an endophyte from the plant Melia azedarach (family Meliaceae). Penicillium brasilianum , a fungus isolated from different sources, such as soil  was isolated from root bark of Melia azedarach as an endophyte following the procedure described by Santos and Rodrigues-Filho (P. brasilianum (strain LaBioMMi 136) was isolated by replication and grew as a bluish-colored culture. For DNA isolation, the fungus was cultivated at a density of 107 conidia ml−1 in a chemically defined medium (Czapeck) composed of glucose (30 g liter−1), NaNO3 (3.0 g liter−1), K2HPO4 (1.0 g liter−1), MgSO4 · 7H2O (0.5 g liter−1), KCl (0.5 g liter−1), and FeSO4 · 7H2O (0.01 g liter−1). Total genomic DNA was extracted using the DNeasy plant minikit (Qiagen) according to the manufacturer’s instructions.The fungus es-Filho . The rooP. brasilianum (strain LaBioMMi 136) genome was sequenced by two platforms, 454 (Roche) and MiSeq paired-end 2 × 150 bp (Illumina) sequencing. The Illumina read length was up to 151 bp (paired end). For the first Illumina run (SRA accession number SRR7995492), the total number of reads after adapter and quality trimming was 12,274,914 with an average length of 117 bp. For the second Illumina run (SRA accession number SRR7995491), the total number of reads after adapter and quality trimming was 11,321,146 with an average length of 129 bp. The 454 FLX+ platform produced reads of up to 1,000 bp; the total number of reads was 1,383,158, and the average read length was 581 bp with a total genome coverage of 140×. The 454 coverage was 40×, and the Illumina coverage was 100×. The 454 reads were quality controlled using GSRunBrowser v2.53 (Roche) with default parameters, and for the Illumina reads, MiSeq reporter v2.2 with adapter trimming and FastQC v0.10.1 (default parameters) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) were used.The de novo assembled using Newbler v2.53 (Roche). The final assembly consists of a genome size of ∼32.9 Mbp covered by 248 scaffolds with an N50 value of 292,589 bp. The GC content of the genome is 47.75%.The genome was P. brasilianum (LaBioMMi 136) was automatically annotated using SABIA v2.0 (The genome sequence of BIA v2.0  with defP. brasilianum (LaBioMMi 136) has the potential to produce a diverse array of natural products. The genome has 42 putative biosynthetic clusters containing, among others, 22 backbone genes, 12 of which are nonribosomal peptide synthetases (NRPSs), 13 polyketide synthases (PKS), 3 terpenes, 2 NRPS/PKS hybrids, 1 hybrid terpene/PKS, and 1 NRPS/terpene, further supporting the great genetic and enzymatic machinery potential for secondary metabolite production.antiSMASH v3.0  analysisP. brasilianum, leading to a better understanding of the host-endophyte chemical interactions and its complex secondary metabolite collection.Our data will contribute to future molecular studies with LJBN00000000. This paper describes the first version of the P. brasilianum genome (LJBN01000000). The first Illumina run is available under the SRA accession number SRR7995492, and the second Illumina run is available under the SRA accession number SRR7995491. The 454 run raw reads are available under SRA accession number SRR7993873.This genome sequencing project was uploaded to GenBank and is available under the accession number


Trichodesmium is an important dinitrogen (N2)-fixing cyanobacterium in marine ecosystems. Recent nucleic acid analyses indicate that Trichodesmium colonies with their diverse epibionts support various nitrogen (N) transformations beyond N2 fixation. However, rates of these transformations and concentration gradients of N compounds in Trichodesmium colonies remain largely unresolved. We combined isotope-tracer incubations, micro-profiling and numeric modelling to explore carbon fixation, N cycling processes as well as oxygen, ammonium and nitrate concentration gradients in individual field-sampled Trichodesmium colonies. Colonies were net-autotrophic, with carbon and N2 fixation occurring mostly during the day. Ten percent of the fixed N was released as ammonium after 12-h incubations. Nitrification was not detectable but nitrate consumption was high when nitrate was added. The consumed nitrate was partly reduced to ammonium, while denitrification was insignificant. Thus, the potential N transformation network was characterised by fixed N gain and recycling processes rather than denitrification. Oxygen concentrations within colonies were ~60–200% air-saturation. Moreover, our modelling predicted steep concentration gradients, with up to 6-fold higher ammonium concentrations, and nitrate depletion in the colony centre compared to the ambient seawater. These gradients created a chemically heterogeneous microenvironment, presumably facilitating diverse microbial metabolisms in millimetre-sized Trichodesmium colonies. Trichodesmium has a ubiquitous distribution throughout tropical and subtropical oceans where it contributes substantial amounts of new nitrogen (N) to the oligotrophic near-surface ocean through dinitrogen (N2) fixation [Trichodesmium is suggested to account for up to half of the biologically fixed N2 [Trichodesmium grows as filaments, referred to as trichomes, which at times aggregate as millimetre-sized spindle-shaped (tufts) or spherical (puffs) colonies [2 fixation [fixation , 2. In tfixed N2 , which gfixed N2 –6 and evfixed N2 –9. Trichcolonies , often fcolonies . These cfixation , 13 but fixation –16.2 to ammonium which, in addition to regenerated ammonium, can be oxidised to nitrite and nitrate (nitrification) or incorporated into biomass (assimilation). In turn, particulate organic N can be recycled to ammonium (ammonification), while nitrate and nitrite can be reduced to ammonium via assimilatory and dissimilatory pathways, or transformed to N2 (denitrification). Additional