Purpose. To investigate the correlation between aqueous and serum levels of pigment epithelium-derived factor (PEDF) and macular choroidal thickness in high myopia patients, both with and without choroidal neovascularization (CNV). Methods. Serum and aqueous levels of PEDF were measured by enzyme-linked immunosorbent assay in 36 high myopia patients (36 eyes) with no CNV (non-CNV group), 14 high myopia patients (14 eyes) with CNV (CNV group), and 42 nonmyopia patients (42 eyes) (control group). Macular choroidal thickness was measured by enhanced-depth imaging optical coherence tomography. Results. Aqueous levels of PEDF were significantly higher in CNV group compared with non-CNV (P < 0.001) and control (P < 0.001) groups. Macular choroidal thicknesses were significantly decreased in the non-CNV and CNV groups compared with the control (P < 0.001) group. A statistically significant difference (P = 0.012) was found between the CNV and non-CNV groups. There was a positive correlation between aqueous PEDF and macular choroidal thickness in the non-CNV group (P = 0.005), but no correlation with the CNV group. No correlation between serum PEDF and macular choroidal thickness was detected in the three groups. Conclusion. Variations in aqueous PEDF levels coincide with changes in macular choroidal thickness in high myopia patients with no CNV, while no such relationship exists in high myopia patients with CNV. High myopia, which accounts for 27–33% of all myopia, is a major cause of legal blindness in numerous developed countries worldwide , with a Pigment epithelium-derived factor (PEDF), a secreted 50 kDa glycoprotein belonging to the superfamily of serine protease inhibitors, was first identified in conditioned media of cultured fetal human retinal pigment epithelial (RPE) cells . PEDF noA new technique was recently implemented for improving depth imaging by optical coherence tomography (OCT). This technique—enhanced-depth imaging- (EDI-) OCT—has been shown to produce reliable images of full-thickness choroid . EDI-OCTWhether aqueous levels of PEDF are reflective of stages of chorioretinal atrophy and CNV in high myopia remains to be elucidated. The aim of our study was to determine how changes in choroidal thickness in high myopia contribute to the formation of myopic lesions in CNV. We investigated variations in aqueous and serum levels of PEDF in high myopia patients (with and without CNV) and correlated these variations with macular choroidal thickness. Results are expected to clarify the role of PEDF in the pathophysiology and morphology of high myopia.This observational, comparative, prospective study was carried out at the Tianjin Eye Hospital, Tianjin, China, between August and November 2013. The study conformed to the Declaration of Helsinki tenets for research involving human subjects and was approved by the Institutional Review Board of Tianjin Eye Hospital. Informed consent was obtained from all participants. Patients who had had surgery on both eyes were only enrolled at the time of the first surgery. non-CNV group comprised 36 high myopia patients (n = 36 eyes) (with no CNV) who were in need of cataract surgery. Inclusion criteria included patients with eyes of axial lengths of ≥26.5 mm  [ CNV group comprised 14 high myopia patients (n = 14 eyes) who were in need of intravitreal injections of ranibizumab . Inclusion criteria comprised patients with eyes of axial lengths of ≥26.5 mm, refractive errors of >6 D, and choroidal neovascularization by fundus fluorescein angiography (FFA) and who were 50–70 years of age. Exclusion criteria were the same as those for the non-CNV group. Patients with secondary choroidal neovascular diseases, for example, angioid streaks and ocular trauma, were also excluded. All cases of CNV were confirmed by FFA. The control group comprised 42 normal patients (n = 42 eyes) who were in need of cataract surgery. Inclusion criteria comprised patients with healthy eyes with spherical equivalents of −3D–+3D. Exclusion criteria were the same as for the other groups.Patients were divided into groups based on myopia with and without CNV. Then, 2001) , refractμL) were collected by aspiration into a 1 mL syringe at the start of cataract surgery or before a single 0.05 mL intravitreal injection of ranibizumab. Serum specimens were collected prior to surgery. The levels of PEDF in aqueous humor and serum were measured using enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instructions .Samples of undiluted aqueous humor , refractive error, intraocular pressure (IOP), and axial length; OCT; dilated fundus examination by indirect ophthalmoscopy; and color fundus photographic assessment of myopic maculopathy. FFA was performed on all high myopia patients to confirm the presence or absence of CNV.The scan protocol of the Cirrus OCT  generates a cube of data through a 9 mm line consisting of 4,096 A-scans around the macular region via a HD 5-line raster mode. EDI-OCT protocols have been described elsewhere . Using tAxial length was measured using the Intraocular Lens Master . Refractive error was measured by autorefractometry . IOP was measured by noncontact tonometry . t-test was used to compare two independent groups with normal distribution; the Mann-Whitney U test was used to compare two independent groups without normal distribution; the Kruskal-Wallis H-test was used to compare variables among different groups; Fisher's exact t-test was used to compare noncontinuous variables; and Pearson's correlation test was used to analyze the correlation between aqueous PEDF concentrations, macular choroidal thickness, and serum PEDF concentrations for the three groups. A P < 0.05 was considered statistically significant.Statistical analyses were performed using version 17.0 SPSS software . All data were described as mean ± standard deviation (SD), with a 95% confidence interval (CI). An unpairedP < 0.001). Diffuse chorioretinal atrophy was present in 28 of 36 (77.8%) highly myopic eyes in the non-CNV group.P = 0.001) and were significantly elevated in the CNV group (17.0 ± 5.8 ng/mL) compared with the other two groups (P < 0.001) . Aqueous levels of PEDF were significantly lower than serum levels of PEDF in all three groups (P < 0.001). Mean macular choroidal thicknesses were 230.6 ± 81.8, 111.1 ± 45.0, and 77.2 ± 26.9 μm in the control, non-CNV, and CNV groups, respectively. Differences were statistically significant for the non-CNV and CNV groups compared with the control group (P < 0.001), while a significant difference (P = 0.012) was also found between the non-CNV and CNV groups (Aqueous levels of PEDF were significantly decreased in the non-CNV group (3.6 ± 1.3 ng/mL) compared with the control group (4.8 ± 1.8 ng/mL) (< 0.001) . Mean seV groups .R2 = 0.211, P = 0.005)  . Also no correlation was found between aqueous and serum concentrations of PEDF for the non-CNV and CNV groups . There were also no correlations between aqueous PEDF, serum PEDF, and macular choroidal thickness for the control group.We studied the correlation between aqueous PEDF levels and macular choroidal thickness for the non-CNV group and found a significant, positive correlation (= 0.005) , while n= 0.214) . ConversPathologic myopia with progressive and excessive elongation of the eyeball results in a number of chorioretinal changes, including posterior staphyloma, chorioretinal atrophy, and pathologic CNV. Although the mechanisms underlying the development of chorioretinal atrophy and CNV remain unknown, dysfunctional RPE and RPE retraction—in the direction of radial traction line from its underlying glial tissue—are important contributors .PEDF is synthesized and secreted by RPE and retinal ganglion cells and diffuses into the vitreous and aqueous humors . In highP = 0.001). Ogata et al. also found the mean aqueous PEDF levels in high myopia to be significantly lower than in eyes with cataract alone [In the present study, we found that levels of aqueous PEDF in non-CNV eyes were significantly lower (3.6 ± 1.3 ng/mL) than in the control group ; meanwhile aqueous levels of VEGF were significantly associated with both macular choroidal thickness (P < 0.001) and axial length (P < 0.001) [Vascular endothelial growth factor (VEGF) and PEGF are two major cytokines in angiogenesis. VEGF not only is the major stimulator of neovascular growth and vascular permeability, but also maintains normal functions in many normal adult tissues. The aqueous level of VEGF has been analyzed in high myopia in another study of our group. We found aqueous levels of VEGF from non-CNV high myopia patients were significantly lower compared with those from control persons (< 0.001) . Interes< 0.001) .P < 0.001). Ogata et al. demonstrated increased expression of both VEGF and PEDF in retinas with experimentally induced CNV [Aqueous levels of PEDF were significantly increased in the CNV group compared with the non-CNV and control groups . In addition, Ikuno et al. reported that eyes with myopic CNV have a thinner choroid in comparison to the contralateral eye without CNV [P = 0.012) between highly myopic eyes with CNV and those with no CNV. Ikuno et al. also found choroidal thinning to be more prominent in eyes with myopic CNV compared with non-CNV eyes [In our study, macular choroidal thicknesses were 111.1 ± 45.0 and 77.2 ± 26.9 hout CNV . Chung ehout CNV . We founCNV eyes . The assCNV eyes .μm every 10 years [To ensure consistency and to minimize any potential influence from diurnal fluctuation, we measured choroidal thickness between 9:00 a.m. and 12:00 p.m. Studies have shown the choroid to be thickest near midnight and thinnest near noon . Using r10 years . Thus, iAlthough the primary regulatory role of the choroid in ocular physiology is well known, the in vivo clinical association of choroidal thickness with aqueous PEDF levels is unclear. We examined scatterplots of aqueous PEDF levels and macular choroidal thickness and found positive correlations for the non-CNV , but notSeveral limitations of our study should be mentioned. First, patients undergoing cataract surgery may differ from patients in general or high myopia patients and, accordingly, it is unclear whether the patients included in our study were representative of normal and high myopia patients. Second, choroidal thickness measurements were performed manually, as there exists no automated device for such measurements. In the present study, two masked readers performed measurements, with open negotiation if there was a difference of >15%. Third, RPE autofluorescence was not evaluated but may be an interesting variable to add to future studies.In summary, variations in aqueous PEDF levels coincide with changes in macular choroidal thickness in high myopia patients with no CNV, while no such relationship exists in high myopia patients with CNV. Our findings suggest that in vivo macular choroidal thickness may be indicative of aqueous PEDF concentration in high myopia with no CNV, but no indication in high myopia with CNV. Nevertheless, based on previous studies showing that age, refractive error, and/or axial length could significantly influence choroidal thickness, the use of macular choroidal thickness as an indirect assessment of aqueous PEDF concentration might be of limited clinical application. Large-scale studies are recommended, particularly to examine the potential prognostic value of aqueous PEDF levels and choroidal changes.


