When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis.Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches ,2 may leThe results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results. However, there are many sources of errors, both when purifying RNA, performing the RT reaction and during the PCR setup ,4. Q-PCRcriteria . In recocriteria ,6-9. We Lepeophtheirus salmonis) starvation experiment using the 2-ΔΔCT method [-ΔΔCT method, our results show that LsTryp1 transcript levels decrease following starvation and return to normal adult level when the louse subsequently gets access to food  were reared as earlier described [Salmo salar) and 3 lice were stored in RNA later (Ambion). The remaining 12 lice were starved in incubators with flowing seawater for 14 days. After starvation, 3 lice were sampled and stored as described above, and the remaining 9 lice were put in a tank with uninfected salmon where they could settle on their salmon hosts and resume feeding. After 15 days on their new hosts 3 lice were sampled and stored as described above. The experimental procedures were carried out in accordance with national regulations for use of animals in scientific research.Salmon lice  or 0.4 (CT = 32) from the most similar parallel at the same dilution. At least 4 dilutions were required for each stage. The resulting data were calibrated to unstarved lice and analysed as described by Kvamme et al.[α = 0.05) from the other dilutions. The signal corresponding to the initial template concentration (R0) was derived using the average PCR efficiency for LsTryp1 and eEF1α when the PCR efficiencies were not significantly different . When the PCR efficiency differed significantly, R0 was calculated using individual gene specific mean efficiencies. The mean R0 for each dilution of LsTryp1 was normalised to corresponding eEF1α values. The normalised R0 values were calibrated to the values for unstarved lice. 95% confidence intervals (CI) were derived from normalised R0 values.The transcript levels of LsTryp1  and the 1 [eEF1α  in 1 sel1 [eEF1α . The RNA1 [eEF1α  and cDNAescribed  and a meescribed . The latescribed . When usme et al.. When usRS and PF conceived the study and designed the Q-PCR assay. RS carried out the assay and analyses, and wrote the first draft of the communication. PF contributed to development of the communication. FN provided expert input for the writing and supervised the study.


Saccharomyces cerevisiae has been associated with human life for millennia in the brewery and bakery. Recently it has been recognized as an emerging opportunistic pathogen. To study the evolutionary history of S. cerevisiae, the origin of clinical isolates and the importance of a virulence-associated trait, population genetics and phenotypic assays have been applied to an ecologically diverse set of 103 strains isolated from clinics, breweries, vineyards, fruits, soil, commercial supplements and insect guts.S. cerevisiae strains that are associated with ecology, geography and virulence. Shared alleles suggest that the clinical isolates contain genetic contribution from the fruit isolates. Clinical and fruit isolates exhibit high levels of recombination, unlike the genetically homogenous soil isolates in which no recombination was detected. However, clinical and soil isolates were more resistant to oxidative stress than any other population, suggesting a correlation between survival in oxidative stress and yeast pathogenicity.DNA sequence data from five nuclear DNA loci were analyzed for population structure and haplotype distribution. Additionally, all strains were tested for survival of oxidative stress, a trait associated with microbial pathogenicity. DNA sequence analyses identified three genetic subgroups within the recombining S. cerevisiae delineated three distinct groups, comprising primarily the (i) human-associated brewery and vineyard strains, (ii) clinical and fruit isolates (iii) and wild soil isolates from eastern U.S. The interactions between S. cerevisiae and humans potentiate yeast evolution and the development of genetically, ecologically and geographically divergent groups.Population genetic analyses of  S. cerevisiae has long been associated with humans as the fermentative agent in the production of bread, beer, and wine. Archeological evidence for the production of fermented beverages in China dates to 7,000 BCE, and molecular evidence demonstrating S. cerevisiae was the fermentative agent has been found in wine jars from ancient Egypt dating to 3,150 BCE S. cerevisiae embraces a wider range than domesticated strains found in the vineyard and the brewery. Wild strains have been isolated from mushroom fruiting bodies as well as oak tree-associated soils and fluxes S. cerevisiae are furthermore a major cause of spoilage of mango fruit and peach puree, and it has recently been identified in surveys of the