complexity to the N cycle is added by dissolved organic nitrogen (DON), which comprises a heterogeneous mixture of N compounds of different lability [Trichodesmium colonies, representing microenvironments distinctly different from the surrounding water in terms of their physico-chemical properties, metabolic functions and phylogenetic composition. For example, oxygen concentrations can decrease from 200% down to 10% air-saturation, and the pH from 8.8 down to 7.5, during a light–dark shift in Trichodesmium colonies [Trichodesmium colonies [Trichodesmium colonies are favourable microhabitats for numerous epibionts in otherwise N depleted waters [The N cycle involves a series of oxidation and reduction processes . Bioavailability . The co-lability , 20. Nonlability , 22. Thecolonies –25. Morecolonies , and newcolonies –29. Thiscolonies , referrecolonies  or trichd waters , 32, 33.Trichodesmium has been recognised for several decades [Trichodesmium holobiont stretches beyond N2 fixation, suggesting an intimate spatial coupling of various N cycling processes. However, this metabolic potential is yet to be confirmed by actual rate measurements.The association of epibionts with  decades –37. Howe decades , 38–41.  decades –16. ThusTrichodesmium holobiont with rate measurements of co-occurring N transformation processes. We used stable-isotope incubations to quantify N gain, recycling and loss processes in field-sampled Trichodesmium colonies. For simplicity, we focused on processes transforming inorganic N speciesc, like N2 fixation, ammonium release, nitrification, denitrification and nitrate reduction to ammonium/nitrite, without including dissolved organic N speciesc or assimilation processes other than C/N2 fixation in our analyses. Moreover, we measured oxygen profiles in individual colonies, and modelled microscale gradients of oxygen, ammonium and nitrate in the trichosphere, to characterise the chemical microenvironment in Trichodesmium colonies. Our quantitative estimates reveal the complexity of N cycling processes and microscale heterogeneity in Trichodesmium colonies.Here, we complement the previously observed metabolic potential in the Trichodesmium colonies were sampled at Station ALOHA (22°45′N 158°00′W) in the oligotrophic North Pacific Subtropical Gyre, in the frame of the Hawai’i Ocean Time-series program (HOT cruise #265), during September 2014. Colonies were collected from 0 to 10 m water depth using a plankton net . Seawater temperature was 27 °C and salinity 34.4. Further biogeochemical properties of the surface water are listed in Supplementary Table Trichodesmium under varying partial pressures of carbon dioxide [Seawater and puff-shaped  dioxide . We thera content, Trichodesmium species composition and epibionts, cell numbers and dimensions, and particulate organic carbon and nitrogen contents (POC and PON) were analysed as presented in Eichner et al. [a was analysed fluorometrically after extraction in 90% acetone. Trichodesmium species were tentatively identified from Lugol-preserved colonies via microscopy based on cell shape and size [Colony characteristics, including chlorophyll r et al. . In brieand size . Heterot13C-dissolved inorganic carbon  and 15N2 (Cambridge Isotope Laboratories), to quantify C fixation, N2 fixation and ammonium release during active N2 fixation. 15N2 was added as an aliquot of 15N2-enriched seawater, which was prepared from 0.2 µm filtered seawater following [2 and C fixation rates in single Trichodesmium cells using secondary-ion mass spectrometry (SIMS), as presented previously [13C-labelling was 4.2 ± 0.2%  and the 15N-labelling was 4.2 ± 0.4% . Incubations #2–6 were enriched with 15N-nitrate, 15N-nitrite or 15N-ammonium  to final concentrations of 0.9 ± 0.3 µM , equal to 15N-labelling of 55, 94 and 79% in the substrate pool, respectively. These final concentrations exceeded typical in situ concentrations, in order to overcome diffusion-limited solute transport into the colonies  into acid-washed Falcon tubes and stored at −20 °C until nutrient analyses following [−1 (precision/accuracy ± 1%). Incubation #5 was enriched with 86 µM N-Allylthiourea  in addition to 15N-nitrate, to inhibit the ammonium oxidation step of nitrification [Stable-isotope incubations were conducted during day-time (6AM–6PM) and night-time (6PM–6AM), with colonies being collected at 5AM (September 16) and 5PM (September 14), respectively. Colonies were individually transferred with an inoculation loop into 0.2 µm filtered water (to remove loosely associated biota), and thereafter into 5.9 ml Exetainer vials  with 0.2 µm filtered surface seawater, adding five colonies per vial. Isotopically labelled substrates were added to measure C fixation and specific N transformation processes . No isotopes were added to incubation #7. The Exetainers were closed headspace-free and placed in an incubator on deck, cooled with flowing surface water and shaded to 50% surface irradiance . The Exetainers were attached horizontally onto thin wires in the water, to allow for gentle movements during water flow and ship movement, thus keeping colonies in suspension and decreasing diffusion-limited solute transport to the colonies, as compared to colonies that would settle onto the vials’ bottom . Incubat13C and ±0.3‰ for 15N, using Vienna PeeDee Belemnite and air as C and N standards, respectively). Rates of N2 and C fixation were calculated following [15N-ammonium.Twenty-five colonies (5 colonies × 5 replicates) from incubation #1 were pooled per GF/F filter . Filters were dried at 50 °C overnight, fumed over HCl, pelletized into tin cups and analysed by EA-IRMS  at the University of Southern Denmark in Odense and the UFZ in Leipzig, Germany. Both GC-IRMS set-ups and the analytical procedure are specified in supplementary