Garcinia kola Heckel (Guttiferae) leaves have received limited scientific attention despite their traditionally acclaimed medicinal properties. The scavenging ability of the methanolic leaf extract (MLE) of G. kola was assayed for hydroxyl radical (OH•), superoxide anion (O2−), 1,1-diphenyl-2-picrylhydrazyl (DPPH), azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS•+), and lipid peroxidation (LP) activity in egg yolk, rat liver, and brain homogenates. Total phenolic and flavonoid contents of the extract were also evaluated. Group I animals were given oral doses of water, whereas Group II and Group III animals received 100 and 200 mg/kg body weight (bw) MLE, respectively, for 14 days. Plasma glucose, magnesium, γ-glutamyltransferase (GGT/γGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and urea were evaluated. Hepatic reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD), LP, and liver histopathological appearance were also assessed. The extract scavenged OH•, O2−, DPPH, and ABTS•+ and inhibited LP in egg yolk, rat liver, and brain homogenates. Furthermore, oral administration of the extract showed no adverse effects on hepatic and renal function tests. Increased hepatic GSH and nonsignificant changes in LP, GPx and SOD activities, and liver histology were observed. These results suggest that G. kola leaves have antioxidant activities which may have application in traditional medicine. Medicinal plants have been used as folklore remedies over the years to treat and manage human ailments. A large body of scientific evidence has been collated to show their immense potential in various traditional cultures . They coGarcinia kola, a medium-sized tree found in most forests, belongs to the family Guittiferae and is commonly referred to as bitter kola. G. kola has been popularized as a “wonder plant” among South Western Nigerians because every part of it has been found to be of some medicinal value [ G. kola is commonly used in folklore remedies for the treatment of ailments such as liver disorders, hepatitis, diarrhoea, laryngitis, bronchitis, and gonorrhoea [ G. kola [ G. kola seeds has been widely reported, including hepatoprotective effects [ in vitro antioxidant potential of the MLE of the leaf of the plant and its in vivo effects in male albino rats.al value . G. kolanorrhoea . It contnorrhoea . Garcini G. kola . The the effects  and trea effects , 10. Eve4), thiobarbituric acid (TBA), sodium dodecyl sulfate (SDS), Folin-Ciocalteu reagent, glacial acetic acid, potassium ferricyanide, quercetin, gallic acid, ascorbic acid, azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS+), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were procured from Sigma Co.  USA. All other reagents used were of analytical grade.Trichloroacetic acid (TCA), ferrous sulphate (FeSOhttp://www.nap.edu). Rats weighing between 180 and 200 g were used for this investigation. The animals were fed with rat chow pellets and water ad libitum and maintained under a 12 h light and 12 h dark photo cycle throughout the period of the experiment.All experiments involving rats were performed in accordance with prescribed ethics of animal use and investigator obligations  were exhaustively extracted with 3000 mL of 70% methanol. Methanolic leaf extract (MLE) was concentrated using a rotary evaporator. The percentage yield of the extract was calculated asThe powdered leaves of G. kola. Blood was obtained through heart puncture and collected into heparinized tubes and the livers of the rats were also removed. A small portion of the liver was fixed in 10% formalin for histological examination. The remaining part of the liver was washed in 1.15% KCl and blotted dry. Rat liver homogenates were prepared in potassium phosphate buffer  using a homogenizer fitted with a Teflon pestle. The homogenates were centrifuged at 9,000 g for 10 min to obtain the postmitochondrial fraction (PMF) which was used to evaluate reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), and lipid peroxidation.Rats for this study were randomized into three groups of six rats each. Groups I, II, and III were given distilled water and extract 100 mg/kg and 200 mg/kg body weight, respectively, through oral gavage for 14 consecutive days. All the animals were sacrificed by cervical dislocation 24 hr after the last exposure to the MLE ofThe hydroxyl radical scavenging capacity of the MLE was evaluated according to an established method of Halliwell and Grootveld . SuperoxγGT, ALT, and AST were determined with kits from Chemelex, S.A. 08420 Canovelles, Barcelona (Spain). Activities of rat hepatic GSH, SOD, and GPx were determined as previously described [The manual methods described by Cheesbrough  were useescribed , Misra aescribed , respectescribed .P < 0.05. The 50% inhibitory concentration (IC50) of MLEs was estimated from regression analysis of concentration-inhibition curves using GraphPad Prism version 5.02 statistical software . In this study, the IC50 value is defined as the concentration of extract required to scavenge 50% free radicals and is inversely proportional to the activity of the extract.Data are expressed as mean ± standard deviation (SD) from the 6 rats in each group. The results were analysed statistically using one-way analysis of variance (ANOVA) followed by Dunnett's test. The minimum level of significance was fixed at The weight of the dried methanolic leaf extract obtained was 223.5 g. The percentage yield of the extract was 22.35%. G. kola showed inhibition of hydroxyl radical scavenging activity with an IC50 value of 748.0 μg/mL compared with an IC50 value of 45.2 μg/mL for quercetin  produced an IC50 value of 40.6 μg/mL. FeSO4-induced lipid peroxidation in rat brain homogenate was similarly inhibited by the extract in a dose-dependent manner as shown in 50 value of 228.0 μg/mL while ascorbic acid showed IC50 value of 36.5 μg/mL.t manner . The IC5 G. kola expressed as μg/mg quercetin equivalent was 0.902 while total phenol content expressed as μg/mg gallic acid equivalent was 1.196.Total flavonoid content of the MLE ofHematological parameters are shown in P < 0.05) decreases in plasma creatinine and magnesium occurred while a significant (P < 0.01) decrease in plasma glucose at 200 mg/kg extract was observed.Biochemical parameters are presented in Administration of the extract to rats caused an increase in GSH levels compared to the control group . The perP < 0.05) increases in the activity of GPx by 1.08% and 7.83%, respectively, compared with the control group (The extract at both doses (100 mg/kg and 200 mg/kg) did not induce significant changes in hepatic SOD activities compared with the control group. However, the extract at 100 and 200 mg/kg showed significant (ol group . The extol group . The res3+)-EDTA, H2O2, ascorbic acid, and deoxyribose. The extent of inhibition of deoxyribose degradation in the presence of an agent gives an indication of hydroxyl radical scavenging ability. The MLE of G. kola exhibited dose-dependent inhibition of hydroxyl radical formation  to a blue formazan product that can be measured spectrophotometrically at 560 nm. The superoxide anion scavenging property exhibited by G. kola may be due to neutralization of O2− radical via hydrogen release and reduction of NBT by various phytochemical constituents in the extract. The inhibition of superoxide anion by the MLE of G. kola leaf may be of biological significance in terms of the cellular damage attributed to superoxide anion radical.Superoxide anion is a relatively weak oxidant generated in aerobic cells with the ability to transform into more reactive species such as H and DNA , and has and DNA . The rib G. kola and ascorbic acid exhibited DPPH radical scavenging activities in a concentration-dependent manner. The DPPH scavenging activity of the extract may be due to its ability to donate hydrogen ions to stabilize free radicals. Thus, the hydrogen donating property is central to the neutralization of free radicals that initiate oxidation processes and termination of radical chain reaction [1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) reacts in the presence of antioxidants to form 1,1-diphenyl-2-picryl hydrazine. The degree of discoloration from deep purple to yellow indicates the extent of radical scavenging potential of the antioxidant . The MLEreaction .•+ radical reactions involve electron transfer and take place at a faster rate compared with DPPH radicals. ABTS•+ generation involves direct production of a blue/green chromophore through reaction between ABTS and potassium persulfate to give an absorbance of 0.750 at 734 nm in 0.1 M sodium potassium buffer (pH 7.4). The MLE of G. kola showed remarkable concentration-dependent inhibition of ABTS•+ which shows that the extract can participate in electron transfer, in addition to its H-donating property as found in DPPH radical scavenging.ABTS G. kola on egg yolk, rat liver and brain homogenates in the presence of FeSO4, a ROS-generating system. The extract showed significant inhibition of lipid peroxidation in all the tissue preparations tested. The inhibition of lipid peroxidation exhibited by the extract, though lower compared to the standard used, could be attributed to the presence of phytochemicals present in the leaves. This implies that the extract has potential to protect the lipid membrane constituents of liver, brain, and egg from Fe3+-induced lipid peroxidation.Malondialdehyde is formed by oxidation of polyunsaturated fatty acids during the lipid peroxidation process and reacts with two molecules of thiobarbituric acid to produce a pinkish red chromogen . This st G. kola leaves in terms of quercetin equivalent was 0.90 ± 0.07 μg/mg QE and total phenol was 1.20 ± 0.07 μg/mg GAE. The antioxidant activities observed in this study may relate to the flavonoid and phenolic constituents in the leaf extract.The antioxidant properties of vegetables and fruits are significantly correlated with flavonoids, a class of secondary plant phenolics . The flaHematological parameters are used to assess the effects of xenobiotics such as plant extracts on the well-being of animals . HematolPlasma AST, ALT, and GGT evaluated in this study show no significant difference in the treated groups compared to the control group. This shows that the MLE at both doses did not induce significant adverse effects on the integrity of hepatic cell membrane. AST, ALT, and GGT are enzymes that are released into circulating blood when the structural integrity of hepatic cells is compromised and therefore are used as biomarkers to evaluate hepatocellular insult . There wγ-glutamyl-cysteinyl-glycine or glutathione), a water-soluble tripeptide containing a thiol group, is a potent reducing agent [ G. kola significantly (P < 0.05) increased hepatic GSH concentration by 13.7% and 20.1% at 100 and 200 mg/kg, respectively. Thus, the leaf extract may modulate glutathione metabolism via secondary plant metabolites (bioflavonoids and xanthones).GSH  in GPx activities were observed in rats treated with the extract at 100 and 200 mg/kg body weight, which shows that the extract at both doses did not adversely affect the activity of GPx. Lipid peroxidation is a chain reaction initiated by free radical through the oxidation of polyunsaturated fatty acid. Malondialdehyde is an accumulated product of peroxidation of long chain fatty acids [ G. kola at both doses (100 and 200 mg/kg) lowered the formation of malondialdehyde in rat liver homogenates compared with the control. This result implies that the leaf extract at both doses maintains hepatic cell integrity.Glutathione peroxidase (GPx) is a selenoenzyme responsible for the elimination of hydrogen peroxide and singlet oxygen . Signifity acids . Lipid pty acids . MethanoHistopathological analysis of formalin fixed and paraffin-embedded liver sections shows that the extract at both concentrations (100 and 200 mg/kg) was well tolerated by the hepatic tissues of rats. in vitro and in vivo effects observed in this study might be linked to the reported phenolic compounds in MLE of G. kola. The mechanisms of the in vitro antioxidant and hypoglycaemic effects of the leaf extracts should be studied further in cell culture systems and animal models to enhance our understanding of the physiological significance of our findings and to clarify the usefulness of the G. kola leaves in traditional medicine practices.The


