The anti-adipogenic effects of yerba mate, YGD, resveratrol and YGD + resveratrol and yerba mate + resveratrol combinations were evaluated in 3T3-L1 cells by Oil Red staining, cellular triglyceride content, and PCR quantitative array. The results demonstrated that all of the tested compounds inhibited adipogenesis. Yerba maté extract significantly down-regulated the expression of genes that play an important role in regulating adipogenesis, such as Adig, Axin, Cebpa, Fgf10, Lep, Lpl, and Pparγ2. In addition, these genes, YGD also repressed Bmp2, Ccnd1, Fasn, and Srebf1. Resveratrol also modulated the expression of Adig, Bmp2, Ccnd1, C/EBPα, Fasn, Fgf10, Lep, Lpl, and Pparγ2. Moreover, resveratrol repressed Cebpb, Cdk4, Fgf2, and Klf15. The yerba maté extract and YGD up-regulated the expression of genes involved in inhibiting adipogenesis, such as Dlk-1, Klf2, and Ucp1. Resveratrol also induced the expression of Klf2 and Ucp1. In addition resveratrol modulated the Ddit3, Foxo1, Sirt1, and Sirt2. The combined effects of these compounds on gene expression showed similar results observed from individual treatments. Our data indicates that the synergy between the compounds favors the inhibition of adipogenesis.We aimed to evaluate the  The inc obesity . Researc obesity ,5.yerba maté (Ilex paraguariensis) as an excellent candidate in the struggle against obesity [yerba maté suppresses body weight gain and visceral fat accumulation and decreases serum levels of cholesterol, triglycerides, LDL cholesterol, glucose, insulin, pancreatic lipase and leptin [In vitro and in vivo studies have shown that yerba maté modulates signaling pathways, which regulate adipogenesis, antioxidant, anti-inflammatory and insulin signaling responses [Several studies have identified  obesity ,15,16,17d leptin ,19,20. Tesponses ,22.yerba maté was also evaluated in humans. In a clinical study, it has been demonstrated that a herbal preparation containing yerba maté, Guarana and Damiana (“YGD”) significantly delayed gastric emptying, reduced the time to perceived gastric fullness, and induced significant weight loss over 45 days in overweight patients [The anti-obesity role of patients . Subsequpatients .Another important candidate to manage obesity is resveratrol , a naturally occurring polyphenol found in different plant species . Resverain vitro and animal studies have demonstrated that resveratrol has an antiobesity potential by inhibiting adipocyte differentiation, decreasing proliferation, inducing apoptosis, decreasing lipogenesis, and promoting lipolysis and FA β-oxidation [in vitro and animal data indicate that the potential antiobesity effects and health promoting effects of resveratrol in the context of obesity should be better comprehended.Several xidation ,40,41,42xidation ,44,45,46the in vitro effects of yerba maté, YGD, and resveratrol on expression level of key genes involved in the process of adipogenesis.Considering the importance of adipogenesis in the obesity, the present study aimed to evaluate yerba maté and resveratrol were not cytotoxic to 3T3-L1 cells. Concerning the YGD + resveratrol and yerba maté + resveratrol mixtures the MTT revealed they were not cytotoxic.The MTT analysis revealed that YGD yerba maté  or resveratrol  to prevent lipid accumulation was examined by Oil Red O staining and evaluation of triglyceride content of 3T3-L1 adipocytes. Our data indicated that all of the tested compounds inhibited adipogenesis, but at different concentrations. YGD inhibited adipogenesis in both methods at a concentration of 100 µg/mL (Yerba maté significantly inhibited adipogenesis at a concentration of 150 µg/mL (yerba maté + resveratrol (150:10 or 200:10 μg/mL) in the inhibition of adipogenesis. Our data also showed that all of the tested combination inhibit the adipogenesis  inhibited adipogenesis at the lowest concentration. It is noted that the level of inhibition of adipogenesis with the combinations is higher than that observed for resveratrol alone. The results are attributable to a synergistic effect obtained from the potent anti-adipogenic effect of resveratrol in combination with the other compounds.The anti-adipogenic effects occurred at concentrations that did not affect cell viability. Regarding d before ,14. The d before ,47. The yerba maté (150 μg/mL), resveratrol (10 μg/mL), YGD + resveratrol (100:10 μg/mL), or yerba maté + resveratrol (150:10 μg/mL) on the expression of several genes that regulate adipogenesis, the Mouse Adipogenesis RT2 ProfilerTM PCR Array (SABiosciences) was utilized. This assay was performed at the lowest concentrations in which an inhibition of adipogenesis by both employed methods was observed. The triplicate samples from each group gave reproducible results.To determine the effect of YGD (100 μg/mL), yerba maté extract modulated the expression of 12% (10/84) the genes. YGD modulated the expression of 17% (14/84) of the genes. Although YGD modulates the expression of more genes than yerba maté, we noted that both compounds act in the same pathways of adipogenesis , Axin (Axin 1), Cebpa (CCAAT/enhancer binding protein (C/EBP) alpha), Fgf10 (Fibroblast growth factor 10), Lep (Leptin), Lpl (Lipoprotein lipase), and Pparγ2 . In addition to those genes, YGD also down-regulated Bmp2 (Bone morphogenetic protein 2), Ccnd1 (Cyclin D1), Fasn (Fatty acid synthase), and Srebf1 (Sterol regulatory element binding transcription factor 1). As observed for yerba maté and/or YGD, resveratrol also modulated the expression of Adig, Bmp2, Ccnd1, C/EBPα, Fasn, Fgf10, Lep, Lpl, and Pparγ2. Moreover, resveratrol repressed Cebpb (CCAAT/enhancer binding protein (C/EBP), beta), Cdk4 (Cyclin-dependent kinase 4), Fgf2 (Fibroblast growth factor 2), and Klf15 (Kruppel-like factor 15). The yerba maté extract and YGD up-regulated the expression of genes involved in inhibiting adipogenesis, such as Dlk-1 (Delta-like 1 homolog), Klf2 (Kruppel-like factor 2), and Ucp1 (Uncoupling protein 1). Resveratrol also induced the expression of Klf2 and Ucp1. Additionally, resveratrol modulated the Ddit3 (DNA-damage inducible transcript 3), Foxo1 (Forkhead box O1), Sirt1 (Sirtuin 1), and Sirt2 (Sirtuin 2) gene expression levels. The combined effects of these compounds on gene expression showed similar results on expression level of targeted genes.The yerba maté and resveratrol modulates adipogenesis by down regulating the levels of pro-adipogenic transcription factors, such as Pparγ2 and Cebpa, in vivo [yerba maté, YGD, resveratrol, and their combinations were able to down-regulate the expression of Pparγ2 and Cebpa. Additionally, Cebpb was repressed by resveratrol, as previously described [Adipogenesis is the developmental process by which a multipotent mesenchymal stem cell differentiates into a mature adipocyte. This process involves a highly regulated and coordinated cascade of transcription factors, including members of the PPAR, C/EBP, and SREBP families, which together lead to the establishment of the differentiated state . In this in vivo ,35,37,38escribed , and by Fasn. Fasn was also down regulated by resveratrol as previously described [Adig and Srebf1, adipogenic markers that mediate the initiation of adipogenesis.SREBP1 is a transcription factor that controls fatty acid synthase, and functions an additional regulator of adipogenesis in parallel with C/EBPα and PPARγ pathways . Here, wescribed . The datKlf2, it was shown that all tested compounds increased the expression level of this gene, which may then contribute to the observed repression of 2Pparγ expression. Similar results were previously observed for yerba maté [Klf15, further contributing to the inhibition of adipogenesis.Numerous effects of the KLF protein family have been reported in different tissues. In the 3T3-L1 cell line model, KLF2 inhibits adipogenesis by inhibiting PPARγ . By asserba maté . On the rba maté . Our genDlk1.It has been reported that this gene contributes to inhibition of adipogenesis via inhibitory actions on C/EBP-β and C/EBP-δ in the pre-adipocyte cell line 3T3-L1. It is believed that down-regulation of Dlk1 is critical for adipogenesis to occur [yerba maté, as previously described [Dlk1, which might be at least in part, responsible for the observed inhibition of adipogenesis. The results presented in this study show that all of the tested compounds down regulated the Pparγ and Cebpa mRNA expression, which leads to further down regulation of other genes such as Lep and Lpl. Similar data were previously observed for yerba maté [In addition, expression of C/EBPs and PPARγ is dependent on the activation of other genes that are essential for adipogenesis such as to occur ,54. We oescribed , and therba maté  and resvrba maté .Bone morphogenetic proteins (Bmps), are members of the transforming growth factor-β (Tgf-β) family, which regulate diverse physiological processes including adipose tissue formation . It has Sirt1, Sirt2 and Foxo1 by resveratrol and its combination with yerba mate or YGD observed in this study might contribute to the exhibited down-regulation of Pparg2.FOXO1 is a transcription factor which negatively regulate the adipogenesis ,58 . It Ddit3  is a dominant-negative member of the C/EBP family. It is initially expressed by growth-arrested preadipocytes and sequesters/inactivates C/EBPβ during 3T3-L1 preadipocyte differentiation. Thus, its down-regulation is required for the complete adipocyte differentiation [Fgf10, which plays crucial roles in adipogenesis by contributing to the expression of C/EBPβ through an autocrine/paracrine mechanism [Fgf10, a direct effect was observed only for resveratrol and mixed compounds. Additionally, Cdk4 activity has been proposed to be required for adipogenesis. Cdk4 participates in the clonal expansion phase of adipocyte differentiation through activation of PPARγ [Cdk4, which might contribute to the Pparg repression observed.It has been described that ntiation ,64. Therechanism . Althougof PPARγ . Our datAxin, which is known to be a negative regulator of the WNT/β-catenin pathway, was down-regulated by yerba maté, YGD and the combination of compounds. Regarding Ccnd1, our data show an inhibition of its expression