text 2, N2O, ammonium, nitrate and nitrite was calculated from the 15N-excess concentrations relative to air, and corrected for the 15N mol fraction in the N pool in control samples. Rates were calculated from the production of each N compound versus time per colony, and tested against controls for statistical significance . Rates not significantly different from controls were defined as not detectable. DIN concentrations were substantially enhanced by 55–94% after 15N-isotope additions, potentially stimulating (or inhibiting) N transformation processes in strictly N limiting water. Thus, all rates (except for N2/C fixation and ammonium release) should be considered as potential rates, and moreover as net rates due to concurrent production and consumption processes.The production of −2 s−1, respectively), as described in [−1, similar to natural floating or sinking velocities) [J were calculated according to Fick’s first law . Fluxes were normalised to entire colonies using the colony surface areas, calculated from the colony radius assuming spherical geometry.Oxygen concentration measurements were done under light and dark conditions (at 1000 and 0 μmol photons mribed in . Single ocities)  of filteocities) . Oxygen ocities) . Oxygen Concentration profiles and distribution fields of oxygen, ammonium and nitrate were simulated for colonies and single trichomes, using a recently developed advection–diffusion–reaction model . This moTrichodesmium colonies have a complex geometry, and are able to ascend, descend or remain neutrally buoyant in the water column [Trichodesmium colonies is therefore challenging and computationally expensive. Moreover, interstitial voids in highly porous phytoplankton colonies and aggregates are filled with viscous polymers [r column . Simulatpolymers  which inpolymers . For simCα (α specifies the considered solute) within the computational domain, with t as time, ∇ as gradient operator, Dα as diffusion coefficient, ε as local porosity and n as local number of cells in the representative elementary volume. The reaction term Rα denotes the solute consumption/release rate by each cell in the colony, accounting for Michaelis–Menten kinetics. Rα for oxygen and nitrate consumption were modelled according to the first-order kineticsKα)mm( the maximum reaction rate for each solute. Rα for oxygen and ammonium release were modelled according to zeroth-order kineticsVα)m( as constant (maximum) reaction rate. Kα)m( for oxygen respiration was set to 1 µM [Vα)m during stable-isotope incubations, normalised to single-cell units , whereas picocyanobacteria, dinoflagellates and fungi were rare, mostly limited to one individual of each per colony. Further characteristics of colonies and cells are listed in Table  in ref. ) at prop2 fixation occurred mostly at day-time, and less at night-time . For th2, and (iii) residual nitrate consumption specified as total decrease of added 15N-nitrate minus (i) and (ii). Approximately 10% of the consumed nitrate was reduced to ammonium and/or nitrite after 12-h incubations during day- and night-time. Complete denitrification was insignificant. Rates of nitrite reduction to ammonium were significantly lower than those for nitrate reduction to nitrite or ammonium . N2 and N2O production were not detected in most cases after 15N-nitrate, nitrite or ammonium additions, except for (i) low N2O production (mass 29) in 15N-ammonium incubations and (ii) N2O production (mass 30) after 15N-nitrate incubations in combination with N-Allylthiourea an inhibitor for nitrification, both during day-time , respectively, at time zero and did not change significantly after 12-h incubations .Bulk concentrations of ammonium, nitrate and nitrite in the control vials  were 0.19 ± 0.15, 0.40 ± 0.03 µmol LJ were 0.039 ± 0.032 nmol cm−2 s−1  during light due to net photosynthesis, and −0.024 ± 0.019 nmol cm−2 s−1  during darkness due to respiration. These fluxes lead to supersaturation of 145 ± 36% (92–203%) and undersaturation of 78 ± 12% (56–98%) in the colony centre, respectively. Rates of oxygen production exceeded those of respiration (Table pCO2 and day-times) during the same field campaign are available in [We recorded 32 oxygen profiles (21× light/11× dark), showing steep oxygen gradients from the ambient water towards the colony centre , morphology (puff-shape) and oxygen profiles were representative for the investigated colonies. Applying the shown porosity .The model output was validated against oxygen profiles measured for one ity Fig. , the modity Fig. . The curhttps://www.pangaea.de/).In addition to oxygen profiles, we modelled those for ammonium and nitrate. During day-time, newly fixed N was partly released as ammonium. Accordingly, the model predicted a steep ammonium concentration gradient, with ammonium concentrations being elevated to 570% (1.1 µM) in the colony centre compared to the ambient water (0.2 µM) Figs.  and 4. BTrichodesmium colonies represent highly heterogeneous microenvironments with dynamic physical, chemical and biological conditions, that change over micrometres [Trichodesmium colonies have often been proposed but rarely measured, since microsensors for relevant N compounds are not available for measurements in seawater. We used stable-isotope incubations in combination with microsensor measurements and computational simulations, to reveal the microscale growth conditions and N transformation processes inside N2-fixing Trichodesmium colonies. Our simulations predicted that ammonium concentrations were almost 6-fold enriched in the colony centre, owing to ammonium release from N2 fixation. Importantly, this enrichment might have been lower during our incubations due to a rapid ammonium turnover within colonies, as discussed below. It may, however, also increase due to enhanced