Chrysanthemum morifolium) breeding; however, cross barriers always exist in these combinations. Many studies have shown that paternal chromosome doubling can often overcome hybridization barriers during cross breeding, although the underlying mechanism has seldom been investigated.Cross breeding is the most commonly used method in chrysanthemum (C. morifolium (pollen receptor) × diploid C. nankingense (pollen donor) and C. morifolium × tetraploid C. nankingense. Seeds were obtained only from the latter cross. RNA-Seq and isobaric tags for relative and absolute quantitation (iTRAQ) were used to investigate differentially expressed genes and proteins during key embryo development stages in the latter cross. A previously performed cross, C. morifolium × diploid C. nankingense, was compared to our results and revealed that transcription factors , hormone-responsive genes (auxin-binding protein 1), genes and proteins related to metabolism  and other genes reported to contribute to embryo development  had higher expression levels in the C. morifolium × tetraploid C. nankingense cross. In contrast, genes related to senescence and cell death were down-regulated in the C. morifolium × tetraploid C. nankingense cross.In this study, we performed two crosses: The data resources helped elucidate the gene and protein expression profiles and identify functional genes during different development stages. When the chromosomes from the male parent are doubled, the genes contributing to normal embryo developmentare more abundant. However, genes with negative functions were suppressed, suggesting that chromosome doubling may epigenetically inhibit the expression of these genes and allow the embryo to develop normally.The online version of this article (doi:10.1186/s12864-016-2939-0) contains supplementary material, which is available to authorized users. Triticum aestivum) is a universally acknowledged textbook example of an allohexaploid derived through hybridization between a domesticated forms of the tetraploid T. turgidum ssp. dicoccoides and the diploid Aegilops tauschii  showed altered expression  involved in cell enlargement and plant growth reported to be regulated by ARF were down-regulated in the abnormal embryos when compared with the normal embryos  was performed on 29 randomly selected genes with differential expression levels. The differential expression patterns in the three samples, and the expression trend of almost all of the genes were consistent with the sequencing data Fig. . Most ofC. morifolium × diploid C. nankingense cross, that might enhance chrysanthemum embryo development, including transcription factors, energy metabolism-related genes and other genes that might function in embryo development. Then, the same comparative analysis was applied to the NE18 libraries. A total of 22 candidates (Table C. morifolium × tetraploid C. nankingense) than in cross II (C. morifolium × diploid C. nankingense). Conversely, genes related to senescence and programmed cell death were down-regulated , a total of 23 differentially expressed proteins (DEPs) (Table In the embryos from cross I (s) Table  were ideC. morifolium × diploid C. nankingense), peptides were searched in a proteomic library. As a result, 10 proteins were aligned with the same peptides, and their expression levels were variable. The proteins associated with energy metabolism, such as pyruvate kinase (gi|92870921), transitional endoplasmic reticulum ATPase (gi|326527541) and cytosolic nucleoside diphosphate kinase (gi|73808794), had significantly higher NE18/AE18 ratios in cross I compared with those of cross II  mutant, the embryos develop abnormally after the globular embryo stage because of a failure of cell elongation .After RNA extraction and DNase I treatment, mRNA was isolated using magnetic beads with Oligo (dT) and fragmented in mixed fragmentation buffer. Then cDNA was synthesized using the mRNA fragments. Short fragments were purified and resolved with EB buffer for end repair and single nucleotide A (adenine) addition. Then, the short fragments were connected with adapters, and the suitable fragments were selected for PCR amplification. The Agilent 2100 Bioanalyses and ABI StepOnePlus Real-Time PCR System were used in quantification and qualification of the sample library . At lasthttp://sourceforge.net/projects/tgicl) was used for de novo assembly of the data by Trinity. This program can splice and remove redundant unigenes from each sample’s assembly to acquire non-redundant unigenes that are as long as possible. Then, the unigenes were divided into two classes by gene family clustering. The first clusters were denoted by the prefix CL and the suffix indicating the cluster number containing several similar unigenes (more than 70 %) in one cluster. The other cluster contained singletons with the prefix representing the unigene.The raw reads produced from sequencing contain adapters and unknown or low-quality bases. Therefore, obtaining clean reads by removing the unqualified reads is necessary. Then, transcriptome de novo assembly was performed with Trinity software . The TGIIn the final step, a BlastX alignment  was performed between databases such as NR, Swiss-Prot, KEGG and COG, and the directions of the unigenes were decided according to the best alignment results. If the results conflicted in the different libraries, the sequence direction was determined by the priority order of NR, Swiss-Prot, KEGG and COG. If a unigene was not aligned to any of the above databases, the software ESTScan  was usedhttp://www.geneontology.org) annotations of the unigenes based on the NR annotations. After GO annotations were obtained for each unigene, WEGO software [First, unigene sequences were aligned to protein databases including NR, Swiss-Prot, KEGG and COG by BlastX and to the nucleotide database NT by BlastN. Then, the putative functional annotations of the unigenes were retrieved. Next, we used the Blast2GO program  to obtaisoftware  was usedTo predict unigene expression levels in different samples, we calculated the unigene expression levels using FPKM . After tIn the GO functional analysis, all of the DEGs were mapped to each term of the GO database and the gene numbers associated with each GO term were calculated. After obtaining a gene list and gene numbers for each included GO term, we used the hypergeometric test to identify significantly enriched GO terms in the DEGs compared to the genome background. In KEGG pathway analysis, pathway enrichment analysis identifies significantly enriched metabolic pathways or signal transduction pathways that involve the DEGs compared with the whole genome background.Elongation Factor 1a (EF1a) gene, which is stably expressed in chrysanthemum [−ΔΔCt method [To ensure the libraries’ reliability, we randomly selected 29 differentially expressed genes and validated the data by qRT-PCR using three biological replicate samples. The qRT-PCR assays were conducted as described by Song et al  on a Masanthemum , 46. Relt method .3/50 mg, Waters Corporation, Milford, MA, USA) to remove the salt buffer and re-dissolved in a low salt buffer [For each sample, approximately 0.5 g of ovules was used for protein extraction by the trichloroacetic acid/acetone method . The extt buffer . Then, tt buffer  for elut4HCO2 in 90 % acetonitrile, pH 10.0, adjusted with NH4OH). The peptides were eluted from the LC column at a flow rate of 200 μL/min. A total of 15 fractions were collected and dried in a vacuum concentrator for the next step.Next, the peptide mixture was re-dissolved in buffer A  and fracMass spectrometric analysis of the iTRAQ-labeled samples was performed on a Nano Aquity UPLC system  connected to an LTQ Orbitrap XL mass spectrometer  equipped with an online nanoelectrospray ion source . The detailed steps were described previously .p-value < 0.05, and the proteins identified with mass tag changes ratio must be ≥ 1.3 or ≤ 0.75.Protein identification and quantification for the iTRAQ experiment were performed with ProteinPilot software version 4.0 (Applied Biosystems) . The resAE18, abnormal embryo at 18 DAP; ARF, auxin response factor; COG, clusters of orthologous groups of proteins; DAP, days after pollination; DEGs, differentially expressed genes; DEPs, differentially expressed proteins; FPKM, fragments per kb per million fragments; GO, gene onotology; KEGG, Kyoto Encyclopedia of genes and genomes; NE12, normal embryo at 12 DAP; NE18, normal embryo at 18 DAP; qRT-PCR, quantitative real-time PCR; TCA, tricarboxylic acid cycle; TEM, transmission electron microscopy