after YGD, resveratrol and mixed compound treatments. These results are in accordance with a previous study, which demonstrated an early effect of resveratrol on Ccnd1 down-regulation. The authors also observed a subsequent inhibition of cell cycle reentry and clonal expansion [It has been proposed that WNT/β-catenin pathway exert a negative regulation on adipogenesis . When thxpansion , which lUcp1 is known to be a significant component of whole body energy expenditure, and its dysfunction contributes to the development of obesity. In this study, we observed that all of the tested compounds were able to up-regulate the expression of Ucp1. Similar results were previously described for yerba maté [rba maté ,7 and rerba maté ,71.yerba maté powder and the patented herb extract formulation YGD  were provided by Natures Remedies, Amersham, UK. Resveratrol and Oil Red O were purchased from Sigma-Aldrich . Cell culture reagents were purchased from Invitrogen .The soluble 2. Forty-eight hours after achieving confluence (day 0), the cells were incubated in differentiation medium . After 96 h (day 4), the cells were then cultured in maturation medium  for 10 days.The 3T3-L1 cell line was purchased from the Rio de Janeiro Cell Bank  and it was cultured to confluence in DMEM supplemented with 10% fetal bovine serum and 10 mL/L penicillin/streptomycin at 37 °C and 5% COyerba maté , resveratrol , YGD + resveratrol (100:10 or 150:10 μg/mL), or yerba maté + resveratrol (150:10 or 200:10 μg/mL) during the differentiation period (from day 0 to day 4).For the MTT and Oil Red O assays the cells were incubated with YGD, 2. The supernatant was discarded and MTT was added (5 mg/mL in PBS). Cells were incubated for 3 h at 37 °C in 5% CO2, after which 100 µL 10% sodium dodecyl sulfate in 0.01 M HCl was added to each well. Samples were then incubated for 18 h at 37 °C and absorbance was measured at 540 nm in a microplate reader . Data are presented as mean ± SEM from three independent experiments (each in triplicate).Cell toxicity was estimated by using the tetrazolium salt reduction test (MTT assay) on 3T3-L1 cells after exposure with the described compounds (at day 4). Briefly, the cells were cultured as described and incubated with the compounds or vehicle (0.1% DMSO) at 37 °C in 5% CODifferentiation was evaluated using an Oil Red O staining assay. Briefly, the cells were incubated with the compounds during the differentiation period (day 0). At the end of the maturation period, the cells were washed with PBS, fixed in 4% paraformaldehyde for 30 min and incubated for 1 h with 1% Oil Red O solution . After mIntracellular triglycerides content was assessed using commercial kit . Briefly, the cells were incubated with the compounds during the differentiation period (day 0) as previously described. At the end of the maturation period, the cells were deprived of fetal bovine serum 24 h before harvest. The absorbance at 550 nm is proportional to the concentration of triglycerides of each sample. All samples were determined in triplicate.The 3T3-L1 cells were cultured as previously described. The material used for RNA extraction was harvested at the end of the differentiation period (day 4). To stabilize and protect the RNA, all samples were stored at −80 °C in RNAlater . Total RNA was isolated using the RNeasy tissue kit . After the extraction, ~100 ng of RNA was used for cDNA synthesis using the High Capacity cDNA Archive Kit  according to the manufacturer’s protocol.2 Profiler™ PCR Array  was utilized to determine the expression profile of 84 key adipogenesis genes.The Mouse Adipogenesis RTRplp1), hypoxanthine phosphoribosyltransferase 1 (Hprt1), ribosomal protein L13A (Rpl13a), lactate dehydrogenase A (Ldha), and beta-actin (Actb)).The gene array profiled the expression of 84 genes, including regulatory genes, PPARγ targets, and genes related to adipogenesis. In addition, the array included controls for human genomic DNA contamination and the reverse transcription step, a positive control for the PCR, and 5 housekeeping genes , and the threshold cycle numbers were determined using RQ Study Software . The reactions were performed in duplicate, and the threshold cycle numbers were averaged. The 50 µL reaction mixtures contained 25 µL Platinum SYBR Green Quantitative PCR SuperMix-UDG  and 100 ng of cDNA. The reactions were cycled through a preliminary UDG treatment for 2 min at 50 °C and 2 min denaturation at 95 °C, which was followed by 45 cycles of denaturation at 95 °C for 15 s, annealing for 15 s, and primer extension at 72 °C for 15 s. Subsequently, a melting point analysis of the double-stranded amplicons was performed that consisted of 40 cycles with a 1 °C decrement (15 s each) in each cycle, beginning at 95 °C. Data are presented as mean ± SD from three independent experiments (each in triplicate). The data were analyzed using web-based PCR data analysis software (SABiosciences) and normalised to the 5 housekeeping genes.p value) of less than 5% was considered to be significant (p < 0.05). For gene expression, significative down-regulation was considered when the fold regulation was ≤−2; significative up-regulation was considered when the fold regulation was ≥+2.The data obtained were analyzed using the SPSS 13.0 statistical Software . Comparisons between groups of data were performed using a one-way ANOVA followed by Dunnett’s Multiple Comparisons. An associated probability (yerba maté, YGD and resveratrol regulate the expression of genes that are involved in adipogenesis. Furthermore, these compounds are postulated to regulate adipogenesis through the WNT pathway, which would result in a significant repression of Pparγ2 and C/EBP-α. The results also showed that resveratrol regulates a greater number of targets than yerba maté and YGD alone. The combination of resveratrol -yerba mate or -YGD indicates that the synergy between the compounds might favors the inhibition of adipogenesis by modulation the expression of all studied genes.This study determined that


The aim of the study was to assess markers of anemia in type 1 diabetes (T1D) children, compare them to results obtained in the control group, and estimate their relation to BMI SDS.  94 (59% ♀) T1D children without other autoimmune disorders, aged 12.5 ± 4.1 years, T1D duration: 4.2 ± 3.6 years, HbA1c 7.3 ± 1.5% (57 ± 12.6 mmol/mol). Sex- and age-matched controls (43 children). In all children, anthropometric measurements, the blood count, iron turnover parameters, and vitamin B12 concentration were taken. p < 0.001), hepcidin , and vitamin B12 concentrations  and lower TIBC  than in the control group. Logistic regression revealed that RDW, TIBC (both p < 0.001), and hepcidin (p < 0.05) significantly differentiated both groups. In T1DM children, BMI SDS negatively correlated with vitamin B12 (p < 0.01) concentration and mean corpuscular hemoglobin concentration (p < 0.05) and positively with TIBC (p < 0.01) and HbA1c (p < 0.001).  T1DM children had significantly higher red cell distribution width (RDW) (13.6 versus 12.6%;  Patients and controls differed especially in terms of RDW and TIBC. In studied T1DM children, BMI SDS was associated to iron metabolism parameters and vitamin B12 concentration. Type 1 diabetes (T1D), because of the rising incidence, remains a challenge for diabetologists as well as for pediatricians and general practitioners, who more and more often face the challenge to take optimal care for young patients with this disease . With tiAccording to our research hypothesis, children with T1D differ from healthy peers in terms of blood count, iron metabolism parameters, and vitamin B12 levels. Therefore, the aim of the study was to assess the abovementioned parameters in T1D pediatric patients with no other autoimmune diseases and compare them to results obtained in a group of healthy children as well as estimate their relation to chosen factors, among others: anthropometric measurements, sex, glycemic control, and disease duration. Confirming the research hypothesis would be a strong premise to monitor red blood cell parameters and—depending on the results—adequate broadening of the diagnostic tests.The study group included 94 children with T1D  . The blood samples were collected during the routine control visit in the Outpatient Clinic. Their mean age at study time was 12.5 ± 4.1 years (SD) (ranging from 3 to 19 years). Mean duration of T1D was 4.2 ± 3.6 years (SD) (0.08–15.7 years). For the purposes of statistical analysis, the percent of life with diabetes was calculated . The mean glycated hemoglobin A1c (HbA1c) at the day of the study is equaled to 7.3 ± 1.5% (57 ± 12.6 mmol/mol).In all patients, physical examination with auxological assessment was performed. Routine laboratory tests (screening for autoimmune concomitant diseases and complications) were performed according to recent Diabetes Poland and ISPAD guidelines , 9.Patients with clinical and/or laboratory signs of other than T1D autoimmune diseases  or infection were excluded from the study. Furthermore, children enrolled for this study, according to their medical records, did not have chronic diabetes complications.The control group comprised 43 children  that were age- and sex-matched to the individuals from the study group. They were recruited from patients admitted to the Clinical Hospital No. 1 in Zabrze for nocturnal enuresis or arterial hypertension diagnostics in whom hospital observation and diagnostic procedures allowed to exclude any organic disease that could influence the parameters assessed in this project. These children had no infection, autoimmune diseases, or family history of such diseases.Anthropometric measurements carried out in all children included weight (kg) and height (cm). Using these values, the body mass index (BMI) was calculated using the standard equation (the body mass in kilograms divided by the square of the body height in meters). To allow comparisons, weight, height, and BMI were expressed as standard deviation scores (SDS).