ammonium production during organic matter remineralisation in decaying colonies, microbial infections or zooplankton grazing [2-fixing cyanobacteria in the Baltic Sea, ammonium concentrations were predicted to be up to 60-fold enriched within their centre compared to the ambient water [rometres –25 and wrometres . Steep n grazing . For insnt water , 59, 60.Trichodesmium colonies may become more heavily colonised by epibionts than free trichomes. In support of this hypothesis, typical epibionts on Trichodesmium colonies include Bacteroidetes and Gammaproteobacteria  [−1), the effective boundary layer thickness would decrease down to ~200–500 µm  , 38, 40,romonas) . Yet, inions see ).2 fixation in Trichodesmium colonies were largely restricted to daylight hours, supporting specific growth rates of 0.10 and 0.02 d−1, respectively  [Trichodesmium can indeed fix N2 and utilise combined N simultaneously when grown in culture [Trichodesmium cells have been shown to fix N2 in nature [Trichodesmium to associated heterotrophic bacteria in the same colonies as studied herein  , 72. Tri culture . Likewisn nature , and newum cells , 75, as um cells .Trichodesmium colonies, which we did not account for in our incubations. In the Caribbean Sea and Atlantic Ocean, Trichodesmium has been shown to release up to half of the recently fixed N2 as DON [−1 h−1 [Trichodesmium colonies; yet, it remains uncertain whether they are preferably assimilated by Trichodesmium or their epibionts [Organic compounds add another level of complexity to the N cycling network in 2 as DON . The rel [−1 h−1 . Such repibionts , 76.Trichodesmium and other associated autotrophs under natural conditions. Our simulations predicted nitrate depletion in the colony centre, even after we added 1 µM of nitrate  were demonstrated to be associated with Bacteroides, whereas those for nitrite reductase (nirB) were ascribed to Gammaproteobacteria in Trichodesmium cultures [About 10% of the consumed nitrate was recovered as ammonium. Whether nitrate was reduced to ammonium via assimilatory (N incorporation) or dissimilatory pathways (N transformation coupled to energy production) cannot be determined from our experimental design. The genetic potential is present for both. Assimilatory nitrate reduction is directly encoded in pibionts , 38. Sticultures .Trichodesmium colonies that were sampled at Station ALOHA a few months prior to our sampling [2-fixing colonies of Nodularia [Nodularia colonies [Nitrification was insignificant although oxygen was plentiful even during darkness, and ammonium concentrations that were predicted to be high within colonies Figs.  and 4. Csampling . Furthersampling , could nsampling , 33. Nitodularia  and decaodularia . Howevercolonies , and nitcolonies . The ratcolonies , 84 are colonies , and phycolonies .15N-nitrate production during our 15N-ammonium incubations. We actually found an indication for partial nitrification in N2O production after 15N-ammonium incubations  have been shown to be present in natural Trichodesmium colonies [15N-nitrite incubations in combination with the measured ammonium release did not yield any significant 29N2 production. Presumably this was due to the generally slow growth rates of anammox bacteria [Trichodesmium colonies ([Trichodesmium cultures [Trichodesmium colonies was 10% air-saturation (~20 µM) [Trichodesmium colonies.Complete denitrification was not detectable, presumably due to the low nitrate availability and high oxygen concentrations (≥50% air-saturation or ≥100 µM) even during darkness. Denitrifiers and denitrification-relevant gene expression  . Given t(~20 µM) –98, compTrichodesmium ecology by quantitatively exploring the potential microbial N cycling network in millimetre-sized colonies. The observed N cycling in puff-type colonies was imbalanced, with a high potential of gain and recycling processes but a low potential for loss by denitrification  appeared to act in concert to preserve new N from N2 fixation in close proximity, to fully benefit from bioavailable N in the oligotrophic near-surface ocean. Trichodesmium colonies may thus be described as consortia, reflecting associations of synergistic, mutualistic or syntrophic lifestyles [We evaluated a long-standing aspect of ion Fig. . Thus, Tfestyles , 100, inSupplementary Information


Similarly, inflammatory condition decreased DNMT1 and SIRT1 expression , as well as total DNMTs  and SIRT1  activities. Interestingly, GOx- and LPS-treated cells exhibited lower LINE-1 methylation compared to controls . We also demonstrated that treatment with 10 μM resveratrol for 24 h counteracted the detrimental effect on DNMT and SIRT1 functions, and LINE-1 methylation, in cells under oxidative and inflammatory conditions. However, further studies should explore the perspectives of resveratrol as a suitable strategy for the prevention and/or treatment of retinal degenerative diseases.The role of epigenetic alterations in the pathogenesis of retinal degenerative diseases, including age-related macular degeneration (AMD), has been pending so far. Our study investigated the effect of oxidative stress and inflammation on DNA methyltransferases (DNMTs) and Sirtuin 1 (SIRT1) functions, as well as on long interspersed nuclear element-1 (LINE-1) methylation, in human retinal pigment epithelial (ARPE-19) cells. Therefore, we evaluated whether treatment with resveratrol may modulate DNMT and SIRT1 functions and restore changes in LINE-1 methylation. Cells were treated with 25 mU/mL glucose oxidase (GOx) or 10 µg/mL lipopolysaccharide (LPS) to mimic oxidative or inflammatory conditions, respectively. Oxidative stress decreased DNMT1, DNMT3a, DNMT3b, and SIRT1 expression ( Oxidative stress and inflammation are shared pathological features across retinal degenerative diseases. Among