Cryptosporidium spp., Giardia spp., and members of Microsporidia are enteropathogenic parasites of humans and animals, producing asymptomatic to severe intestinal infections. To circumvent various impediments associated with current detection methods, we tested a method providing multistage purification and separation in a single, confined step. Standard real-time PCR was used as a detection method. Samples spiked with C. parvum and G. intestinalis were split for comparison to standard Method 1623. Results were equivalent to immunomagnetic procedures for Cryptosporidium, and Giardia. Overall percent recovery for Cryptosporidium with Method 1623 averaged 26.89%  and was similar but less variable for qPCR method at an estimated average of 27.67 . For Giardia, Method 1623 had an overall average recovery of 27.11% , while multistage purification and qPCR had an estimated lower overall recovery at 18.58% . Microsporidia were also readily detected with an estimated recovery of 46.81% overall  for E. intestinalis and 38.90%  for E. bieneusi. Cryptosporidium, Giardia, and Microsporidia are enteropathogenic parasites of humans and animals, producing asymptomatic to severe intestinal infections [ Cryptosporidium and Giardia. These methods are hampered by cost and their time consuming nature [ Cryptosporidium and Giardia species [ Cryptosporidium and Giardia, and though Microsporidia and other waterborne pathogens are listed in various contaminate lists, no cost and time-effective method for their detection exists.fections , 2. Detefections . Currentg nature  and requ species . Problem species  and poor species . These mIt is well known that molecular techniques have been developed that are more effective than immunofluorescence microscopy in detecting specific pathogens . NotablyTo circumvent problems associated with IMS, a basic method combining filter dissolution and separation of targets in a centrifugation device (CFD) was evaluated. The procedure was coupled with downstream qPCR for a rapid, cost-effective approach to detecting these pathogens. This report describes the procedure and presents data regarding sensitivity and efficacy in a comparative evaluation to use of IMS microscopy performed in a NELAC certified laboratory. Cryptosporidium parvum oocysts (CpAZ strain) and G. intestinalis  cysts (gamma-irradiated) used for spiking suspension in all trials (Method 1623 and qPCR) and standards (qPCR). Pathogens were flow cytometry-sorted and suspended in 0.75 mL of reagent water with 0.01% Tween 20, which had been inactivated and preserved . Encephalitozoon intestinalis were supplied as purified suspensions in phosphate-buffered (PBS) solution  and Enterocytozoon bieneusi spores were supplied as unpurified suspensions . All oocysts, spores, and cysts were purchased 3-4 weeks prior to trials and stored at 4°C. All parasites are evaluated for general quality and intactness under differential interference contrast microscopy.Inactivated C. parvum oocysts and G. intestinalis cysts . To standardize turbidity treatments and to ensure the absence of pathogens in water samples prior to spiking, water sample volumes having turbidities > 1 were created by adding a purified loam of soil to Milli-Q purified water . Soil was autoclaved twice at 121°C for 20 min at 24 hr intervals prior to mixing with water. Water used for spiking with turbidity standards < 1 NTUs (potable water) was provided at the City of Tampa Water Department (Biol. Dept).For a comparative evaluation with IMS-microscopy , bulk v C. parvum and G. intestinalis. Prior to spiking, representative aliquots from carboys were tested for turbidity using a turbidimeter . Carboys were spiked with gamma-irradiated C. parvum oocysts and G. intestinalis cysts according to the manufacturer's instructions . Additionally, carboys were spiked with predetermined counts of E. intestinalis and E. bieneusi for evaluation of separation via the CFD and detection using qPCR. Estimates for spiking suspensions and for qPCR standard curves were done via hemocytometer counts. The standard suspensions of Microsporidia were prepared 1 week prior to each trial. Method blanks, in which no pathogens were added, were included in trial 1.Water of varying turbidities was divided into separate polycarbonate carboys (20–25 L). In each of 2 trials, sample water of 3 turbidities (3 replicates/turbidity) was spiked with 100 (oo)cysts each ofSpiked samples evaluated using Method 1623 were filtered through FiltaMax filters (Idexx) with flow volumes recorded using a digital rate meter . Sample volumes evaluated using filter dissolution combined with qPCR were collected on 142 mm mixed cellulose ester  membranes using a filter tower (5 mm Hg vacuum). MCE filters were folded, placed into zip-lock bags, and labeled accordingly. Filters were transported to UWF on ice and processed within 48 hrs.μm MCE filters which were shipped overnight to UWF. The volumes were not equal but provided additional testing material.Environmental samples were collected from the Pensacola and the Tampa Bay region. A few additional samples were collected from rivers and waste water treatment facilities (WWTF) in St. Petersburg and Auburndale, Florida. Samples from the Pensacola region were collected into polycarbonate carboys and transported back to the lab for filtering and processing. Unused portions of samples processed by routine testing at the City of Tampa Water facility by Method 1623 were used for evaluation via qPCR. Unused volumes were filtered onto 1.2 Samples were processed as described for the IMS microscopy method  at the μm; Nuclepore Track-Etched, Whatman) was placed upon the mesh screen of the lower section  were placed upon mesh supports to remove larger particulates.The study utilized a multitiered centrifugation-filtration  device  which coFor dissolution, the MCE filters were folded using sterile forceps and inserted into the primary chamber . Lysis buffer was added and filters were subjected to 3 freeze-thaw (liquid nitrogen/65°C) cycles. Tubes were agitated using a FastPrep (ThermoSavant) or a PowerLyzer homogenizer (MoBio) using 45 second bursts at setting 4.5 and S3500, respectively. Genomic DNA extraction was done according to the manufacturer's instructions and stored at −20°C. G. intestinalis cysts and C. parvum oocysts consisted of those targeting the β-Giardin gene and the gene for the Cryptosporidium oocyst outer wall protein , 0.50 μM each primer, 0.04 U of FastStart Taq DNA polymerase (Roche), 0.2 mM PCR Nucleotide MixPlus (Roche), 0.025% bovine serum albumin , and 0.3 μM of each hybridization probe. Cycling conditions were consistent with those of Guy et al. 2003. Fluorescence was measured at the end of each cycle. For detection of E. bieneusi spores, primers, and hybridization probe targeting the ITS region of the 18S rRNA gene were utilized [ Encephalitozoon spp.  spores were from Wolk et al. 2002 [ E. bieneusi and Encephalitozoon spp. were performed separately in 15 μL total volumes containing 3.0 mM MgCl2 (Roche), PCR buffer (FastTaq 10x/Green), 0.40 μM each primer, 0.04 U of FastStart Taq DNA polymerase (Roche)/l, 0.2 mM PCR Nucleotide MixPlus (Roche), 0.025% bovine serum albumin , and 0.3 μM of each hybridization probe. Thermal conditions for both reactions consisted of 5 min at 95°C followed by 50 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C. Fluorescence was measured at annealing step of each cycle.Primer-probe sets which were used to detectn . PCRs wutilized . Primersal. 2002 . ReactioA minimum of 8–10 replicate reactions were used for all samples and results pooled. To ensure purity of reagents, “no template controls” (NTCs) were run with every PCR reaction series. Standard suspensions containing 100 and 500 cysts and oocysts and 1000 and 5000 spores were used to develop standard curves. Standards were subjected to gDNA extraction methodology as stated previously. The projected number of pathogens was calculated based on the portion of gDNA analyzed using the following formula: number of pathogens  in each sample =  × /. All runs and data analysis were performed using Rotor-Gene 3000 (Corbett) real-time PCR machines and software. Pathogen presence and species identification in unknowns were verified using electrophoresis on 2% agarose gels and subsequent sequencing  according to manufacturer's instructions. Data were aligned against existing sequences for known pathogens . C. parvum oocysts and G. intestinalis cysts, parallel processing of split samples directly compared Method 1623 and qPCR. Data for the methods comparison are listed and presented  and was similar but less variable for the CFD + qPCR method at an average of 27.67 . For Giardia, Method 1623 had an overall average recovery of 27.11% , while the CFD + qPCR had a lower overall recovery at 18.58% .Using inactivated (gamma-irradiated)resented . Overall E. intestinalis, and E. bieneusi spores were added to samples as well as C. parvum and G. intestinalis. The estimated number of E. intestinalis and E. bieneusi spores detected using CFD + qPCR and associated percent recovery for each sample are listed  for E. bieneusi  is often expensive, time-consuming, and intimidating to those who need it . Most ap Cyclospora cayetanensis). Regardless, a combination of molecular and microscopy-based analyses should ultimately increase effectiveness of waterborne pathogen detection.In the event of large outbreaks, analysis of numerous field samples to provide spatial and temporal assessments is necessary . Arguabl Cryptosporidium spp. oocysts [ Cryptosporidium oocysts and of 63.1% for Giardia cysts have also been reported [ in situ hybridization (FISH) [The purification approach in this study is an adaptation of the cellulose-acetate filter dissolution, having previous reported rates of recovery ranging from 70–79% for the recovery of oocysts , 23. Meareported . The metreported  as well reported  and fluon (FISH) . However C. parvum oocysts were reported to be viable in mice following use of the methodology [Concerns over providing information on the viability and infectivity of protozoan pathogens have been made , 28. Howhodology . These ihodology . In any  G. intestinalis  andoocysts, –35) and oocysts, ; ~50 mL,oocysts, ). C. parvum was detected in a sample of raw sewage using qPCR, and other samples for sewage and sewage treatment plant effluents were positive for Microsporidia by qPCR. E. bieneusi was detected in a few of the samples collected from various freshwater sources. This pathogen is prevalent throughout the world and is found in a wide variety of hosts including pigs, humans, and other mammals [The methodology was successful in detection of pathogens in the spiked samples as well as in environmental samples. mammals , 39. The mammals . Cryptosporidium [ Giardia [μm to 12–15 μm prescreens may improve the overall performance of the method. Nytex was used since, at the time, no stainless steel mesh prescreen was available. Additionally, gamma-irradiated, flow cytometry-sorted C. parvum and G. intestinalis suspensions  were used in order to provide precision with regard to matrix spiking but also to provide a degree of safety during the development and evaluation of the assay. The effect of the gamma-irradiation on the performance of PCR is an unknown factor.The number of samples and analytical replicates affects detection of pathogens in water by both microscopy and PCR . A minimporidium  and Giar Giardia  currentlThis report has demonstrated the capability of combining filter dissolution with qPCR for the direct detection of human enteric pathogens with results that are comparable to IMS microscopy, at substantial reductions in time and expense. Assay times were roughly 3–3.5 hr  from initial filtration of samples to completion of qPCR. The method was straightforward and minimized sample manipulation.