μg). The blood samples were taken between 7 and 9 am while the children were on fasting. Peripheral blood count (RBC: red blood cells (10 > 6/μl), MCV: mean corpuscular volume (fl), MCHC: mean corpuscular hemoglobin concentration (g/dl), HGB: hemoglobin (g/dl), HCT: hematocrit (%), RDW: red cell distribution width (%) measured using Sysmex XT-2000i ) and biochemical parameters  and the cobas 6000 device ) were measured in the Central Laboratory of the Clinical hospital No. 1 in Zabrze, Poland. Normal ranges, lower limits of detection (LLD), indirect precision (IP), and repeatability (R) of the measured biochemical parameters are the following: vitamin B12 >150 pg/ml , transferrin 2–3.6 g/l , ferritin 20–200 μg/l , iron 5.83–24.5 μmol/l , and TIBC 55–75 μmol/l . Hepcidin concentration was determined using an enzyme-linked immunosorbent assay ; normal range: 0.187 to 12 ng/ml . HbA1c assessment was carried out using a DCCT  reference method; normal range < 6% (<42 mmol/mol).Patients and controls had blood samples taken to perform the blood count (1.6 ml) and carry out biochemical measurements (5 ml)—iron, ferritin, transferrin, hepcidin, vitamin B12 concentrations, and total iron-binding capacity (TIBC). Additionally, children with T1D had their HbA1c and fetal hemoglobin (HbF) measured .Statistical analyses were performed using the R software (N), median, upper and lower quartile (Q1 and Q3), minimal (min) and maximal (max) value, mean, and standard deviation (SD).For all of the analyzed parameters, the following descriptive statistics were determined: number  or Student's t-test depending on the group number. In cases of other types of distribution, the nonparametric ANOVA Kruskal-Wallis and Mann–Whitney U test were employed. For post hoc comparisons, we used the Tukey-Kramer test or its nonparametric alternative. The logistic regression technique with the forward feature selection procedure was applied during the construction of the binary classifier. Akaike information criterion supported by Bayes factor principle adjudged on the final model (BF > 3.1623) [χ2 or likelihood ratio G test. Statistical significance was considered at p < 0.05.Tukey's criterion was used to identify outlying values. Lilliefors parametric test was used to assess the normality of the distribution. The variance homogeneity hypothesis was tested by means of  3.1623) . Correlap < 0.001) (p < 0.001) and vitamin B12 concentrations  versus 397  p < 0.01) as well as significantly lower TIBC values  versus 68.15  p < 0.001) in T1D patients (p < 0.001), and hepcidin (p < 0.05) as parameters significantly differentiating the study and the control group with 89% sensitivity and 77% specificity  . Analysipatients . Logistir = 0.27, p < 0.01). There was also a negative relation between RDW and HGB  and positive correlation between iron concentration and age . HbA1c was positively associated with MCV  and negatively with MCHC . Additionally, statistical analysis revealed some significant correlations of the analyzed biochemical and blood count parameters with BMI SDS in T1D patients. Vitamin B12 concentration   were inversely related to BMI SDS. Furthermore, a positive correlation was determined with TIBC  .Among patients with T1D, hepcidin correlated positively with RDW ( < 0.01)  and MCHC < 0.01)  and HbA1Neither T1D duration nor the percent of life with T1D was found to be related to BMI SDS.Until present, there have not been many studies assessing the prevalence of anemia among children with T1D. There is a range of publications regarding patients with type 2 diabetes and anemia, usually accompanying renal complications , 12.None of the investigated children in this study had anemia. Thomas et al. estimated that the prevalence of anemia (based on the hemoglobin concentration) in a group of adults having T1D for a mean of 20 years is equaled to 14%. Risk factors included disturbed renal function and albuminuria  . The menIn an analysis of 200 children with T1D conducted in Egypt, anemia was diagnosed in 37% of cases. Among patients with anemia, 54.7% had iron deficiency, 18.7% had folic acid deficiency, 18.7% were diagnosed with thalassemia minor, 4% with celiac disease, and in 24% anemia developed as a consequence of a parasitic infestation . Mean agIn another investigation, coming from Poland, patients with newly diagnosed T1D revealed a tendency for iron deficiency anemia in comparison to children with longer lasting T1D (>1 year) .The mean values of iron concentration, iron metabolism parameters, and vitamin B12 levels were within normal ranges in our patients. Normal vitamin B12 and iron concentrations in children with T1D were also described by other authors , 16.Adequate blood count results, iron concentrations, iron metabolism parameters, and vitamin B12 levels in our patients can be explained by optimal nutrition and regular control visits at the outpatient clinic as well as continuous reeducation. Despite results remaining within normal ranges, we revealed statistically significant differences in terms of RDW, TIBC, and hepcidin in comparison to the control subjects, which partially confirms the initial research hypothesis. RDW is a sensitive indicator of a developing anemia. Its normal value reflects isocytosis. Increased RDW (anisocytosis) occurs not only in cases of iron deficiency but also in anemia related to inflammation, although the values are higher in iron deficiency anemia . Hepcidi2) [Because of the increasing global problem concerning improper body weight in children, also those with T1D , as well2) . The aut2) . Taking 2) . The res2) .It is also a known fact that T1D patients with improper body weight often do not have optimal glycemic control , 25. In Expanded diagnostic tests conducted in a representative group of children and adolescents with T1D allowed to construct a reliable database. Another strength of this study is the enrollment of patients without other concomitant autoimmune diseases which could interfere the final results as well as the comparison with a healthy control group. The analysis of the iron metabolism parameters was precise and complex, including the assessment of hepcidin, which is not often measured in T1D children. As a limitation, the quite wide range of diabetes duration (recently diagnosed to long lasting) should be mentioned.The assessed blood count, iron metabolism parameters, and vitamin B12 results remained in the studied patients within normal ranges. Therefore, single assays of the abovementioned basic parameters may not be sufficient indicators of anemia in children with T1D.However, results remaining within reference ranges do not exclude a patient with T1D from being at risk of the development of anemia. Basing on statistically significant differences among respective parameters of the blood count and iron metabolism in children with T1D as compared with the control group, it is indicated to monitor trends, and the interpretation of results should be holistic and simultaneous.In the schedule of the health care of children with T1D, in addition to monitoring development of celiac disease and thyroid dysfunction, it should also be considered to follow alteration in the blood count, iron metabolism, and vitamin B12 status.Moreover, in children with T1D and abnormal body weight, coexisting deficiencies of vitamin B12 and iron should be taken into account.


To examine the incidence of atrial fibrillation in individuals with type 2 diabetes compared with age- and sex-matched controls from the general population and its variation in relation to glycaemic control and renal function.A total of 421,855 patients with type 2 diabetes from the Swedish National Diabetes Registry and 2,131,223 controls from the Swedish Population Registry, matched for age, sex and county, were included and followed from January 1, 2001 to December 31, 2013.Overall, 8.9% of individuals with type 2 diabetes and 7.0% of controls were diagnosed with atrial fibrillation during follow-up, unadjusted incidence risk ratio (IRR) 1.35 (95% 1.33–1.36). Women < 55 years old with type 2 diabetes had an IRR of 2.36 (95% CI 2.10–2.66), in relation to controls, whereas the corresponding value for men < 55 years old with type 2 diabetes was IRR 1.78 (95% CI 1.67–1.90). In the fully adjusted Cox regression, the risk of type 2 diabetes on incident atrial fibrillation was 28% greater vs controls, hazard ratio (HR) 1.28 (95% CI 1.26–1.30), p < 0.0001. The excess risk of atrial fibrillation in individuals with type 2 diabetes increased with worsening glycaemic control and renal complications. For individuals with HbA1c ≤ 6.9% (≤ 52 mmol/mol) and normoalbuminuria the excess risk vs controls was still increased, adjusted HR 1.16 (95% CI 1.14–1.19); p < 0.0001.Individuals with type 2 diabetes had an overall 35% higher risk of atrial fibrillation compared to age- and sex-matched controls from the general population. The excess risk for atrial fibrillation increased with renal complications or with poor glycaemic control. Individuals with type 2 diabetes with good glycaemic control and normoalbuminuria had slightly increased risk. Type 2 diabetes is a metabolic disorder, with an increased risk of micro- and macrovascular complications such as thromboembolism, stroke and mortality . At the There are conflicting findings on whether type 2 diabetes is an independent risk factor for atrial fibrillation –7. Howev2DS2-VASc-score used to identify high-risk patients with atrial fibrillation that may benefit from anticoagulation treatment in order to prevent stroke from thromboembolism , had atrial fibrillation before January 1, 2001 or before index date  or no NDR visits after January 1, 2001 [7712 (0.28%)] were excluded . Controls who died before January 1, 2001  or had atrial fibrillation before January 1, 2001 or before index date  were also excluded.2), low-density lipoprotein (LDL) cholesterol, systolic and diastolic blood pressure, smoking, renal function (eGFR), and albuminuria.Variables assessed were sex, age, country of birth, educational level, HbA1c (glycated haemoglobin), diabetes duration, BMI  method in mmol/mol and converted to levels according to the National Glycohemoglobin Standardization Program for dual reporting.Renal complications were categorized as normoalbuminuria, microalbuminuria, macroalbuminuria, and stage 1–5 chronic kidney disease (CKD). Microalbuminuria was defined as two positive tests from three urine samples taken within 1 year, with an albumin/creatinine ratio of 3–30 mg/mmol (~ 30–300 mg/g) or U-albumin of 20–200 μg/min (20–300 mg/L), and macroalbuminuria as albumin/creatinine ratio > 30 mg/mmol (~>300 mg/g) or U-albumin > 200 μg/min (> 300 mg/L). Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology (CKD-EPI) equation . Renal fThe study outcome is atrial fibrillation and flutter, the ICD codes 427D (ICD-9) and I48 ICD-10), with both primary and secondary diagnosis. International Classification of Disease 9th (ICD-9) and 10th (ICD-10) codes were used to define study comorbidities coronary heart disease, admission for heart failure, valve disease, stroke, and cancer diagnosis before the first and baseline registration , and by gender as events per 1000 person-years with 95% confidence intervals estimated using Poisson regression. The effects of time-updated mean HbA1c categories , time-updated albuminuria categories , and time-updated eGFR categories  versus the reference group on time to incident atrial fibrillation were analyzed using Cox regression in three different models. In model 1, adjustments were made for time-updated age and sex. In model 2, adjustments were made for country of birth, education, and baseline comorbidities . In model 3, additional adjustments were made for time-updated diabetes duration. The effects of diabetes group overall and of HbA1c, albuminuria and eGFR categories versus the normal population were obtained for 10th, 25th, 50th, 75th, and 90th percentile of diabetes duration, corresponding to 1, 3, 7, 13 and 19 years. Main analyses were shown for median diabetes duration of 7 years.In individuals with diabetes, overall and for women and men separately, the association of a 1.0% (10 mmol/mol) increase in time-updated mean HbA1c was evaluated with incidence of atrial fibrillation. Adjustments in models 1, 2, and 3 correspond to the main analyses adjustments. Further adjustments were done in models A, B, C, and D. Model A, adjustment was done for time-updated mean systolic blood pressure, time-updated mean body mass index, time updated smoking status and time-updated status about blood pressure lowering medication. In model B, adjustments for time-updated mean high-density lipoprotein, time-updated mean low-density lipoprotein, time-updated status about lipid lowering medication were done. Model C were additionally adjusted for time-updated insulin method. In model D, adjustments for time-updated albuminuria categories were done. These analyses have been described earlier . The proAll tests were two-tailed with p-values < 0.05 considered significant. All analyses were performed using SAS software version 9.4 .A total of 421,855 patients with type 2 diabetes and 2,131,223 controls were included. Patient characteristics at baseline are presented in Table The mean duration of follow-up was 5.0 years for individuals with type 2 diabetes and 5.3 years for controls. Between January 1, 2001, and December 31, 2013, a total of 37,590 (8.9%) individuals with type 2 diabetes and 149,231 (7.0%) of controls were diagnosed with atrial fibrillation. Overall, for individuals with type 2 diabetes, the incidence rates of atrial fibrillation were 15.99 cases per 1000 person-years (95% CI 15.83–16.15) compared to 11.89 cases per 1000 person-years (95% CI 11.83–11.95) for the control group  higher mean HbA1c in individuals with type 2 diabetes Table . For indThis large population-based cohort study showed a 35% increased risk of atrial fibrillation in individuals with type 2 diabetes in comparison to matched controls from the general population. The excess risk increased with poorer glycaemic control and severity of renal complications. In individuals with type 2 diabetes, good glycaemic control, and normoalbuminuria, there was a slight yet significantly increased excess risk. The absolute risk of atrial fibrillation was higher for men with type 2 diabetes, but the relative risk was greater for women with type 2 diabetes compared to controls. Women < 55 years with type 2 diabetes had a twofold increased risk, and individuals with type 2 diabetes and CKD stage 5 had a threefold increased risk for atrial fibrillation compared to controls. Additionally, the increased risk of atrial fibrillation in relation to 1.0% (10 mmol/mol) higher mean HbA1c evaluated in persons with diabetes was estimated to range from 1.0 to 4.0%.Our study is in line with others demonstrating the positive association between diabetes and the risk of atrial fibrillation. In two different meta-analyses including both observational and cohort studies, it was concluded that individuals with type 2 diabetes had a 30.0–40.0% increased risk of atrial fibrillation, even after adjustments for BMI, smoking, hypertension, kidney disease, coronary heart disease, and hyperthyroidism , 14. In Although one should be cautious to make direct comparisons between different studies, it is of interest to notice that the excess risk of heart failure , major cThe risk of atrial fibrillation among individuals with type 2 diabetes was also evaluated in relation to 1.0% (10.0 mmol/mol) higher mean HbA1c. HbA1c had a relatively modest impact on atrial fibrillation with a risk increase of 1.0–4.0% per 10 mmol/mol higher mean HbA1c compared to 12.0–19.0% for heart failure, acute myocardial infarction and CHD death , 23. FroSince rates of diabetes mellitus and atrial fibrillation are increasing worldwide and are strong risk factors for stroke and cardiovascular mortality due to thromboembolic risk, it is important to consider early prevention. In a prospective observational study, cardiac rhythm abnormalities were common in patients with moderate to severe CKD not requiring dialysis . TherefoThe link between diabetes mellitus and atrial fibrillation may be explained by several possible mechanisms. The metabolic syndrome characterized by obesity, hypertension, insulin resistance, and dyslipidaemia has been proposed to be associated with increased risk of atrial fibrillation . It has Strengths of our study include the population-based design including the absolute majority of individuals with type 2 diabetes in Sweden. Additionally, well-known risk factors were available in persons with type 2 diabetes. Limitations include the absence of information on risk factors such as smoking, BMI, blood pressure, and lipid levels in the control population. Another limitation is that we cannot exclude the possibility that atrial fibrillation was detected to a somewhat higher extent in individuals with type 2 diabetes because it is a chronic disease requiring regular visits.Type 2 diabetes remains a risk factor for atrial fibrillation, increasing risk by approximately 35% on a group level. Persons with advanced renal complications have substantially higher excess risks with a threefold excess risk at CKD-5. Glycaemic control likely has a relatively limited direct effect on the development of atrial fibrillation, but since it is essential for the development of renal complications it may have an effect over long time periods. Screening for atrial fibrillation should be considered in persons with increased risks including elderly and those with advanced renal complications among persons with type 2 diabetes, especially since the procedure is relatively simple.Additional file 1: Table S1. Cox regression for time to atrial fibrillation-overall, men and women. Table S2. Adjusted hazard ratios for atrial fibrillation and 95% confidence intervals for time-updated mean HbA1c categories, albuminuria categories and eGFR categories versus the reference group examined by Cox regression–Men. Table S3. Adjusted hazard ratios for atrial fibrillation and 95% confidence intervals for time-updated mean HbA1c categories, albuminuria categories and eGFR categories versus the reference group examined by Cox regression–Women. Table S4. Adjusted hazard ratios for atrial fribrillation and 95% confidence intervals for time-updated mean HbA1c categories together with albuminuria and eGFR versus the reference group examined by Cox regression–Men. Table S5. Adjusted hazard ratios for atrial fibrillation and 95% confidence intervals for time-updated mean HbA1c categories together with albuminuria and eGFR versus the reference group examined by Cox regression–Women. Table S6. The impact of time-updated mean HbA1c categories on time to first atrial fibrillation using Cox regression analysis among type 2 diabetes patients–Men. Table S7. The impact of time-updated mean HbA1c categories on time to first atrial fibrillation using Cox regression analysis among type 2 diabetes patients–Women.