these, age-related macular degeneration (AMD) is the most common cause of blindness in developed countries, with a prevalence that ranges from 2% to 20% among elderly people . Overall2O2) treatment is one of the most commonly used models to test oxidative stress susceptibility in vitro and oxidative damage in vivo. However, the effect of H2O2 is dose-dependent and ranges from cell proliferation, migration, survival, and differentiation [2O2 concentration, is alternatively used. In vivo studies also demonstrated that ocular injection of lipopolysaccharide (LPS) in rats stimulates the synthesis and release of proinflammatory mediators, such as nitric oxide, platelet-activating factor, tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β), and other cytokines [Hydrogen peroxide , out of which only DNMT1, DNMT3A, and DNMT3B are catalytically active . In mammSirtuin 1 (SIRT1), one of the seven mammalian homologs (SIRT1–SIRT7) of yeast silent information regulator 2, is a NAD+-dependent histone deacetylase with multiple roles in aging, apoptosis, DNA repair, inflammation, and oxidative stress . AlthougResveratrol , a flavonoid associated with the cardiovascular benefits of red grapes and wine, has been shown to significantly increase SIRT1 activity through allosteric interaction, increasing SIRT1 affinity for both NAD+ and the acetylated substrate ,38. MoreThe present study investigated the effect of oxidative stress and inflammation on retinal DNMT and SIRT1 functions, as well as on LINE-1 methylation levels, in ARPE-19 cells. Therefore, we evaluated whether treatment with resveratrol may modulate DNMT and SIRT1 functions and restore changes in LINE-1 methylation.2O2, which leads to reactive oxygen species (ROS) production and a cytotoxic effect of less than 50% [p = 0.004) and increased ROS production by 50.1% compared to untreated cells (p < 0.001) (ARPE-19 cells were treated with GOx to mimic a condition of oxidative stress through the continuous production of Hthan 50% . Consistthan 50%  treatmen< 0.001) .Escherichia coli, serotype 0127:B8) to mimic an inflammatory condition [p = 0.035). Interestingly, treatment with 10 µg/mL LPS for 24 h significantly increased ROS production by 32.6% compared to untreated cells (p = 0.004)  . Accordip-values < 0.05). In addition, total DNMT activity was reduced by 28.5% in GOx-treated cells compared to untreated cells (p < 0.0001). Since DNMT functions, especially DNMT1, are regulated by SIRT1 [p = 0.002) and activity  compared to untreated cells (p < 0.0001)  .p = 0.004), while DNMT3A and DNMT3B expression seemed to be unaffected. In addition, treatment with LPS reduced total DNMT activity by 14.9% (p = 0.007). Compared to untreated cells, we also showed that LPS treatment decreased both SIRT1 expression  and activity  (p < 0.0001)  . In line 0.0001) .p-values < 0.05 vs. GOx- or LPS-treated cells).We also aimed to demonstrate the antioxidant and anti-inflammatory effect of resveratrol against GOx- and LPS-induced changes in ARPE-19 cells. First, we determined viability of cells exposed to various concentrations of resveratrol (1–10 μM) for 24 h. In line with a previous study , we founp = 0.039 for DNMT1; FC = 0.86, p = 0.005 for DNMT3A; FC = 0.85, p = 0.008 for DNMT3B; FC = 1.07, p = 0.003 for SIRT1) and activity  of DNMTs (p = 0.002) and total DNMTs activity  (p = 0.009) and activity  (p < 0.001) and inflammatory  conditions  , as well= 0.005)  in cellsnditions .Efforts to understand the mechanisms underpinning the multifactorial nature of retinal degenerative diseases have led us to explore the DNA methylation process in RPE cells under oxidative stress and inflammation, which interplay in retinal degeneration . In suppTo our knowledge, the present work demonstrated for the first time that oxidative stress and inflammatory conditions reduced DNMT and SIRT1 functions, as well as LINE-1 methylation levels, in RPE cells. The slight but significant difference (≈3%) in LINE-1 methylation levels between treated and untreated cells could be partially due to reduced DNMT function. Decreased LINE-1 methylation leads to genomic instability and plays a crucial role in the development of chronic degenerative diseases ,32,33,34IL17RC gene encodes for an essential subunit of the IL-17 receptor complex that modulates activity of proinflammatory IL-17A and IL-17F. Although these findings were not confirmed in a subsequent study [Several lines of evidence proposed the relationship of oxidative stress and inflammation with DNA methylation in retinal degenerative diseases, especially in AMD. A previous study, comparing DNA methylation between AMD patients and age-matched controls, revealed that glutathione S-transferase isoforms mu1 (GSTM1) and mu5 (GSTM5) undergo epigenetic repression in AMD RPE/choroid via promoter hypermethylation, which, in turn, decreased mRNA and protein levels . These ent study , the putRecently, several lines of evidence suggested that SIRT1, a NAD+-dependent histone deacetylase, protects RPE cells against apoptosis and counteracts changes in RPE functions induced by oxidative stress and chronic inflammation. In fact, SIRT1 might be involved in retinal degeneration via modulating cell senescence, DNA damage repair, and apoptosis . PreviouBeyond the antioxidant and anti-inflammatory properties, recent literature reported the antiaging effect of resveratrol in epithelial, endothelial, and corneal wound healing ,55,56. I2. Medium was changed every 48 h.ARPE-19, purchased from the American Type Culture Collection , were maintained in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum , 100 U⁄mL of penicillin, and 100 µg⁄mL of streptomycin (Gibco BRL). Cells between 6–10 passages were used in all experiments and incubated at 37 °C and 5% COEscherichia coli, serotype 0127:B8; Sigma