Advances in the knowledge of renal neoplasms have demonstrated the implication of several proteases in their genesis, growth and dissemination. Glutamyl-aminopeptidase (GAP) (EC. 3.4.11.7) is a zinc metallopeptidase with angiotensinase activity highly expressed in kidney tissues and its expression and activity have been associated wtih tumour development.In this prospective study, GAP spectrofluorometric activity and immunohistochemical expression were analysed in clear-cell (CCRCC), papillary (PRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytoma (RO). Data obtained in tumour tissue were compared with those from the surrounding uninvolved kidney tissue. In CCRCC, classic pathological parameters such as grade, stage and tumour size were stratified following GAP data and analyzed for 5-year survival.GAP activity in both the membrane-bound and soluble fractions was sharply decreased and its immunohistochemical expression showed mild staining in the four histological types of renal tumours. Soluble and membrane-bound GAP activities correlated with tumour grade and size in CCRCCs.This study suggests a role for GAP in the neoplastic development of renal tumours and provides additional data for considering the activity and expression of this enzyme of interest in the diagnosis and prognosis of renal neoplasms. Clinical data support the fact that renal cell carcinomas (RCCs) are neoplasms with high prevalence and mortality rates . The 200At present, there is no clinical marker to detect the disease in the asymptomatic potentially curable phase or to reliably predict the clinical course of every case. Only classic pathological parameters such as histological subtype, tumour stage and grade may contribute to that purpose. However, depending on the clinical setting and other circumstances, many renal tumours escape the expected behaviour and this means it is necessary to discover more predictable parameters .Recent findings have revealed the implication of several proteases in the genesis, growth and dissemination of renal neoplasms. Significant effort has been made towards understanding the role of matrix metalloproteinases -7. In paGlutamyl-aminopeptidase (GAP), also known as aminopeptidase A (EC. 3.4.11.7), is a membrane-bound zinc metallopeptidase that removes N-terminal acidic residues from peptides such as angiotenin II, its best known natural substrate . GAP hasImmunoassay and semiquantitative enzymatic studies in renal cell carcinoma cell lines and in primary renal cancers have shown altered GAP expression and activity in these neoplasms ,9,17. Ithttp://www.biobancovasco.org). All patients were informed about the potential use for research of their surgically resected tissues, and accepted this eventuality by signing a specific document approved by the Ethical and Scientific Committees of the Basque Country Public Health System (Osakidetza) (CEIC 11/51).The authors declare that all experiments carried out in this study comply with current Spanish and European Union legal regulations. Samples and data from patients included in this study were provided by the Basque Biobank for Research-OEHUN  areas were obtained in all cases. For activity studies, tissue samples were stored at −80°C until the enzyme assays were performed. In addition, selected tissue samples were formalin-fixed and paraffin-embedded for histopathological studies. The 2004 WHO histological classification of adult renal cell tumours  were useAlthough GAP has been classically reported as a cell-surface peptidase , severalg for 35 min. The resulting supernatants were used to measure cytosolic (soluble) enzyme activities and protein concentrations. To avoid contamination with soluble enzymes, the resulting pellets were washed three times by suspension in 10 mM Tris–HCl buffer at pH 7.4. Pellets were then homogenised in 10 mM Tris–HCl buffer at pH 7.4, and centrifuged at low speed (1500 g) for 3 min to purify the samples. The supernatants thus obtained were used to determine membrane-bound enzyme activities and protein concentrations. All the aforementioned steps were carried out at 4°C.Explanted tissue samples were homogenised in 10 mM Tris–HCl buffer at pH 7.4, for 30 seconds at 800 rpm using a Heidolph PZR 50 Selecta homogeniser, and ultracentrifuged in a Centrikon T-2070 Kontron Instruments apparatus at 100,000 The enzyme activity of tumour and non-tumour tissue samples from 50 patients with CCRCC , 10 patients with ChRCC  and 8 patients with RO  was analysed by spectrofluorometric methods. GAP activity was measured by a modified version of Tobe et al.’s method , using GProtein concentration was measured in triplicate by the Bradford method , using BFormalin-fixed and paraffin-embedded tumour tissue from 12 CCRCC ; 6 PRCC , 4 ChRCC  and 4 RO  were immunostained with a rabbit polyclonal antibody specific for glutamyl-aminopeptidase . The immunostaining process was performed following routine methods in an automatic immunostainer (Dako Autostainer Plus). In short, endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide in absolute methanol for 10 minutes. Antigen retrieval was carried out in citrate buffer  for 15 minutes at 100°C in a microwave oven. The primary antibody was applied for 1 hour at room temperature. A subsequent reaction was performed with secondary antibodies and biotin-free HRP enzyme labelled polymer of the EnVision-Flex detection system . Nonspecific IgG was used as a negative control. A positive reaction was visualized with diaminobenzydine solution followed by counterstaining with haematoxylin. Two independent observers analysed the immunohistochemical slides separately assigning staining intensity according to a semiquantitative scale . Minor disagreements were reconciled under a multihead microscope.ENPEP mRNA was performed to assess the transcription levels of this enzyme. The total RNA of tumour tissue samples from 30 CCRCC patients  was isolated following the standard protocol previously described  and stage [pT1/2 (organ confined) vs. pT3/4 (non-organ confined)]. Spearman’s correlation (ρ) test was performed to evaluate the correlation between GAP activity, patient age and gender, and tumour size of CCRCC. A value of Data obtained in the GAP activity assays across the different tumour types and in stratified CCRCC are reported in Figures P < 0.001), and drastically in ChRCC  and RO . The soluble GAP activity in renal tumours .Figure Table P < 0.05). Analyses of grading for membrane-bound GAP and staging for both soluble and membrane-bound GAP activities were not statistically significant.After stratification by grade, soluble GAP showed higher activity in CCRCCs with a high Furhman’s grade in comparison to those clear cell carcinomas in a lower grade   Figure .P > 0.05)  Figure A and B.Figure ENPEP mRNA levels in CCRCCs with different Fuhrman’s grade [G1/2 (low) vs. G3/4 (high)] and stage [pT1/2 (organ confined) vs. pT3/4 (non-organ confined)]. ENPEP mRNA levels were higher in low grade CCRCCs than in high grade tumours , in a subset of renal tumours and in their non-tumour adjacent tissues. GAP activity of both membrane-bound and soluble fractions was markedly decreased in all tumour subtypes when compared with normal tissues. Interestingly, the decrease in membrane-bound GAP activity revealed a gradient along the different phenotypes of renal neoplasms. Thus, while the activity was twice lower in CCRCC than in normal tissue, the decrease in ChRCC and RO was 16-fold and 34-fold respectively. In addition, we recently demonstrated that this enzyme’s activity decreased 8-fold in PRCC . This fiIn terms of immunohistochemistry, GAP was strongly expressed in the membrane of proximal tubule cells, whereas tumours from this origin showed only a mild membrane positivity (CCRCC) or even very weak staining (PRCC). This result is in accordance with previous works ,9 and suOur previous studies demonstrated that several aminopeptidase activities are significantly increased in high stage and high grade CCRCCs ,12,29,31GAP mRNA levels in renal tumours are similar or even higher than in normal renal tissue ,11. In tThe exact role that GAP plays in renal neoplasms still remains to be clarified. This enzyme has been reported to play a functional role as a regulator of angiotensin II-mediated tumour growth and invasiveness via conversion of locally produced angiotensin II (ang II) in several solid tumours . The dowGAP has been commonly described as a membrane-bound peptidase, but the activity of soluble isoforms has also been reported in normal and tumour tissues -23. Our In this context, the role of soluble peptidases in the regulation of proliferative diseases is less clear than that of membrane-bound peptidases. Nevertheless, recent findings suggest that a number of peptides and hormones, commonly called “intracrine”, act in the intracellular space after either internalisation or retention in their cells of synthesis ,45. FurtThis study demonstrates marked decreases in activity and mild to weak expression of GAP in the four most common histological subtypes of renal neoplasms , and a positive correlation between the soluble GAP and CCRCC aggressiveness. These results favour the possibility of a metabolic imbalance among intrarenal angiotensins and a role of GAP in renal neoplastic diseases. A better understanding of the pathophysiological role of GAP in these proliferative disorders will be helpful for designing effective diagnostic, prognostic and therapeutic tools for renal neoplasms.GAP: Glutamyl-aminopeptidase; RCCs: Renal cell carcinomas; CCRCC: Clear cell renal cell carcinoma; PRCC: Papillary renal cell carcinoma; ChRCC: Chromophobe renal cell carcinoma; RO: Renal oncocytoma; UP: Units of peptidase, pmol of product/min.All the authors declare no competing interests.GL, LB and JIL: research conception and design, edition, revision and approval of the final version. GL, LC and JG: statistical analysis. LB, CES, BS and IP: sample preparation, enzyme assays and protein determinations. FMP, MLC, LB and IP: qRT-PCR assays. JIL: provision of the surgical material and clinical follow-up data of the patients. JIL: selection of appropriate material for immunohistochemical analysis and interpretation of the obtained results. BS and IP: prepared figures and table. GL, LC, LB and JIL: collection and interpretation of final results. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/386/prepub