A good substrate–product correlation between CO2 and CH4 concentrations indicates rapid in-dump CH4 oxidation with only insignificant amounts of CO formed. Other gas components include hydrogen, and C3–C6 saturated and unsaturated hydrocarbons. Decreasing oxygen content in the gases is temperature-dependent, and O2 removal rapidly increased at > 70 °C. Emission differences between both basins are minor and most probably reflect the higher maturity of coal waste organic matter in the Lower Silesia dumps causing its higher resistance to temperature, or/and a higher degree of overburning there.Gaseous emissions from seven self-heating coal waste dumps in two large coal mining basins, Upper and Lower Silesia (Poland), were investigated by gas chromatography (GC-FID/TCD), and the results were correlated with on-site thermal activity, stage of self-heating as assessed by thermal mapping, efflorescences, and surface and subsurface temperatures. Though typical gases at sites without thermal activity are dominated by atmospheric nitrogen and oxygen, methane and carbon dioxide are present in concentrations that many times exceed atmospheric values. On average, their concentrations are 42.7–7160 ppm, respectively. These are levels considered harmful to health and show that coal waste fire can be dangerous for some years after extinction. At thermally active sites, concentrations of CHThe online version of this article (10.1007/s10653-018-0153-5) contains supplementary material, which is available to authorized users. Coal waste dumps, a common landscape feature in coal mining regions, are a potential source of hazardous substances emitted to the atmosphere and leached to surface and ground waters  are odorless. The gases also contain NOx, NH3, SOx, and H2S from the thermal decomposition of sulfide minerals, HCl, light aliphatic compounds up to C10, aromatic compounds such as benzene and its alkyl derivatives, styrene, alcohols, PAHs, and heavy metals, e.g., Hg, As, Pb, and Se  to examine the variability in occurrence and distribution of the main gas components emitted from self-heating dumps in Upper and Lower Silesia, (b) to establish whether differences in gas distributions are related to thermal stage, (c) to compare the activity and dynamics of self-heating in the two basins, and (d) to assess levels of hazardous gas emissions in both. Preliminary research on gas compositions performed in Upper Silesia  of which and is ~ 50 m high. Planned capacity was > 3 × 106 m3  and contains 10.2 mln tonnes of waste . The thermal activity stage was established mainly on field observations and temperature measurements.How coal waste dumps are affected by fire that depends on a variety of factors, e.g., fire duration, oxygen access, volume of burning waste, the nature of organic material, its content, and petrographic composition. Gas sampling sites were chosen where signs of thermal activity were evident, namely open fire, smoke, odors, charred vents, efflorescences, a lack of vegetation, or the presence of moss or mullein  in places where fire was beginning to encroach on cool coal waste. Here, temperatures are high, smoke, and odors noticeable, and blooming organic efflorescences prominent. Nine samples are from sites of ‘waning’ thermal activity marked by lower temperatures and a lack of efflorescences. In addition, five samples are from sites with no current thermal activity; these were never touched by fire or past activity had ceased.Thermal maps help to reveal the self-heating history of coal waste dumps. They can aid the location of current hot spots, their migration paths, and variations in intensity with time. Regretfully, such archival data are rarely available for dumps. For this study, a series of Landsat 5, 7, and 8 images with snow covering was used. The thermal mapping procedure used is detailed in Nádudvari . Hot spoT values are 6–14 °C higher than background surface temperatures (Table S1). Cold and frosty weather can induce a marked decrease in hot-spot surface temperatures . Thus, fires falling below these resolutions, or have low surface temperatures, evade detection.In general, most coal waste hot spots where intensive fire is present can be detected when res Fig. . Where ires Fig. . The resEight Landsat images from 1987 to 2015  clearly show continuous thermal activity during that period in the coal waste dumps in Nowa Ruda  include 12 from Wełnowiec, 10 from Rymer Cones, 5 from the Anna dump and 5 from Czerwionka-Leszczyny in the Upper Silesian Coal Basin, and 21 from Słupiec, 3 from Nowa Ruda, and 1 from Przygórze in the Lower Silesian Coal Basin . Numbers of samples reflected numbers of active sites and their intensity. Only at the Wełnowiec dump was it possible to clearly distinguish all three stages of thermal activity. Two sites (W2 and W3) relate to the initial stage, one (W4) to ongoing activity, and two (W5 and W6) to waning fire . Sites W1, W7, and W8 that had seen fire in the past were inactive on the day of sampling (13.01.2014). Rymer Cones was sampled on 14.04.2011  and, four years later, on 16.03.2015  at sites of ongoing thermal activity. Samples from the Anna dump and Czerwionka-Leszczyny were collected on 12.11.2016 from sites of thermal activity that had been waning since September 2016 at least. Sampling took place at Słupiec on 13.12.2013 and at Nowa Ruda and Przygórze on 28.5.2014 . At Słupiec, ten thermally active sites from the top and slopes of the dump were sampled at varying depths (S1–S10) and a reference sample (S11) was collected at 1 m depth at a site where thermal activity had never been noted. Though the Nowa Ruda and Przygórze dumps had been very active in the past, at Nowa Ruda, only two sites are still active (N2 and N3) and any in the Przygórze dump has ceased (P1).3) were collected a few centimeters subsurface and deeper (< 1.5 m) using syringe samplers. A 1.5-m steel pipe protected by a clinch was hammered as deeply as possible into vents or heated spots. The clinch was removed, and the gas was collected using a plastic pipe with an attached syringe fixed to the steel pipe. The clinches were abandoned.Samples (100 cmTemperature was measured using a pyrometer coupled with a K-probe which enabled measurement up to 0.3 m subsurface (Table S2). Surface temperatures at thermally inactive sites reflected the weather and ambient air temperatures.Efflorescences blooming at fissures were identified using SEM–EDS and XRD. The morphologies of samples on carbon tape were examined using a Philips XL 30 ESEM/TMP scanning electron microscope coupled to an energy-dispersive spectrometer  at the Faculty of Earth Sciences, University of Silesia. Analytical conditions were: accelerating voltage 15 kV, working distance ca 10 mm, and counting time 40 s. In addition, powdered samples were examined using a Bruker AXS D8 ADVANCE diffractometer in the X-Ray Diffraction Laboratory, Institute of Geological Sciences, Polish Academy of Sciences and an X-ray Philips PW 3710 diffractometer at the Faculty of Earth Sciences, University of Silesia. Efflorescence phase compositions are given in Table S2.4, C2H6, C3H8, iC4H10, nC4H10, C5H12, C6H14, C7H16, unsaturated hydrocarbons, CO2, O2, H2, N2) were determined on an Agilent 7890A gas chromatograph equipped with a set of columns, and flame ionization (FID) and thermal conductivity (TCD) detectors. This GC is configured to do an extended natural gas analysis up to C14. The analyzer is a three-valve system using three 1/8-inch packed columns  and a GS-Alumina capillary column (50 m × 0.53 mm). The system consists of two independent channels. The channel using the FID for the detailed hydrocarbon analysis is a simple gas sampling valve injecting the sample into the GS-Alumina column. The second channel using packed columns is for determination of methane, ethane, and non-hydrocarbon gases. The GC oven was programmed as follows: initial T of 60 °C held for 1 min., then to 90 °C at 10 °C/min., then to 190 °C at 20 °C/min., and finally held for 5 min. The front detector (TCD) was operated at 150 °C, and the back detector (FID) at 250 °C. Helium was used as a carrier gas flowing through the TCD channel at cm3 min−1 and through the FID channel at cm3 min−1.To assess the variability of gas compositions, several dominating compounds were selected based on previous research . In terms of the temperatures measured at sampling sites (Table S2), and of the self-heating stage, the gas samples may be grouped into samples from (1) from sites with no thermal activity, i.e., with no known fire history or where thermal activity has ceased, (2) sites with self-heating ongoing; these subdivide into gases (a) from the initial stage of self-heating, (b) emitted during intense heating, and (c) from sites of waning activity.2 and N2, two further main components are CH4 and CO2, both toxic and regarded as the main greenhouse gases . Unsaturated hydrocarbons were typically present in lower amounts than were their saturated analogues. To compare the highly variable gas distributions, the following relative percentage concentrations were calculated: (1) The relative percentage compositions of gases present together with atmospheric N2 and O2 (Table 4 (Table 4. As there are significant differences in emitted gas compositions between the Upper (US) and Lower Silesia (LS) basins, both are treated separately below. Apart from Oases Kim . Absolutculated:  The rela2 and N2 , namely 1.085 (US) and 0.151 (LS) % vol. However, at some inactive sites CO2 was absent. The atmospheric gases are accompanied by small amounts of organic compounds, among which, CH4 (0.0061 and 0.0040% vol.) and ethylene (0.0030 and 0.0011% vol.) predominate. Heavier aliphatic hydrocarbons from cis-2-butene to n-hexane occur in much lower amounts (0.0001–0.0005% vol.).The typical gas at a site with no thermal activity is dominated by atmospheric O2 Tables  and S3. 2 content, being < 5.5 times lower than in the atmosphere. Apart from the major atmospheric gases, CO2 is the predominating component, averaging 3.7350 (US) and 5.2447 (LS) % vol. The organic gases also include CH4 (1.3233 (US) and 0.1432 (LS) % vol.), saturated aliphatic hydrocarbons including ethane, propane, n-butane, n-pentane, n-hexane, n-heptane, iso-butane, and iso-pentane, together with unsaturated aliphatic hydrocarbons including ethylene, acetylene, propylene, and trans- and cis-2-butene. Typically, concentrations decrease with increasing molecular weight but, in some LS gases , elevated contents of propane and n-butane were noted. In sample A1, the relative content of propane exceeds that of CH4 (Table S3). Thermal activity also results in elevated H2 contents, i.e., 0.2125 (US) and 0.0186 (LS) % vol. (Table 2 concentrations (0.0000055% vol.). The unsaturated hydrocarbons and H2 are pyrolytical products of self-heating; they are common in refinery and coal pyrolysis gases , (3) reducing gases (CH4 and H2), (4) saturated aliphatic hydrocarbons in the range C2–C7, and (5) unsaturated aliphatic hydrocarbons in the range C2–C4.In the dump emissions, the classes of gases distinguished include (1) main air components, (2) oxygenated compounds . Apart from its significance as a greenhouse gas, CO2 is also toxic. The normal CO2 concentration outdoors is ca 300–350 ppm or 0.54–0.63 g/m3 (Killops and Killops 3). The highest CO2 concentration registered at thermally active sites was 291.5211 g/m3 or 161,666 ppm. This is > 500 times the normal atmospheric level, and > 1.5 times the level (100 000 ppm) that leads to loss of consciousness and, ultimately, death . Thus, CO2 emissions from inactive dumps at ca 7000 ppm are