Chemical, Missouri, MO, USA) for 24 h, respectively. The concentrations of GOx and LPS were chosen according to previously published studies that used the same cell line [To mimic conditions of oxidative stress and inflammation, cells reaching 80–90% of confluence were starved in serum-free DMEM and treated with 25 mU/mL GOx or 10 µg/mL LPS  assay. Treated and untreated cells were briefly seeded at a density of 2.0 × 104 cells/well in a 96-well plate and incubated for 24 h. Then, cells were incubated with MTT (1.6 mg/mL) at 37 °C for 4 h. After removing the solution, cells were resuspended in 100 µL of dimethyl sulfoxide and optical density was read at 540 with an optional reference wavelength of 670 nm. Cell viability was reported as percentage of control.Intracellular ROS levels were determined using the Abcam Cellular ROS Detection Assay Kit according to manufacturer’s instructions . In brief, cells were seeded at a density of 2.0 × 104 cells/well in a dark, clear bottom 96-well microplate. Cells were rinsed with 100 μL/well of 1× Buffer and stained by adding 100 μL/well of the redox-sensitive fluoroprobe 2′,7′–dichlorofluorescin diacetate (DCFDA) for 45 min at 37 °C in the dark. After removing DCFDA, 100 μL/well of 1× Buffer were added and fluorescence was measured at Ex/Em = 485/535 nm. ROS production was reported as percentage of control.Nuclear proteins were extracted using the Nuclear Extraction Kit according to manufacturer’s instructions . Briefly, cell pellet (2 × 106 cells) was obtained by trypsinization and centrifugation of cells at 70–80% of confluence following standard protocols. Cell pellet was resuspended in 200 µL of pre-extraction buffer and incubated on ice for 10 min. After centrifugation, nuclear pellet was resuspended in 400 µL of extraction buffer and incubated on ice for 15 min. Finally, the suspension was centrifuged for 10 min at 14,000 rpm at 4 °C and the supernatant was transferred into a new vial to measure the protein concentration of the nuclear extract. Nuclear proteins quantification was performed by the Qubit Fluorometer  using the Qubit Protein Assay Kit according to manufacturer’s instructions.Total DNMT activity was quantified using the colorimetric DNMT Activity Quantification Kit  according to manufacturer’s instructions. Briefly, 7.5 ng of nuclear extracts were diluted in 50 μL/well of reaction solution and incubated at 37 °C for 120 min, including blank and positive control. After removing the reaction solution, each well was rinsed with wash buffer for three times, and 50 μL/well of the diluted capture antibody was added. The plate was covered with aluminum foil and incubated at room temperature for 60 min. After removing the capture antibody, each well was rinsed with wash buffer for three times, and 50 μL/well of the diluted detection antibody was added. The plate was covered with aluminum foil and incubated at room temperature for 30 min. After removing the detection antibody, each well was rinsed with wash buffer for four times, and 50 μL/well of the enhancer solution was added. The plate was covered with aluminum foil and incubated at room temperature for 30 min. After removing the enhancer solution, each well was rinsed with wash buffer for five times, and 100 μL/well of the developer solution was added. Finally, the plate was covered with aluminum foil and incubated at room temperature for 10 min, away from direct light. When the positive control turned to medium blue, 100 μL/well of stop solution was added to stop the reaction. OD was read within 2–10 min at 450 nm with an optional reference wavelength of 655 nm. DNMT activity was reported as percentage of control.2O, 5 μL fluorosubstrate peptide, 5 μL NAD, 5 μL developer, and 7.5 ng nuclear extract was mixed thoroughly, and the fluorescence intensity was measured at Ex/Em = 350–450 nm for 30 to 60 min at 1–2 min intervals. SIRT1 activity was reported as percentage of control.SIRT1 activity was quantified using an SIRT1 Activity Assay Kit  according to manufacturer’s instructions. The reaction mixture containing 30 μL ddH® Reagent  and reverse transcribed to single-stranded cDNA using the SuperScript III Reverse Transcriptase  according to the manufacturer’s protocols. mRNA levels were determined by qPCR with TaqMan Gene Expression Assays  using the QuantStudio™ 7 Flex System (Applied Biosystems). Specific primers were used to detect DNMT1 (assay No. Hs00945875_m1), DNMT3a (Hs01027162_m1), DNMT3b (Hs00171876_m1), and SIRT1 (Hs01009006_m1). Data were normalized to the values of GAPDH expression (Hs02758991_g1). All samples were analyzed in triplicate using the 2C−ΔΔt method [Total cellular RNA was extracted using Trizolt method .DNA was extracted using the DNeasy Blood and Tissue Kit  and quantified using the Qubit dsDNA High Sensitivity Assay Kit  according to the manufacturer’s protocols. Methylation analysis of three CpG sites in the LINE-1 promoter (GeneBank accession No. X58075) was performed by pyrosequencing of bisulfite-converted DNA using the PyroMark Q24 Instrument , as previously reported . BrieflyResults were reported as MD ± SE unless otherwise indicated. Statistical differences in the data were evaluated by Student’s t-test using the control group and/or GOx- and LPS-treated cells as reference. All the analyses were conducted using GraphPad Version 6.0 with a significance level of 0.05.In conclusion, we demonstrated for the first time that oxidative stress and inflammatory conditions negatively affect both DNMT and SIRT1 functions, and LINE-1 methylation in RPE cells. Interestingly, treatment with 10 μM resveratrol for 24 h counteracts these detrimental effects. Further studies should be encouraged to explore the perspectives of resveratrol as a suitable strategy for the prevention and/or treatment of retinal degenerative diseases.