Backgrounds. VitB6 deficiency has been associated with a number of adverse health effects. However, the effects of VitB6 in metabolic syndrome are poorly understood. Methods. VitB6 (50 mg/kg/day) was given to Apoe−/− mice with hkdigh-fat diet (HFD) for 8 weeks. Endothelial dysfunction, insulin resistance, and hepatic lipid contents were determined. Results. VitB6 administration remarkably increased acetylcholine-induced endothelium-dependent relaxation and decreased random blood glucose level in Apoe−/− mice fed with HFD. In addition, VitB6 improved the tolerance of glucose and insulin, normalized the histopathology of liver, and reduced hepatic lipid accumulation but did not affect the liver functions. Clinical and biochemical analysis indicated that the levels of VitB6 were decreased in patients with fatty liver. Conclusions. Vitamin B6 prevents endothelial dysfunction, insulin resistance, and hepatic lipid accumulation in Apoe−/− mice fed with HFD. Supplementation of VitB6 should be considered to prevent metabolic syndrome. Vitamin B6 (VitB6) includes pyridoxal, pyridoxine, and pyridoxamine, which function as essential cofactors for enzymes involved in various metabolic activities, which include amino acid, fat, and glucose metabolism . The phoNutrient overload is associated with high incidence of chronic metabolic diseases, including obesity, insulin resistance, and type 2 diabetes . ProlongApoe−/− mice fed with high-fat diet. Clinically, deficiency of VitB6 should be considered as a high risk factor of NAFLD.The identification of the link between VitB6 and metabolic syndrome including insulin and NAFLD might help to define novel nutritional and pharmacological approaches for the treatment of diabetes, obesity, and insulin resistance. Here, we reported that administration of VitB6 prevents endothelial dysfunction, insulin resistance, and hepatic lipid accumulation in β (GSK3), forkhead box protein O (FOXO), and GAPDH were purchased from Santa Cruz Biotechnology . The secondary antibodies were obtained from Jackson ImmunoResearch Laboratories . VitB6, acetylcholine (ACh), sodium nitroprusside (SNP), and phenylephrine were from Sigma-Aldrich Company. All drug concentrations are expressed as final working concentrations in the buffer.Human recombinant insulin was purchased from Sigma-Aldrich . Antibodies against Akt (pAkt), GLUT4, glycogen synthase kinase-3Apoe−/− mice were purchased from Hua-Fu-Kang Animal Company . All animals were housed in temperature-controlled cages with a 12-hour light-dark cycle and given free access to water and normal chow. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.Male Model of hyperlipidemia was induced by feeding mice with HFD containing 0.21% cholesterol and 21% fat . This diet was administered at 6 weeks of age and continued for 8 consecutive weeks. At 6 weeks of age, VitB6 (50 mg/kg/day) was also added to the drinking water for 8 weeks. The animal protocol was reviewed and approved by the Animal Care and Use Committee of Hunan Normal University.Blood was sampled from mice for determination of total bilirubin (TB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and albumin (ALB). Serum levels of TB, AST, ALT, AP, and ALB were determined by commercial kits .In vivo or ex vivo organ chamber study was performed as described previously [4, 0.6; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; EDTA, 0.026; pH 7.4 at 37°C; and they were gassed with 95% O2 plus 5% CO2, under tension of 1.0 g, for 90-minute equilibration period. During this period, the Krebs solution was changed every 15 min. After the equilibration, aortic rings were challenged with 60 mM KCl. After washing and another 30-minute equilibration period, contractile response was elicited by phenylephrine (1 μM). At the plateau of contraction, accumulative ACh  or SNP  was added to induce the relaxation.eviously . Mice weAs described previously , glucoseμm) were stained with either hematoxylin or eosin. Degree of severity of liver fibrosis was derived from blind analysis of each of the animals in each group. To determine hepatic lipid accumulation, frozen liver sections were stained with 0.5% Oil Red O for 10 min, washed, and counterstained with Mayer's hematoxylin for 45 sec. Data for Oil Red O staining were presented as the mean percentage of stained area to a total hepatic region in 10 fields from each liver section. Quantitative analysis was performed using analySIS-FIVE program .Histological specimens were taken at the end of the study period for all mouse groups as described previously . For eacμg proteins were loaded to SDS-PAGE and then transferred to membrane. Membrane was incubated with 1 : 1000 dilution of primary antibody, followed by 1 : 2000 dilution of horseradish peroxides-conjugated secondary antibody. Protein bands were visualized by ECL . The intensity (area × density) of the individual bands on western blots was measured by densitometry . The background was subtracted from the calculated area. The average of density for the bands in control group is considered as 100%.The protocol for western blot was described as previously with some modifications . Liver tLipids in mouse liver were extracted as described by Folch et al. , 16. Chot-test was used for two groups' comparison. P < 0.05 was considered significant.The results were expressed as mean ± SEM. One-way ANOVA followed by Apoe−/− mice with HFD [Apoe−/− mice dramatically increased serum levels of triglyceride, cholesterol, and LDL, indicating that the model is successfully established. Importantly, the random level of blood sugar was also increased in Apoe−/− mice fed with HFD. However, treatment of these mice with VitB6 did not alter the levels of triglyceride, cholesterol, and LDL, except for random level of blood glucose.Endothelial dysfunction has been identified as an early hallmark of CVD, such as atherosclerosis and hypertension –20. We fwith HFD . As indiThe endothelial function was determined by using ACh. As shown in Apoe−/− mice. As shown in Apoe−/− mice. The peak level of BG was about 600 mg/dL after 30 minutes. The level of BG was back to the basal level after 90 minutes. However, administration of VitB6 delayed and lowered the peak levels of BG (470 mg/dL). After 90 minutes of glucose injection, the level of BG was also back to the basal level. These data indicate that VitB6 increases the tolerance of glucose.Insulin resistance is a high risk factor of endothelial dysfunction in CVD . We nextApoe−/− mice, the levels of BG were reduced to 40% of basal level at the 60th minute and then went back to 80% of basal level at the 120th minute. However, VitB6 further reduced the level of BG at the 90th minute to 25%. After the 120th minute, the level of BG was 55% of the basal level. Collectively, this suggests that VitB6 enhances insulin sensitivity in mice.The protective effect of VitB6 on glucose metabolism was further confirmed by measuring the sensitivity of insulin . By injeApoe−/− mice, the levels of pAkt, GSK3, GLUT4, and BG were increased and the level of FOXO was reduced in HFD-fed Apoe−/− mice with VitB6, further supporting the notion that VitB6 improves insulin resistance in mice.The beneficial effects of VitB6 on insulin resistance were further examined by assaying the hepatic levels of pAkt, GSK3, GLUT4, and FOXO, which are proteins related to glucose metabolism . As depiApoe−/− mice with or without VitB6 treatment. Histological analysis of HE staining in liver sections from mice at the end of the experiment , cystathionine-γ-lyase (CGL), cytoplasmic and mitochondrial serine hydroxymethyltransferase (cSHMT and mSHMT), and glycine decarboxylase (GDC) in the mitochondria [α-ketobutyrate, ammonia, and cysteine. Because both CBS and CGL require PLP as a coenzyme, inadequate VitB6 status might lead to impaired regulation of cellular homocysteine concentration. High levels of homocysteine impair endothelial function and cause metabolic syndrome including insulin resistance and lipid accumulation in liver. HHCY might play a role in the pathogenesis of vascular disorders and is considered as an independent risk factor for atherosclerosis [The major discovery in the present study is that VitB6 produces several beneficial effects to prevent metabolic syndrome, such as insulin resistance and NAFLD. Traditionally, VitB6, in the form of PLP, is the coenzyme of 5 enzymes in these metabolic pathways: cystathionine-chondria . In thisclerosis . From ouWe also identified VitB6 deficiency as a new risk factor of NAFLD. Obesity, metabolic syndrome, and type 2 diabetes mellitus are strictly related and are key pathogenetic factors of NAFLD, the most frequent liver disease worldwide. NAFLD is a clinicopathological syndrome including a wide spectrum of liver damage instances, ranging from hepatic steatosis to nonalcoholic steatohepatitis (NASH) to cirrhosis . EpidemiApoe−/− mouse is suitable for studying atherosclerosis resulting from hypercholesterolemia. Additionally, this mouse has several intriguing characteristics. First, Apoe−/− mice show obesity-resistant phenotype, resulting in remarkable insulin sensitivity. Second, this mouse has hepatic steatosis due to impairment of VLDL secretion from liver. Third, this mouse basically possesses endothelial dysfunction damaged from excess beta lipoprotein. It would be better to investigate the metabolic effects of vitamin B6 on wild-type mice with diet-induced metabolic disorders, such as C57B16 strain.A limitation of this study is that In summary, the results of this study have shown that low VitB6 status has substantial effects on metabolism including glucose and fatty acid. The results of this study also demonstrate that the deficiency of VitB6 might be a risk factor of NAFLD.