close to levels at which adverse health effects might be expected  and 4.4556 (LS) g/m Pauluhn . This su2 also shows inverse correlations with CH4 (below) when the emitted gas results from self-heating sites are recalculated to relative percentages, omitting N2 and O2 , were a few tens g/m3. There are two possible explanations for these high CH4 levels, namely (i) the compound was released from still-decaying urban wastes originally dumped there, or (ii) that, during their combustion, the top cover of coal waste limited CH4 oxygenation. Conspicuously, lower CH4 emissions  from other Silesian dumps are still 1000 times higher than atmospheric levels (Table S3).Methane predominates among organic compounds, occurring in amounts > 80%, in some cases, < 99.91% rel. in sites of current thermal activity  and 0.05163 (LS) g/m3 . These levels are 4–40 times atmospheric levels. However, they pale in comparison with thermally active sites where CH4 emissions average 6.350585 (US) and 1.031258 (LS) g/m3 . Carras et al. . Here, CH4 may be a biogenic product of microorganisms living on coal waste. It is usually considered that methanogens live in wet anaerobic conditions not seen in coal waste dumps  and between values of oxygen decrease (OD) and CO2 relative contents , and sites with ongoing heating show no significant differences and CO2 production prevails (Table S2). However, where fire is beginning to wane (W5 and W6) CO2 relative contents decrease, whereas those of CH4 increase. No CO2 is expelled in thermally inactive sites.Relative percentage contents of CO6 and C7 hydrocarbons are absent. Both normal and branched compounds occur. Apart from W1, W8, R1–4, S1a–c, S6, and S11, ethane predominates in the saturated gas fraction.Saturated aliphatic hydrocarbons occur in the range from ethane to heptane though, in most gases, C2–C4 and acetylene. Due to its relatively high reactivity, acetylene was found in only a few samples . Other compounds with triple bonds are absent. Among unsaturated hydrocarbons, ethylene dominates though typically comprising < 1.0% of total organic compounds. However, ethylene contents increase significantly in sites showing particularly elevated temperatures, e.g., to > 25% of all organic compounds in W3b (t = 690 °C at 50 cm). Contents of all other unsaturated hydrocarbons decrease with increasing carbon atom numbers in a molecule. Surprisingly, ethylene is also a significant component  of total organic components in gases at sites of waning thermal activity, e.g., W1, W7, W8, S1b, S11, and N1 with measured temperatures close to ambient. Possibly, as with methane, ethylene is degassed from pores even after self-heating ends or it is produced by bacteria growing on coal waste surfaces; many soil bacteria species, e.g., many chemolithotrophs, can produce ethylene  where temperatures have waned to near ambient. Ethylene together with COHydrogen was found only at thermally active sites, despite being a product of low-temperature oxidation of bituminous coals . N2 is assumed to be inert; it neither reacts with coal waste nor is released. The OD value reflects O2 consumption during heating.Oxygen decrease (OD) calculated as NThe ratio of saturated to unsaturated hydrocarbons (S/UnS). It is assumed that unsaturated hydrocarbons are the products of organic matter macromolecule cracking. This parameter reflects the thermal destruction of organic matter.Carbon dioxide/methane, the ratio of two major components of gas emissions from coal waste dumpsTo assess thermal activity in the coal waste dumps, and to compare its development in different dumps, the following ratios were calculated Table .Table 3COxygen decrease is caused by oxidation of organic matter due to self-heating. Thus, the OD value reflects the process intensity; the higher the value, the more intense the self-heating. OD values are arbitrarily designated as follows: 1.0–1.7 (low self-heating or none), 1.8–3.0 (moderate heating), > 3.0 (intense heating).2 in the total gas composition; the substrate–product relationship is confirmed by inverse correlations (r = − 0.89 for US and − 0.98 for LS) of relative contents (vol.) of CO2 versus O2 and positive correlations (r = 0.87 for US and − 0.93 for LS) between OD values and CO2 relative contents  between OD and CO2 for the Wełnowiec gas samples probably reflect firefighting activity; during sampling, the dump was opened to cool burning waste, increasing O2 access, and intensifying combustion and elevating temperatures (< 700 °C). The strong correlation indicates that most O2 was consumed by CO2 production with other oxides playing only a very minor role.Samples with very low OD values are W1, W2a, W4a, W7, W8, all Rymer Cones gases taken in the later series , and A2–5, CzL1–5, S1a–c, S 6, S10b, S11, P1, and N1–3. Moderate values characterize only six samples, i.e., W3b, R1a and b, R5a, A1, and S10a. The highest values pertain to W2b, W3a, W4b, W5, W6a–b, R4a, R6a, S1d–g, S2–5, and S7–9 , NR1–4, NRS1a, NRS7, and NRS10b (Table There are three factors which should be considered as influencing gas composition: (1) temperature, (2) the stage of self-heating , and (3) characteristics of coal wastes organic matter and minerals. Differences in the chemistry of gas emitted from dumps in LS and US are related to all three factors, but their relative importance varies.At the time of sampling, at the LS sites, only mild thermal activity prevailed with temperatures < 70 °C, i.e., below the threshold temperature above which intense self-heating begins Sokol . Thus, o4/CO2 which, at the LS sites, are similar to those of the US inactive sites. S/UnS follows a similar pattern. Thus, gas composition seems to mainly reflect self-heating stage and temperature level, particularly whether the threshold temperature (60–80 °C) is exceeded or not  are much higher in the active US sites, e.g., CH4 (× 6), C2–C4 saturates (× 2–7), propylene and acetylene (× 5) and H2 (× 8). The lower resistance and rank of the US organic matter are also reflected in more pronounced temperature effects on gas compositions in active and inactive sites, e.g., much higher contents of CH4 and C2H6 hydrocarbons  in US than in LS sites .However, correlations between COnts Fig.  in the ints Fig. . The org4 predominates at all thermally active sites, the compositions of heavier hydrocarbons, i.e., C2–C7, correlate better with self-heating stages . Densities are particularly high in the areas where ca 200 US and 130 LS coal waste dumps are located, i.e., 2000–3000 person/km2; communities clustered around the mines and associated smelters. For example, the Wełnowiec dump lies within 2.5 km of three Katowice districts, Koszutka, Bogucice, and Dąb, with 10000, 14000, and 7000 inhabitants, respectively. The Osiedle Tysiąclecia area slightly further away houses ca 21 000 inhabitants. The worst impacts probably affect settlements such as Skała, Bunczowiec, and Bułowiec (the Rydułtowy districts with ca 8000 inhabitants) located 300 m from the Szarlota dump, 500 m from the Anna-Pszów dump, and ca 2 km m from the Marcel and Rymer Cones dumps, or the Niedobczyce residential area (ca 12000 inhabitants) located 100–200 m from the Rymer Cones. Both regions are characterized by high degrees of citizens mobility to and from homes and working places every day which makes the real impact difficult to assess. However, the fact that incidences of lung cancer and other lung and cardiovascular illnesses generally are much higher in Silesia than elsewhere in Poland may be an additional indicator of exposure to self-heating pollutants  or reflects long-term leakage from rock pores. Even thermally inactive coal waste dumps should be deemed a long-term environmental hazard.Gas emissions at inactive sites comprise CHThe main light hydrocarbons produced during self-heating are saturates. Their dominance over unsaturated hydrocarbons increases with temperature. Acetylene is rare and other alkynes were not found, possibly due to their higher chemical reactivity.2 and O2 indicates that organic matter oxidation, not the formation of other oxides (including inorganic oxides), consumes most of the oxygen budget.Oxygen decrease in the gases is mostly temperature-dependent with a threshold temperature of ca 70 °C. Whenever this level is reached, a significant decrease in oxygen content is registered. A strong substrate–product correlation between COThe distribution of heavier hydrocarbons in the dumps is influenced by the stage of self-heating attained. Initial and waning stages show similar gas compositions, whereas sites with ongoing self-heating show greater emission of heavier hydrocarbons, possibly related to higher temperatures. On a regional scale, the minor differences between emissions in the two Silesian coal basins are also mostly related to the self-heating stage pertaining or to differences in the thermal maturity of coal waste organic matter in both basins. The higher rank of LS organic matter makes it less prone to expelling hydrocarbons when heated. Critically, in Lower Silesia, self-heating is on the wane and most dumps already overburnt.Since self-heating of coal waste dumps exposes large population in Upper and Lower Silesia, precautionary measures against any health dangers should be undertaken, e.g., monitoring of internal temperatures and initial-stage gases. The low threshold temperature (ca 70 °C) means that quick and relatively inexpensive cooling of the damp is possible before the beginning of intense self-heating. Otherwise, temperatures will increase rapidly up to ignition temperature over a few weeks. Unfortunately, it is not easily possible to dismantle coal waste dumps. Due to poor mechanical quality of Silesian coal wastes, their reuse is limited to overburnt material. To limit population exposure to harmful emissions, limiting access to dumps may be advisable, particularly those with ongoing heating.Supplementary material 1 (DOCX 1068 kb)Supplementary material 2 (DOCX 1314 kb)Supplementary material 3 (DOCX 1288 kb)Supplementary material 4 (DOCX 36 kb)Supplementary material 5 (DOCX 14 kb)Supplementary material 6 (DOCX 46 kb)Below is the link to the electronic supplementary material.