fungal diversity in beetle guts S. cerevisiae ecological diversity, encompassing domesticated and wild isolates, has spurred interest in the life history and population genetics of this species. The population biology of an organism so tightly associated with humans is challenging to study due to sampling bias, limited sampling sizes, human influence, and non-random sampling S. cerevisiae have mainly been addressed using strains from grape berries, vineyards and other industrial applications. Fay and Benavides analyzed approximately 7 kb of coding and non-coding DNA sequences in 81 strains from vineyards, fermentation of sake, palm wine, ragi and cider, fruit sources, including lychee, fig, and mushrooms, oak tree and surrounding soil from New Jersey, and patients in the U.S. and Europe. This extensive analysis resulted in the recognition of domesticated, i.e. human-associated, and wild populations in S. cerevisiaeThe breadth of S. cerevisiae's has been reported as an emerging opportunistic pathogen S. cerevisiae causing infections S. cerevisiae and its commercially available preparations known as S. boulardii, that are used to treat antibiotic-related diarrhea, have been shown to cause a wide variety of infections, ranging from cutaneous infections and vaginitis to systemic infections of the bloodstream and vital organs in immunocompromised and immunocompetent individuals Candida albicans, the most common human fungal pathogen C. albicans and its status as genetics model system, S. cerevisiae, the benevolent baker's yeast, has acquired enhanced scientific value as a model pathogen in the study of virulence-related traits In addition to investigations into the origin and consequences of domestication, S. cerevisiae include growth at high temperature S. cerevisiae isolates from spoiled peach pure and grape juice were heat resistant Microbial virulence-related traits promote host invasion, colonization and virulence. One of these is the ability to survive oxidative stress, exerted by radical oxygen species (ROS), an integral component of mammalian host defenses, is associated with virulence in various bacteria S. cerevisiae, little is known about the emergence of this versatile yeast as a pathogen and the role of selection and/or adaptation in this evolutionary process. This study aims to (i) test for genetic differentiation between strains of a broad range of origins, (ii) investigate the evolutionary origin of clinical isolates, and (iii) identify an association between a virulence-related trait and pathogenicity. The targeted virulence trait is survival of oxidative stress. To investigate population structure and evolution of pathogenicity, an ecologically diverse sample of 103 S. cerevisiae isolates, comprising seven populations, was analyzed. Clinical isolates with confirmed virulence were obtained from the California Institute for Medical Research in Stanford and Duke University Medical Center in Durham, NC S. boulardii strains from France, Italy and Germany, complete the sampling Despite progress in understanding the population structure of domesticated and wild S. cerevisiae, expanding the previous findings. (ii) Clinical isolates are genetically similar to the isolates from fruit, which supports the hypothesis that monoculture or fermenting fruits may serve as a natural reservoir for the evolution of clinical isolates. (iii) Strains from the clinic and Pennsylvania soil tolerate oxidative stress better than any other group. However, the clinical isolates are genetically diverse while the soil isolates are identical at all loci. This strongly suggests that resistance to ROS is an adaptive property of the clinical strains.The results confirm previous findings and generated several novel conclusions. (i) At least three divergent groups representing different evolutionary trajectories were detected in S. cerevisiae, as confirmed by ITS sequencing and all strains were determined by FACS analysis to be diploid (data not shown). To ensure correct estimates of population genetic parameters haplotype phase within each locus was determined. For the majority of isolates the correct locus haplotype phase could be determined by visual analysis of sequence alignments. For 22 strains the correct phase of one ore more loci was identified using PHASE 2.1.1. All isolates in this study belong to the species S. boulardii form group C. Note that two fruit isolates in group C stem from grapes, two fruit isolates from Holly and a papaya. Interestingly, all group C vineyard isolates have been collected from Vitis vinifera in Australia or North Carolina. The two identical NC vineyard isolates that are outside the major clusters were isolated from V. rotundifolia grapes, the Muscadine grapes that are native to the southeastern United States. Seven of the nine isolates collected in Adelaide (AU) are identical. As measure of genetic differentiation