After the implementation of the universal salt iodization (USI) program in 1996, seven cross-sectional school-based surveys have been conducted to monitor iodine deficiency disorders (IDD) among children in eastern China.This study aimed to examine the correlation of total goiter rate (TGR) with average thyroid volume (Tvol) and urinary iodine concentration (UIC) in Jiangsu province after IDD elimination.Probability-proportional-to-size sampling was applied to select 1,200 children aged 8–10 years old in 30 clusters for each survey in 1995, 1997, 1999, 2001, 2002, 2005, 2009 and 2011. We measured Tvol using ultrasonography in 8,314 children and measured UIC  and salt iodine  using methods recommended by the World Health Organization. Tvol was used to calculate TGR based on the reference criteria specified for sex and body surface area (BSA).TGR decreased from 55.2% in 1997 to 1.0% in 2009, and geometric means of Tvol decreased from 3.63 mL to 1.33 mL, along with the UIC increasing from 83 μg/L in 1995 to 407 μg/L in 1999, then decreasing to 243 μg/L in 2005, and then increasing to 345 μg/L in 2011. In the low goiter population (TGR < 3.9%), TGR was positively associated with average Tvol (r = 0.99); UIC showed a non-linear association with average Tvol, and UIC > 300 μg/L was associated with a smaller average Tvol in children.After IDD elimination in Jiangsu province in 2001, lower TGR was associated with smaller average Tvol. Average Tvol was more sensitive than TGR in detecting the fluctuation of UIC. A UIC of 300 μg/L may be defined as a critical value for population level iodine status monitoring. Universal salt iodization (USI), defined as the fortification of all salt used for human and animal consumption with iodine, is the main strategy recommended by the World Health Organization (WHO) to control iodine deficiency disorders (IDD) worldwide . Since 1TGR and Tvol vary with iodine intakes among populations , 5. The This study aimed to examine the correlation of TGR with average Tvol and iodine intake in Jiangsu province where USI has been implemented for 16 years.The survey was developed by Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu CDC) and approved by its Ethics Committee. The surveys were performed according to the guidelines laid down in the Declaration of Helsinki. Written informed consents were obtained from school teachers and guardians of each participating student. The Jiangsu CDC conducted all surveys, including field data collection and laboratory measures, with logistic support from local CDCs in selected areas.According to the WHO’s guideline for monitoring IDD in very large populations , we seleThe Tvol of each subject was measured except in 1995. Household salt samples were collected from the houses of the selected children. Casual spot urinary samples of at least 12 students randomly selected from a school (40 children) were collected, with at least two males and two females selected from each age group . Samples of urine and salt were separately collected by different trained personnel to prevent iodine contamination between salt samples and urine samples.The iodine concentration in the salt samples was determined by iodometric titration with sodium thiosulfate using starch as an external indicator , accordiThe iodine concentration in urine (UIC) samples were measured based on the Sandell-Kolthoff reaction (WS/T 107–2006) . Aliquot0.425 × height (cm)0.725 × 71.84 × 10−4.Tvol was determined by real-time echography with Medison mycolor 202  using a 7.5 MHz linear array transducer. Two technicians (QZ and LS), trained by the National Institute of Iodine Deficiency Disorders in Harbin, performed all the measurements. Longitudinal and transverse scans were taken, allowing measurement of the depth, width, and length of each lobe. The volume of the lobe was calculated using the following formula: volume (mL) = 0.479 × depth (cm) × width (cm) × length diameter (cm). The lobe volumes were then summed; the volume of the isthmus was not included. The intra-observer variabilities, evaluated by the different values of our two trained technicians performing repeated measurements to 4% of the samples, were -0.064 to 0.155 and -0.115 to 0.158. The inter-observer variability evaluated by duplicate measurements of approximately 4% of samples was -0.178 to 0.201. The weight and height of each child wearing light clothing without shoes or hats were measured. Body surface area (BSA) was calculated using the following formula: BSA = weight (kg)Median UIC was calculated for each survey as the iodine intake value of each survey. Subjects with missing UIC data were not included in the statistical analysis of UIC and its association with average Tvol. According to the WHO guideline, the term “goiter” refers to a thyroid gland that is enlarged, so goiter was defined using sex- and BSA-specific reference criteria for Tvol . TGR wasTable 1) . The geometric mean value of Tvol with or without BSA-adjustment showed no significant difference in each survey . The geometric mean of Tvol decreased from 3.63 mL in 1997 to 1.97 mL in 2001 and then maintained at approximately 2.10 mL from 2002 to 2005, followed by a remarkable decrease in 2009 to 1.33 mL. Generally, average Tvol decreased over time, with a slight increase observed between 2001 and 2002 and between 2009 and 2011 . There was no significant difference between 2001 and 2002 (P = 0.11) or 2009 and 2011 (P = 0.15), but there was a significant reduction from 2005 to 2009 (P < 0.001).TGR significantly decreased from 1997 to 2011 (P < 0.001) Fig 3 shows a close correlation of average Tvol with TGR  from 2001 to 2011 after IDD was eliminated.We measured 4,767 urine samples for UIC. There were a total of 17 missing UIC data across eight surveys , which leaked during transport. As shown in We observed a non-linear change of average Tvol with UIC , and UICThis study is the first to reveal a positive correlation between average Tvol and TGR in a population after the elimination of IDD. Zimmermann  showed dWe also observed that the fluctuations in average Tvol were associated with UIC if TGR < 3.9% and IDD was eliminated. Tvol with UIC showed a three-stage relationship, and a UIC of 300 μg/L was observed to be an important cut-off point. First, if UIC was less than 300 μg/L (in 2002 and 2005), UIC showed no effects on average Tvol. The result was supported by Zou  and a stThe salt iodine concentration in Jiangsu peaked before 2000. However, no similar exposure was observed in Shenzhen, where the median UIC was 207.1 μg/L in 2011 and 232.2 μg/L in 2009 , or in ZOur results also showed the solid progress toward the implementation of USI in Jiangsu province. Great achievements in USI have decreased the goiter rate from 55.2% to 1%, with the use rate of adequate iodized salt reaching and maintaining approximately 95% . The achThe findings of this study are subject to several limitations. First, we used a cross-sectional design, and the subjects belonged to different birth cohorts, which might cause unquantifiable bias. Second, because we were limited by the relatively high UIC observed throughout the surveys, we could not observe the correlation of average Tvol with UIC < 243 μg/L. In a survey conducted in Shenzhen of China, decreasing TGR was observed when the median UIC dropped from 278.8 μg/L to 207.1 μg/L, which suggested that a possible association between average Tvol and UIC < 300 μg/L might still exist. Third, the association between Tvol and UIC is based on two surveys and may possibly be affected by the lag period. Subsequent surveys will provide additional observations. However, this report is the first study examining the relationship among UIC, average Tvol and TGR with periodic surveys during 16 years after USI.IDD has been eliminated by USI in Jiangsu province since 2001. As IDD has been persistently eliminated as evidenced by UIC, TGR and use of salt iodization, lower TGR is associated with smaller average Tvol, and average Tvol is a more sensitive indicator than TGR to the fluctuation of UIC. Our findings suggest a UIC of 300 μg/L may be held to be a critical value at the population level for iodine status monitoring and program evaluation. Iodine concentration in salt should be evaluated periodically to maintain UIC in the optimal range in the context of overall socioeconomic development.S1 Table(XLSX)Click here for additional data file.


The physiologic stress induced by physical activity is reflected in immune system perturbations, oxidative stress, muscle injury, and inflammation. We investigated the effect of astaxanthin (Asx) supplementation on salivary IgA (sIgA) and oxidative stress status in plasma, along with changes in biochemical parameters and total/differential white cell counts. Forty trained male soccer players were randomly assigned to Asx and placebo groups. Asx group was supplemented with 4 mg of Asx. Saliva and blood samples were collected at the baseline and after 90 days of supplementation in preexercise conditions. We observed a rise of sIgA levels at rest after 90 days of Asx supplementation, which was accompanied with a decrease in prooxidant-antioxidant balance. The plasma muscle enzymes levels were reduced significantly by Asx supplementation and by regular training. The increase in neutrophil count and hs-CRP level was found only in placebo group, indicating a significant blunting of the systemic inflammatory response in the subjects taking Asx. This study indicates that Asx supplementation improves sIgA response and attenuates muscle damage, thus preventing inflammation induced by rigorous physical training. Our findings also point that Asx could show significant physiologic modulation in individuals with mucosal immunity impairment or under conditions of increased oxidative stress and inflammation. Astaxanthin (Asx) is a red-orange carotenoid mainly produced by micro- and macroalgal species and accumulated in many marine organisms, such as shrimps, crabs, trout, and salmon. The polyene system gives astaxanthin its distinctive molecular structure, chemical properties, and light-absorption characteristics . The preThe mucosal immune system functions as the first line of defense against pathogen invasion by preventing the attachments of infectious agents to mucosal surfaces . An impo in vitro and animal studies [Intensive and sustained physical activity elevates the generation of free radicals and reactive oxygen species (ROS), thus creating an imbalance between ROS and antioxidants and leading to oxidative stress that not only causes lipid peroxidation and protein oxidation but also may have a negative impact on immune function . Asx pos studies . These s studies –17.No published human studies exist regarding the influence of Asx ingestion on exercise-induced mucosal immunity dysfunction. One human study showed that Asx could enhance immune response and decrease a DNA oxidative damage and inflammation in healthy females . TherefoForty trained male soccer players, among young selection of soccer club “Partizan,” Belgrade, were recruited as experimental subjects. The participants met the following exclusion criteria: smokers, subjects with recurrent respiratory infections, subjects taking antibiotics, subjects taking immunosuppressive drugs, and alcohol abuse. Athletes as well as their parents gave the written consent after having been explained the purpose, demands, and possible risks associated with the study. The study was conducted according to the guidelines laid down in Declaration of Helsinki. Experimental procedures were approved by the Ethical Committee of Sports Medicine Association of Serbia. They were involved in a controlled training program during a 90-day period over competitive season, in which they had 5 to 7 training sessions per week with an average weekly training of 10 to 15 hours and took participation in national championship. They also had different aspects of trainings, including strength, resistance, cardio, flexibility, and proprioceptive training.N = 21) or placebo  groups. Under double-blind procedures, subjects received Asx (4 mg per day) or placebo supplements for 90 days. The Asx used in this study was a homogenized and spray dried biomass of the green unicellular alga Haematococcus pluvialis. Both Asx and placebo capsules were manufactured by Bioreal (Sweden) and generously donated by Oriflame, Serbia. The dietary supplementation comprised of one capsule daily taken orally in conjunction with a meal.Soccer players were randomly assigned to Asx (2). VO2max was measured on a motor driven treadmill , using an indirect calorimetry system  during an incremental exercise test to volitional fatigue. Basic demographic and training data were obtained through a questionnaire. Subjects were instructed to restrain themselves from making any drastic changes in the diet. They agreed to avoid the use of vitamin/mineral supplements, antioxidant supplements, nutritional supplements, ergogenic aids, herbs, and medications known for their effect on immune function, 1 month before and during the study. Subjects recorded food intake in a 4-day food record before the first exercise test session. The food records were analyzed using CRON-O-Meter v0.9.6. software.Two weeks before the first test session, subjects reported to the laboratory for orientation, measurement of body composition, and cardiorespiratory fitness. Body weight and total body fat were measured using the BC-418, 8-contact electrode device , which uses bioelectrical impedance analysis for body composition analysis. Height was measured to the nearest 0.1 cm with a portable stadiometer. Body mass index (BMI) was calculated by dividing the weight in kilograms by the square of the height in meters , by placing the cotton swab under the tongue. All saliva collections  were made after players had been sitting quietly for a few minutes, leaning forward, with their heads tilted. Immediately after collection, the obtained saliva samples were separated from the cotton by centrifuging at 1500 ×g for 15 minutes. The supernatant fluid was frozen at −80°C for later analysis of sIgA. Before and after saliva collection, salivettes were weighted on an analytical balance. The amount of saliva in grams was converted to milliliters assuming that the specific gravity of saliva is 1 and divided by 2 to express salivary flow in mL/min.