AD) is a devastating neurodegenerative disorder that impairs memory and causes cognitive and psychiatric deficits. New evidences indicate that AD is conceptualized as a disease of synaptic failure, although the molecular and cellular mechanisms underlying these defects remain to be elucidated. Determining the timing and nature of the early synaptic deficits is critical for understanding the progression of the disease and for identifying effective targets for therapeutic intervention. Using single‐synapse functional and morphological analyses, we find that AMPA signaling, which mediates fast glutamatergic synaptic transmission in the central nervous system (CNS), is compromised early in the disease course in an AD mouse model. The decline in AMPA signaling is associated with changes in actin cytoskeleton integrity, which alters the number and the structure of dendritic spines. AMPA dysfunction and spine alteration correlate with the presence of soluble but not insoluble Aβ and tau species. In particular, we demonstrate that these synaptic impairments can be mitigated by Aβ immunotherapy. Together, our data suggest that alterations in AMPA signaling and cytoskeletal processes occur early in AD. Most important, these deficits are prevented by Aβ immunotherapy, suggesting that existing therapies, if administered earlier, could confer functional benefits.Alzheimer's disease ( In add2.4Act1, Actn2, Actn3, Actn4, Dbn1, Cfl1, Camk2a, Camk2b, Camk2 g, Camk2n1, Rac1, Cdc42, Rhoa, Rock1, Rock2, Pfn1, Pfn2, Pfn3, Pfn4, Cit, Pkn1, Prok1, Rhpn1, Rhpn2, Gsn, Cttn, Wasf1, Cdk5, Baiap2, Cldn5, Nefl, Fn1, Aamp, Cspg5, Clstn3, Mog, and Ncan), including upregulation or downregulation, in the hippocampi of 7‐month‐old 3xTg‐AD mice compared to Ntg animals   in synapses. Western blot analysis in hippocampal synaptosomes showed that the human amyloid precursor protein (APP) and C‐terminal fragments (CTFs) (recognized both by the 6E10 antibody) were significantly elevated in 3xTg‐AD mice, with no expression observed in Ntg mice Figure a,b. More2.6We next sought to determine whether the synaptic changes observed in the hippocampus of 3xTg‐AD mice are associated with the Aβ accumulation that this mouse model presents. First, we used a cell culture approach to determine whether Aβ oligomers are capable of altering the levels of plasticity‐related proteins. We observed increased protein levels in the pre‐ and postsynaptic markers synaptophysin and PSD‐95 1 h after cLTP stimulation in primary hippocampal cells  in the ipsilateral hippocampi compared to the contralateral uninjected hippocampi  are highly potent synaptotoxic molecules. In an important way, these deficits are prevented by Aβ immunotherapy, suggesting that preventive and/or earlier treatment could effectively combat synaptic dysfunction. The identification of the cellular and molecular mechanisms that impair synaptic and cognitive function during earlier stages of the disease is critical because it will increase the likelihood of developing effective therapies to stop further synaptic damage before it becomes irreversible in patients with AD.44.1Swe and the human tauP301L (both under the control of the mouse Thy1.2 regulatory element) were co‐microinjected into single‐cell embryos harvested from homozygous mutant PS1M146V knockin (PS1‐KI) mice  mice aged 3 and 7–8 months were used in the current study. Only male mice were used in the current study to avoid the effect of the estrous cycle on plasticity changes of hippocampal AMPA receptors , histological , and biochemical  techniques used in the current manuscript is added as supplemental data , followed by Bonferroni's comparisons, was applied using GraphPad Prism 5® software . The significance was set at 95% of confidence. All values are presented as mean ± SEM.The biochemical data were quantitatively analyzed using ImageJ 1.36b software. For comparison between two groups, Student's DBV and FML conceived and designed the experiments. DBV, GAP, AL, SF, CRO, KI, LTE, JCD, and ACM performed the experiments. DBV, GAP, AL, and CRO analyzed the data. DBV, GAP, AL, SF, CRO, RA, RM, LTE, ACM, MK, JCD, CC, AG, and FML contributed to the writing of the manuscript.No potential conflict of interests relevant to this article were reported. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Citrus plants contain large amounts of flavonoids with beneficial effects on human health. In the present study, the antioxidant and anti-inflammatory potential of bioavailable polyphenols from Citrus sinensis was evaluated in vitro and ex vivo, using the murine macrophages cell line J774A.1 and primary peritoneal macrophages. Following simulated gastro-intestinal digestion, the in vitro bioavailability of Citrus sinensis polyphenolic extract was assessed using the human cell line Caco-2 grown as monolayers on a transwell membrane. Data demonstrated a relative permeation of its compounds (8.3%). Thus, the antioxidant and anti-inflammatory effect of polyphenolic Citrus sinensis fraction (Cs) was compared to the bioavailable one (CsB). Results revealed that Citrus extract were able to reduce macrophages pro-inflammatory mediators, including nitric oxide, iNOS, COX-2 and different cytokines. Moreover, the effect of Citrus sinensis polyphenols was associated with antioxidant effects, such as a reduction of reactive oxygen species (ROS) and heme-oxygenase-1 (HO-1) increased expression. Our results provide evidence that the bioavailable polyphenolic constituents of the Citrussinensis extract accumulate prevalently at intestinal level and could reach systemic circulation exerting their effect. The bioavailable fraction showed a higher anti-inflammatory and antioxidant potential compared to the initial extract, thus highlighting its potential nutraceutical value. Inflammation is a biological process mainly supported by the immune cells to protect the host from microbial infection or tissue injury. Although inflammation is an essential response to eliminate aggressors, its development has to be controlled over time to monitor its intensity and avoid deleterious chronic inflammatory response .Citrus is one of the largest species among plants; it consists of 40 species which are distributed in all continents and its fruits, which are consumed mostly fresh, have been used as a herbal medicine or additive or food supplement [Citrus such as anticancer, antimicrobial, antioxidant and anti-inflammatory [Citrus flavonoids bioavailability [Citrus flavonoidic profile and on their antioxidant and anti-inflammatory effect after GI digestion has not been investigated so far.The oxidative response is deeply involved in the regulation of the inflammatory processes, mediating many aspects of inflammatory-induced tissue damage and dysfunctions ,3. Nowadpplement . A wide ammatory , deeply ammatory . Since sammatory ,14. Similability ,16,17. OCitrus sinensis polyphenols using an extended in vitro model, including Caco-2 cell permeation of the intestinal digesta. The polyphenolic profile of Citrus at the different stages of simulated digestion was compared to those natively present, while the impact of GI digestion on the antioxidant and anti-inflammatory properties of the Citrus extract was determined using an in-cell inflammation model.For these reasons, in the present study we focused on the antioxidant and anti-inflammatory activity of the bioavailable fraction from Citrus species [Identification was carried out on the basis of standard retention time, UV spectra, and by comparing molecular formulas generated by the analysis of accurate MS and MS/MS spectra with those present in literature ,19. Nine species . As it c species ,19.Citrus sinensis juice extract were detected in the salivary phase at levels ranging from 75.1% to 99.2% of their native patterns, thus evidencing a good stability to the biochemical conditions in the mouth  or with the bioavailable extract (CsB) in the culture media. Our data indicated that cellular viability was not significantly affected either by Cs or CsB (data not shown).To evaluate if Cs or CsB administration could affect J774A.1 or mouse peritoneal macrophages viability, cells were exposed to p < 0.001 vs. LPS). At the same concentrations CsB proved to be significantly more effective than Cs , indicating their inhibitory effect both on the iNOS enzyme expression and on its activity. CsB, also in these experimental conditions, was more effective in comparison with Cs . Moreover, COX-2 appeared to be more strongly inhibited by CsB . It’s worth noting that in this experiment, as observed before, CsB exerted a stronger effect than Cs  significantly inhibited ROS production in a concentration range of 250–25 µg/mL both when added 1 h before  could be considered responsible for the higher antioxidant and anti-inflammatory activity showed by the bioavailable fraction [The antioxidant properties of flavonoids are deeply dependent on their chemical structure. Free radical quenching has been related, for example, to the ability of the flavonoids aromatic hydroxyl group to donate the hydrogen delocalizing the unpaired electron around the aromatic system . In addifraction . This obfraction . Moreovefraction .2O) was obtained by a Milli-Q Direct 8 system , methanol, acetonitrile (ACN) and formic acid (HCOOH) LC-MS grade were purchased from Sigma Aldrich . For the quantitative and qualitative analysis of flavonoids two columns were employed respectively: a Kinetex C18 150 × 4.6 mm (100 Å), packed with 2.6 µm particles, and a Kinetex C18 150 × 2.1 mm, 2.6 µm column . Both columns were protected with C18 precolumns (Phenomenex).Ultra-pure water  and polymetoxyflavones (tangeretin) were purchased from Sigma Aldrich. For cell culture unless stated otherwise, all reagents and compounds were purchased from Sigma Chemicals Company .Citrus sinensis juice var. Tarocco was provided by the company “Agrumaria Corleone” , which used fruits from plants cultivated in Sicily . In order to remove fibers, 100 mL of hand squeezed juice were centrifuged at 15,000× g for 15 min, then lyophilized for 24 h, by setting the condenser temperature at −52 °C and the vacuum value at 0.100 mBar. From 100 mL of Citrus sinensis juice, 400 mg of dry extract was obtained. The powder was extracted with MeOH and the procedure was repeated three times for the complete recovery of polyphenolic compounds. The methanolic extracts were combined and the organic solvent was removed by vacuum evaporation at 50 mBar and 40 °C. Sample was stored at 4 °C, then solubilized in methanol to a concentration of 1 mg/mL, subjected to ultrasonication and filtered prior to injection on 0.45 µm nylon membrane (Millipore).The R5 degasser, an SPD-M20A photo diode array detector (equipped with a 2.5 µL detector flow cell volume), a CTO-20A column oven, a SIL-30AC autosampler. The UHPLC system was coupled online to an LCMS–IT-TOF mass spectrometer through an ESI source (Shimadzu). LC-MS data elaboration was performed by the LCMSsolution® software .UHPLC analyses were performed on a Nexera UHPLC system  consisting of a CBM-20A controller, two LC-30AD dual-plunger parallel-flow pumps, a DGU-20 A2O v/v (A) and 0.1% HCOOH/ACN v/v (B). Analysis was performed in gradient elution as follows: 0–2.00 min, 10–15% B; 2–10.00 min, 15–20% B; 10–12.50 min, 20–65% B; 12.50–17.50 min, 65–75% B. Flow rate was 1.8 mL/min. Column oven temperature was set to 40 °C. Injection volume was 2 µL of extract. The following PDA parameters were applied: sampling rate, 40 Hz; detector time constant, 0.160 s; cell temperature, 40 °C. Data