The rats in the model group were injected with complete Freund's adjuvant (FCA), while the treatment group received FCA combined with QLT treatment. After 27 days, the gut flora was profiled by 16S rRNA gene sequencing. The levels of cadherin-11, IL-17α, TLR2, and TLR4 proteins in the synovial tissues were detected by western blotting (WB). The results showed that QLT treatment significantly inhibited raw swelling during the 15–27 d period compared with the model group. QLT treatment reversed the ten altered bacterial genera in the model group, and three families  were closely related to QLT treatment based on linear discriminant analysis (LDA). Functional prediction showed seven types of predicted functions were related to the QLT treatment, and WB results showed that QLT treatment reversed the increased expression levels of cadherin-11, IL-17α, TLR2, and TLR4 in synovial tissues significantly. The expression levels of cadherin-11, IL-17α, and TLR2 correlated negatively with the abundance of Staphylococcus and Candidatus_Saccharimonas. Therefore, RA development was related to gut dysbiosis, and QLT effectively ameliorated RA with decreased inflammatory responses regulated by the gut flora.Rheumatoid arthritis (RA) is a common chronic systemic autoimmune disease. Recent studies show that gut flora plays an important role in regulating the systemic immune response, and gut dysbacteria are linked with systemic chronic inflammation in the development of RA. Our previous results found that Qingluo Tongbi decoction (QLT) can treat RA effectively. The present study explored the effect of QLT on gut flora in an adjuvant-induced arthritis (AA) rat model. Thirty rats were divided randomly into three groups: a control group, a model group, and a treatment group ( Rheumatoid arthritis (RA) is a common chronic systemic autoimmune disease mainly typified by inflammatory cell infiltration, continuous synovial hyperplasia, and cartilage and bone destruction and is accompanied by joint dysfunction and even disability in severe cases . As an aAccording to the TCM clinical reorganization, RA is classified as an “arthralgia syndrome.” Professor Zhou, a national Chinese medicine master, and his colleagues discovered that RA commonly manifested itself clinically as “yin deficiency and collateral heat” based on long-term clinical experience. They managed to treat RA effectively using Qingluo Tongbi decoction (QLT) which nourished yin, cleared heat, alleviated arthralgia, and reopened collaterals, with satisfactory outcomes and mild adverse reactions .Panax notoginseng (Burk.) F. H. Chen, Sinomenium acutum, Bombyx batryticatus, Rehmannia glutinosa (Gaertn.) Libosch, Tripterygium wilfordii Hook. f., Taxillus chinensis (DC.) Danser, Sigesbeckia orientalis L., and Trachelospermum jasminoides (Lindl.) Lem.). Early animal studies in AA rats showed that the QLT granule relieved the decreased bone density of hind limb joints and inhibited the destruction of the cartilage markedly _coprostanoligenes_group (3.8 ± 2.7%), Desulfovibrio (3.6 ± 2.8%), Lactobacillus (3.4 ± 6.4%), Prevotellaceae_NK3B31_group (3.4 ± 2.3%), Ruminococcaceae_UCG-014 (2.9 ± 1.6%), Ruminiclostridium_9 (2.6 ± 1.1%), Ruminococcaceae_NK4A214_group (2.4 ± 1.4%), Ruminococcaceae_UCG-005 (2.0 ± 3.1%), Ruminococcus_1 (1.9 ± 1.2%), Oscillibacter (1.6 ± 0.7%), Roseburia (1.5 ± 1.2%), Alloprevotella (1.0 ± 1.5%), [Eubacterium]_xylanophilum_group (1.0 ± 0.9%), Ruminiclostridium (1.0 ± 0.6%), Ruminococcus_2 (1.0 ± 0.9%), and Ruminiclostridium_6 (1.0 ± 1.0%)  could represent the AA rat's symbolic flora.Moreover, LEfSe analysis was conducted to assess the effects of QLT on the gut flora of RA rats from the phylum to the genus levels . The resU test) was conducted to comparatively analyze the relative abundance of gut bacterial genera between two arbitrary groups. Ten genera were collected from the total of 128 genera identified because the abundance of each genus was significantly different between the control and model groups and between the model and treatment groups; however, they did not differ significantly between the control and treatment groups. In comparison with the control group, the relative abundance of six genera  significantly decreased (P < 0.05) in the model group, while the relative abundance of four genera  distinctly increased (P < 0.05). QLT treatment significantly changed the abundance of genera in AA rats back to their abundance in the control group, and statistical analysis showed that the relative abundance of the above 10 genera in the treatment group was not statistically different from that in the control group (P > 0.05)  .P < 0.05) and between the model and treatment groups (P < 0.05) were observed, but no significant difference between the treatment and control groups (P > 0.05) was found. A total of 32 predictive functions were screened. Compared with the control group, the model group had 21 significantly enhanced functions and 11 significantly weakened ones (P < 0.05) (P < 0.05) (P < 0.05) were downregulated  to levels similar to those of the control group (P > 0.05)  . QLT sig < 0.05) , whereasegulated . Further > 0.05) .