μL of sulfuric acid, and the absorbance of solution in each well was determined at 450 nm by using ELx800 Absorbance Microplate Reader . Regression analysis using the relation of standard sIgA concentrations and amount of absorbance (nm) was used to interpolate the concentration of sIgA in the samples. sIgA is expressed as absolute concentration and secretion rate. sIgA secretion rate (μg/min) was determined by multiplying the absolute sIgA concentration (μg/mL) with saliva flow rate (mL/min).The sIgA concentration was measured by enzyme-linked immunosorbent assay (ELISA). Briefly, the test was performed on 96-well microtiter plates, which were coated with goat anti-human IgA  in coating buffer  and kept overnight at 4°C. On the day of analysis, plates were incubated for 60 min at room temperature before being washed with TTBS buffer  and then blocked with 3% gelatin in TTBS for 2 h at room temperature. Samples were thawed and then diluted (1 : 200) with a sample diluent (1% gelatin in TTBS). Known concentrations of human secretory IgA  and controls were used to construct a standard curve. The plate was then washed before standards and samples were added to wells (in duplicate) and incubated for 1 h at room temperature. The plate was washed again before addition of diluted goat anti-human horseradish peroxidase conjugates . After incubation, the plate was washed again before addition of substrate tetramethylbenzidine  and incubated for 30 min in the dark. The colorimetric reaction was stopped via addition of 100 For determination of oxidative status parameters, the venous blood was collected into heparin evacuated tube . The blood samples were centrifuged at 1500 g for 10 min, and aliquots of plasma were stored at −80°C for later analysis.2+–o-dianisidine complex reacts with a standardized solution of H2O2 by a Fenton-type reaction, producing hydroxyl radicals (OH∙). These potent ROS oxidize the reduced colorless o-dianisidine molecules to yellow-brown colored dianisidyl radicals at low pH. Antioxidants in the sample suppress the oxidation reactions and color formation. This reaction can be monitored by spectrophotometry. The reaction rate is calibrated with Trolox  and was incorporated into Ilab 300 plus autoanalyser . The TAS value of the samples tested is expressed as mmolTrolox equiv./L.Total antioxidant status (TAS) was determined using an automated method developed by Erel . A stand2O2 and incorporated into Ilab 300 plus autoanalyser . The results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (μmolH2O2 equiv./L).Total oxidant status (TOS) was determined according to Erels' method . This as2O2, as a representative of hydroperoxides, which is an indicator of total oxidant status, with 3 mmol/L uric acid (in 10 mmol/L NaOH), as a representative of the antioxidant capacity. This photometric comparison was carried out using an ELISA reader.Prooxidant-antioxidant balance (PAB) was measured according to a previously published method . This meμL of TMB/DMSO was added in 20 mL of acetate buffer , and then 70 μL of fresh chloramine T (100 mM) solution in distilled water was added. The mixture was incubated in a dark place for 2 hr at room temperature, after which 25 units of peroxidase enzyme solution were added into 20 mL TMB cation, dispensed in 1 mL, and put at −20°C. To prepare the TMB solution, 200 μL of TMB/DMSO was added into 10 mL of acetate buffer . The working solution was prepared by mixing 1 mL of TMB cation with 10 mL of TMB solution. The mixture was incubated for 2 min at room temperature, in a dark place, and it was used immediately. The sample (10 μL), standard, or blank (distilled water) were mixed with 200 μL of working solution, in each well of a 96-well plate, which was then incubated in a dark place at 37°C for 12 min. At the end of incubation time, 100 μL of 2N HCl was added into each well and measured in ELISA reader at 450 nm, with a reference wavelength of 620 or 570 nm . A standard curve was provided from the values relative to the standard samples. The PAB values of the unknown samples were calculated using the standard curve and expressed in arbitrary HK units, which represent the percentage of H2O2 in standard solution, multiplied by 6.TMB powder (60 mg) was dissolved in 10 mL of DMSO. For preparation of TMB cation, 400 Hematological analysis was performed in the blood samples collected into K-EDTA tubes , using an automated hematology analyzer Cell-Dyn 3700 . To examine biochemical parameters, blood samples were drawn from an antecubital vein into sample tube with serum separator gel  and then left to coagulate for 30 min to separate the serum, and the obtained samples were stored at −80°C for later analysis. The following items were included in the general biochemical examination: aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LDH), uric acid (UA), creatinine (Cre), high sensitivity C-reactive protein (hs-CRP), total cholesterol (CHOL), HDL cholesterol (HDL-C), and triglycerides (TG). Biochemical analysis was performed using an ILab 300 Plus autoanalyser  employing commercial kits . The concentration of LDL cholesterol (LDL-C) was calculated using the formula of Friedewald et al. . t-test. The effects of supplementation and training were analyzed by two-way analysis of variance (ANOVA) with repeated measures to test for the two main effects and for the interaction between them. When a significant p value was obtained, Bonferroni post hoc comparison test was employed to determine the differences between groups. Two-tailed p values are given throughout. Due to the fact that the distributions of sIgA absolute concentrations and secretion rate, AST, ALT, CK, and hs-CRP were skewed, logarithmic transformation of these values was performed before statistical comparisons were made.Statistical analyses were performed using the PASW Statistics version 18.0 and MedCalc  software. All data were assessed for normality (Kolmogorov-Smirnov tests). The results are expressed in terms of mean values and standard error (SE) for normally distributed variables. Where distribution differed from a normal distribution, geometric means and 95% confidence intervals were given. Subjects' baseline characteristics and nutritional parameters between treatment groups were compared using independent-sample2max .Physical characteristics for the 40 soccer players randomized to Asx and P groups are summarized in p > 0.05), According to 4-day food records analysis using CRON-O-Meter v0.9.6 software, estimated daily energy and nutrient intake in two experimental groups were similar (p > 0.05). The obtained values were unchanged after 90 days of training and supplementation  on sIgA absolute concentration as well as on sIgA secretion rate  , while there were no significant changes in P group.ANOVA repeated measures showed a significant interaction effect of supplementation and training . TOS decreased in both, the supplemented and the P group, after 90 days of regular training in comparison to baseline values. Basal TAS was unaffected by soccer training and supplementation (p > 0.05). There was a significant decrease in PAB in Asx group after 90 days in comparison to baseline values , as a result of continuous training and supplementation. No significant changes were found in P group.Oxidative stress status is reported in F = 4.528, p < 0.05; main effect of training: F = 3.989, p < 0.05) but not in Asx group, as presented in F = 4.875, p < 0.01). These changes were not detected in supplemented group.After 90 days of training and supplementation, ANOVA analysis showed a significant increase in leukocyte count in P group , CK , and LDH  plasma levels. Post hoc comparison showed a significant decrease in CK activity in Asx group after 90 days compared to baseline values (p < 0.01), while a decrease in P group was not statistically significant. After 90 days of continuous training, AST and LDH decreased significantly in both Asx  and P group . In addition, a significant main effect of supplementation was observed on AST  and LDH  levels, with lower activity in Asx compared to P group.Biochemical parameters are shown in F = 5.528, p < 0.05; Cre main effect of training, F = 4.429, p < 0.05). ANOVA analysis also showed a significant supplementation and training interaction effect  on hs-CRP levels. Namely, after 90 days of regular soccer training, a 57% increase in hs-CRP levels may be observed in P group, but not in the Asx group. Analysis of lipid profile revealed no effect of training, supplementation, or supplementation and training interaction.UA and Cre levels significantly decreased in both groups after 90 days of observational period . Also, the increase in neutrophil count and hs-CRP level recorded in placebo group was not apparent in supplemented group, indicating a significant blunting of the systemic inflammatory response in the subjects taking Asx.α [The observed increase in sIgA levels after supplementation could indicate the effect of Asx on sIgA synthesis. Previous studies reported modulatory actions of Asx on humoral immune response such as induced production of polyclonal antibodies G and M in murine spleen cells , increasα , which iα . The benα . Immune α . ROS oveα , where NTherefore, under conditions of increased oxidative stress , dietary antioxidants become critical in maintaining a desirable oxidant/antioxidant balance . In our However, we showed that Asx supplementation resulted in decreased prooxidant-antioxidant balance (PAB) during regular competitive season in young soccer players. Therefore, we believe that deleterious effects caused by the excess of ROS produced during physical activity can be additionally attenuated by the Asx. Asx induced a better redox balance favoring all cellular functions which depend on adequate amounts of ROS, thereby supporting the mucosal immune system. Our finding is in accordance with the study of Park et al.  who examThe magnitude of muscle damage, evaluated by CK and LDH in our study, was reduced significantly over the observational period, probably by adaptation and conditioning of the muscle through regular training, as shown previously by Powers and Jackson . Asx supIn this study, regular soccer training during the competitive season led to certain changes in leukocytes count only in placebo group. Over the time, total leukocytes count, neutrophil count, and hs-CRP levels had increased. The observed elevation in hs-CRP due to the regular intensive training was similar to the levels that have been associated with increased risk of coronary vascular disease . These cEvidence suggests that Asx has potential health-promoting effects in prevention and treatment of various diseases, such as chronic inflammatory diseases, metabolic syndrome, diabetes, cardiovascular disease, neurodegenerative diseases, exercise-induced fatigue, or male infertility , which lAccording to the results presented in this study, Asx supplementation improved sIgA response and attenuated muscle damage, probably due to restoring redox balance, thus preventing inflammation induced by rigorous physical training. Although our study focused on narrow population regarding the age, gender, and life style, nevertheless, it showed the modulating effects of Asx on the examined parameters. Our findings also indicate that Asx could show significant physiologic modulation in other individuals with mucosal immunity impairment or under conditions of increased oxidative stress and inflammation. Although the currently available data and recent findings are very encouraging, more extensive, well-controlled clinical trials are suggested for other threatened categories.