acquisition was set in the range 190–400 nm and chromatograms were monitored at 280 and 330 nm at the maximum absorbance of the compounds of interest.The optimal mobile phase consisted of 0.1% HCOOH/H−1) were prepared in methanol, and the calibration curves were obtained in a concentration range of 0.5–100 µg·mL−1 with seven concentration levels and triplicate injections of each level were run. Peak areas of each standard were plotted against corresponding concentrations (µg·mL−1). The amount of the compounds in the sample was expressed as milligram per gram of extract, linear regression was used to generate calibration curve, R2 values were ≥0.995.For the quantification of flavonoids, eight compounds were selected as external standards: diosmetin 6,8-di-C-glucoside, neohesperidin, eriocitrin, isoquercetin, narirutin, diosmetin, hesperetin and tangeretin. Stock solutions (1 mg·mL2O and (B) ACN both acidified by formic acid 0.1% v/v. Analysis was performed in gradient elution as follows: 0–2.50 min, 5–15% B, 2.50–10.00 min, 15–25% B, 10–12.00 min, 25–55% B, 12–14.50 min, 55–65% B, 14.50–17.00 min, 65–70% B. Flow rate was 0.5 mL/min. Column oven temperature was set to 40 °C. Injection volume was 2 µL. The UHPLC system was coupled on-line to a hybrid IT-TOF instrument, flow rate from LC was splitted 50:50 prior of the ESI source by means of a stainless steel Tee union . Resolution, sensitivity, and mass number calibration of the ion trap and the TOF analyzer were tuned using a standard sample solution of sodium trifluoroacetate. After the calibrant had flowed, cleaning operation of the tube and ESI probe was carried out by flowing acetonitrile . MS detection was operated both positive and negative ionization mode with the following parameters: detector voltage, 1.55 kV; CDL (curve desolvation line) temperature, 200 °C; block heater temperature, 200 °C; nebulizing gas flow (N2), 1.5 L/min; drying gas pressure, 100 kPa. Full scan MS data were acquired in the range of 200–800 m/z . MS/MS experiments were conducted in data dependent acquisition, precursor ions were acquired in the range 150–800 m/z; peak width, 3 Da; ion accumulation time, 60 ms; CID energy, 50%; collision gas, 50%; repeat = 1; execution trigger (BPC) intensity, at 95% stop level.Mobile phases were: (A) H2PO4 (0.7 M), Na2SO4 (0.4 M), NaCl , NaHCO3 (1.0 M), urea (0.4 M) and 290 mg of α-amylase. The pH of the solution was adjusted to 6.8 with HCl 0.1 M. The mixture was introduced in a plastic bag containing 40 mL of water and homogenized in a Stomacher 80 Microbiomaster  for 3 min. Immediately, 0.5 g of pepsin  dissolved in HCl 0.1 M were added, the pH was adjusted to 2.0 with HCl 6 M, and then incubated at 37 °C in a Polymax 1040 orbital shaker  for 2 h. After the gastric digestion, the pancreatic digestion was simulated as follows: the pH was increased to 6.5 with NaHCO3 0.5 M and then 5 mL of a mixture pancreatin (8.0 mg/mL) and bile salts , dissolved in 20 mL of water, were added and incubated at 37 °C in an orbital shaker (250 rpm) for 2 h. After each step of digestion, 10 mL of the obtained extract were centrifuged at 1700× g and 4 °C for 1 h: before each following step, the digestion procedure was started over again. To determine the polyphenolic profile, the supernatants were extracted with an acetonitrile–water  mixture, and then analysed by UHPLC.The assay was performed according to the procedure described by Tenore et al. . GI dige2/air (5:95) and passaged every 7 days by trypsinization.The human colon carcinoma cell line Caco-2 (HTB-37) was obtained from the American Type Culture Collection . Cells were cultured routinely in HEPES buffered Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose and supplemented with 12.5% heat-decomplemented fetal calf serum (FCS), 1% nonessential amino acids, 5 mM L-glutamine, 40 U/mL penicillin, 100 µg/mL gentamycin, and 40 µg/mL streptomycin (DMEMc). Cells were maintained at 37 °C in a humidified atmosphere of CO4 cells/cm2 in 15 mL DMEM containing 10% FCS and cultured for was changed every 21 days. The integrity of the monolayers was evaluated by measurement of the transepithelial electrical resistance (TEER) using a Millicell-ERS device  before and after the treatments. To evaluate transepithelial permeability, the medium was removed from the apical and basal sides of the cultures and replaced by 2 mL of the transport solution consisting of Hanks’ balanced salt solution (HBSS) and Citrus sinensis juice flavonoids deriving from gastrointestinal digestion at pH 6 or 7.4. After 4 h of incubation at 37 °C, apical and basal solutions were collected and to determine the polyphenolic profile, aliquots (5 mL) were immediately mixed with 1 mL of methanol and filtered on 0.45 µm Millex-HV filter units. Samples were stored at −20 °C until HPLC analysis.Caco-2 cells were seeded in transwells at 6 × 102 atmosphere.J774A.1 murine monocyte macrophage cell line , was grown adherent to Petri dishes with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (FCS), 25 mM HEPES, 2 mM glutamine, 100 μ/mL penicillin and 100 mg/mL streptomycin at 37 °C in a 5% CO2 atmosphere. Subsequently, non-adherent cells were removed and RPMI 1640 medium with 10% FBS was added, as previously reported [Female C57BL/6 mice  were fed a standard chow diet and housed under specific pathogen-free conditions at the University of Salerno Department of Pharmacy. All animal experiments were performed under protocols that followed the Italian and European Community Council for Animal Care (DL No. 116/92). Peritoneal cells were harvested by means of peritoneum lavage with 5 mL of EDTA 0.5 mM plated and allowed to adhere for 2 h at 37 °C in a 5% COCitrus sinensis (Cs) or bioavailable Citrus sinensis (CsB) (250–10 μg/mL) alone for 1 h and then simultaneously to lipopolysaccharide from E. coli  for the time described below. In some experiments, in order to evaluate the effect Cs and CsB after inflammation induction, J774A.1 macrophages were firstly treated with LPS and then with Cs or CsB for further 24 h, always in presence of LPS, before the evaluation of pro-inflammatory and pro-oxidant parameters.Macrophages, both J774A.1 and primary macrophages, were incubated with raw 3/well and 5 × 104/well respectively) and treated with serial dilutions of Cs or CsB (250–10 μg/mL) for 24, 48 and 72 h. Cell viability was assessed using the MTT assay as previously reported [v/v) N,N-dimethylformamide, 20% (w/v) SDS with an adjusted pH of 4.5. The optical density (OD) of each well was measured with a microplate spectrophotometer  equipped with a 620 nm filter. Cell viability in response to treatment with Cs and CsB, was calculated as: % dead cells = ((OD treated/OD control) × 100).Macrophages were plated on 96-well plates (8 × 10reported . Briefly2DCF-DA) as previously reported [2DCF-DA is a non-fluorescent permeant molecule that passively diffuses into cells, where the acetates are cleaved by intracellular esterases to form H2DCF and thereby trapped it within the cell. In the presence of intracellular ROS, H2DCF is rapidly oxidized to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). Briefly, J774A.1 (3.0 × 105/well) and mouse peritoneal macrophages (3.0 × 105/well) were plated into 24-well plates and treated with Cs, or CsB (250–10 μg/mL) and LPS as described above, for 24 h. Cells were then collected, washed twice with phosphate buffer saline (PBS) buffer and then incubated in PBS containing H2DCF-DA (10 µM) at 37 °C. After 45 min, cells fluorescence was evaluated using a fluorescence-activated cell sorting  and elaborated with Cell Quest software .ROS formation was evaluated by means of the probe 2′,7′-dichlorofluorescin-diacetate (Hreported . H2DCF-D5/well) were seeded on coverslips in 12 well plate and treated with Cs or CsB (150–50 µg/mL) and LPS (1 µg/mL), as described above, for 20 min in order to detect NF-kB nuclear translocation.For immunofluorescence assay, J774A.1 cells  for 1 h at room temperature. The slides were then washed with PBS for three times and fluorescein-conjugated secondary antibody (FITC) was added for 1 h, DAPI was used for counterstaining of nuclei. Coverslips were finally mounted in mounting medium and fluorescent images were taken under the Laser Confocal Microscope  .4/dish) and allowed to adhere for 4 h. Thereafter, the medium was replaced with fresh medium alone or containing serial dilutions of Cs or CsB (250–10 μg/mL) alone and then co-exposed to LPS (1 μg/mL) for further 24 h to detect nitrite (NO2−), iNOS, COX-2 and HO-1 expression and nitrotyrosine formation. NO production was measured as NO2−, index of NO released by cells, in the culture medium 24 h after LPS stimulation by Griess reaction, as previously reported [Macrophages J774A.1 were plated in 96 well plate (5.0 × 10reported .w/v) sulphanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphtylethylenediamine-hydrogen chloride (HCl) and incubated at room temperature for 10 min, and then the absorbance was measured at 550 nm in a microplate reader Titertek . The amount of NO2−, as μM concentration, in the samples was calculated by a sodium NO2− standard curve. In order to evaluated the iNOS, COX-2 and HO-1 expression and nitrotyrosine formation were evaluate by cytofluorimetry, cells were then collected, washed twice PBS and then incubated in fixing solution for 20 min at 4 °C and then incubated in Fix Perm Solution for 30 min at 4 °C. Anti-iNOS, anti-COX-2, anti-HO-1 and anti-nitrotyrosine (Millipore) were then added for further 30 min. The secondary antibody was added in Fix solution and cells fluorescence was evaluated using a fluorescence-activated cell sorting  and elaborated with Cell Quest software as previously reported [Briefly, 100 mL of cell culture medium were mixed with 100 mL of Griess reagent~equal volumes of 1%  and LPS (1 μg/mL) for 18 h, as previously described, were assessed by an Enzyme-Linked Immuno Sorbent Assay (ELISA) assay by using a commercial kit, for murine TNF-a or IL-6, according to manufacturer’s instruction .4/well). Peritoneal macrophages were allowed to adhere for 24 h at 37 °C in a 5% CO2 atmosphere before experiments. Thereafter the medium was replaced with fresh medium and cells were treated with Cs and CsB (250–10 μg/mL) then exposed simultaneously to LPS (1 µg/mL) as reported above. NO, iNOS, COX-2, nitrotyrosine, ROS and HO-1 and formation evaluation was detected as reported for macrophages.Mouse peritoneal macrophages were plated into 96-well plates  values of at least three independent experiments. Statistical analysis was performed by analysis of variance test, and multiple comparisons were made by Bonferroni’s test. A Citrus sinensis extract. Bioavailability studies were carried out by simulated GI digestion and Caco-2 cells permeation model. Data highlighted how most of the flavonoids are already bioavailable after the salivary and gastric stages, whereas the intestinal environment is mainly responsible for their degradation, in accordance with previous studies [Citrus sinensis juice, that were mainly based on the antioxidant protection provided in vitro, revealing the positive rather than negative influence of digestion processes. Further in vivo studies are necessary to explore the bioavailability of the Citrus flavonoids along with their antioxidant and anti-inflammatory effects, but in vitro models have been often well-correlated to animal and human studies [In the present study, we investigated the antioxidant and anti-inflammatory effect of bioavailable flavonoids from  studies . Moreove studies . Thus, i studies .