α, TLR2, and TLR4 in the synovial tissues correlated negatively with Staphylococcus, while Candidatus_Saccharimonas correlated negatively to a significant degree with the expressed levels of cadherin-11, IL-17α, and TLR2 (P < 0.05).To explore the key gut flora which may trigger the inflammation of synovial tissues, Spearman's correlation analyses were conducted among the 15 known gut genera  [Haemophilus spp. was negatively correlated with serum autoantibody levels, and the relative abundance of Lactobacillus salivarius was significantly increased in patients with active RA [As a common autoimmune disease, RA can be relieved by combating inflammation and suppressing immunity, and it has been speculated that the inflammatory reaction seen in RA is triggered by complicating factors before the onset of RA, such as gut dysbacteria, opportunistic infection, and unhealthy lifestyle. The human flora participates in regulating physiological metabolism and immune balance, indicating that pathogens that cause RA may exist therein. Jussi et al. found that the abundance of the gut genus omyalgia . In addiethacin) . A recenctive RA . Based oRuminococcus_1, Clostridium_sensu_stricto_1, Roseburia, Atopostipes, Ruminococcaceae_UCG-013, and Turicibacter) significantly decreased in abundance in the model group, while QLT treatment could upregulate the relative abundance of the six genera to the point of having no differences from those in the control group. Furthermore, the relative abundance of four distinct genera  that was decreased in the model group was also reversed by QLT treatment. Thus, the above ten genera are possible therapeutic markers for QLT. Notably, Clostridium_sensu_stricto_1 is associated with cellulose digestion and degradation [Roseburia significantly decreases in patients with Crohn's disease to protect colonic epithelial cells from inflammatory injury [Blautia and Parasutterella significantly increases in patients with functional constipation [Herein, the gut flora of AA rats was changed significantly in control rats and was partly restored by QLT treatment. In comparison with the control group, six genera  were related to the redox state and active inflammation , 24, andα is well known for its origin in Th17 cells, which has been shown in recent years to play a significant role in the pathogenesis of chronic destructive arthritis [Among the significantly increased functions, there was particular focus on membrane proteins because of the distinctive effect of QLT in inhibiting paw swelling. Thus, the inflammation-related membrane proteins, TLR2 and TLR4, were selected as they play important roles in the immunity of bacterial infection . Cadherirthritis . Here, wα, were markedly increased in the AA rats' synovium and that QLT treatment can downregulate the expression levels of these proteins. Firstly, cadherin-11 plays key roles in synovial formation and inflammation, as well as in cartilage destruction, which were related to the severity of synovial inflammation [α and IL-17 played essential roles. Additionally, IL-17 exacerbated joint inflammation and destruction by increasing IL-1β and IL-6 and also facilitated synovial expression of TLR-2, -4, and -9 in autoimmune arthritis [α and TLR2- and TLR4-related mucosal immune signaling pathways in AA rats.The results showed that the four molecules, TLR2, TLR4, cadherin-11, and IL-17ammation . After aammation . Secondlrthritis . Finallyrthritis , damaginStaphylococcus and Candidatus_Saccharimonas, which had the highest abundance in the control group. Gut Candidatus_Saccharimonas was reported to be decreased in rats' acute necrotizing pancreatitis [Candidatus_Saccharimonas plays an important role in maintaining normal intestinal function. Members of the genus Staphylococcus have intimate relationships with their hosts, and Staphylococcus aureus in particular has been the focus as the pathogenic species in most research including RA patients [Staphylococcus species are paid considerable attention because 71 species have been reported to date and little is known about their functions [The correlation analysis showed that the levels of the four proteins were negatively related to the abundance of reatitis , which ipatients , 36. Nowunctions , 38. TheIn summary, QLT exerted remarkable therapeutic effects on the inhibition of paw swelling in AA rats, which correlated with the alteration of gut microbiota and with the inhibition of synovial inflammation. The present study paves the way for elucidating the biological mechanism of the role of QLT treatment in RA. Of course, there were two limitations in the present study: (1) a fourth healthy group was not prepared for examining whether QTL exerts an effect on gut microbiota in healthy rats and (2) TCM treatments characteristically have multiple targets; thus, the detailed active constituent and underlying pharmacological mechanism need further study in order to fully understand the effects of QLT therapy on rheumatoid arthritis.