pairwise Fst values were calculated for groups A, B, and C. Fst values showed significant genetic differentiation between the three groups and analysis of molecular variance (AMOVA) indicated that 25% of the observed variance occurs within these groups and 62% between them. The index of association (IA), measuring the extent of linkage equilibrium, was significantly different from zero for group A but not for B and C.Principal component analysis (PCA), a transformation method that reduces multidimensional data sets to lower dimensions, was employed to detect putative structure among 87 isolates for which complete sequence data for five loci could be obtained . ConfideA), nucleotide diversity (π), and deviation from Hardy-Weinberg equilibrium (HWE) were calculated , recombination, linkage equilibrium  confirmed significant differences in oxidative stress between groups of N≥10 (P<0.0001). Clinical isolates differ significantly (P<0.001) from all groups but PA soil  Low levels of recombination and genetic diversity among soil populations (group B) suggest a predominantly clonal life style that contributes to differentiation from clinical and domesticated groups and maintenance of a wild lineage that also exhibits geographic structure because all of its isolates were collected in the eastern United States. Interestingly, the soil isolates share four of five haplotypes with beetle gut isolates, suggesting that insects acquire S. boulardii were not as resistant to ROS as the clinical strains. Indeed, strains of S. boulardii were less virulent compared to a clinical (YJM145) and a laboratory (Y55) strain S. boulardii isolates used here were cultured from commercial products sold in France, Italy and Germany. Their close association with vineyard strains suggests that S. boulardii may have originated from vineyard strains.The clinical and PA soil populations exhibited the highest mean resistance to ROS. Therefore ROS resistance in the clinical isolates arose either independently or was contributed by the soil isolates. The genetic diversity among the clinical isolates suggests multiple origins of the genetic material required for ROS resistance in these strains. Regardless, fungal resistance to ROS offers protection from oxidative host defenses and is undoubtedly an advantageous pathobiological property. Consistent with this conclusion, the probiotic strains of Most recently, a study conducted by Kvitek, Will and Gasch combined investigations into genetic diversity with stress response and identified a distinct sake fermentation clade but no discrete clades for clinical or oak-associated isolates S. cerevisiae beyond its prosaic service as a scientific tool in the laboratory or an agent of fermentation. As a species, S. cerevisiae entered two new life history trajectories while continuing its life in the soil and on decomposing fruit. Some strains of S. cerevisiae became domesticated in fermentation and brewing, and while others became pathogenic. With the accumulation of ecological and population genetic data, this versatile microbe becomes an invaluable model for evolutionary biology and population genetics. The current report offers a new paradigm for studying pathogenesis by identifying correlation(s) among the virulence traits of isolates and the ecology of their ancestral reservoirs. These correlations will identify the genotypes associated with pathogenic strains or the potential for pathogenicity and elucidate the evolution of pathogenicity.This investigation presents an exciting and provocative demonstration of the complex life history of S. cerevisiae strains were isolated from S. cerevisiae infected patients (N = 15), soil in Pennsylvania , soil in North Carolina , vineyards in NC (N = 10) and Australia , various fruits (N = 18), brewery (N = 16), commercial S. boulardii preparations (N = 4), and the insect gut (N = 2) . DNA was extracted using the CTAB buffer method  (N = 2) . All str96Cleanup Kit (Millipore) or the ExoSAP protocol and their concentration determined electrophoretically Genomic DNAs were diluted 1∶100 and 2 µl added to a 25 µl reaction from TaKaRa Ex Taq kit with a final primer concentration of 0.6 µM . The PCRA were calculated in GenAlEx 6, DnaSP 4.50.3, and MultiLocus 1.3 A). Due to limited sampling size S. boulardii and beetle gut isolates were not included in HWE, Fst, π and IA calculations. An UPGMA tree based on a pair-wise genetic distances between isolates included in PCA was build in PHYLIP 3.68 MLS1, ACT1, ADP1, PHD1 and RPB1 in PAUP* 4.0b10 applying the parsimony criterion, conducting heuristic searches, and using tree-bisection-reconnection (TBR) as branch swapping algorithm. These five loci were chosen for analysis based on their prior employment in phylogenetic studies of the Saccharomycetales, population genetic analysis of S. cerevisiae and C. albicans and their differential expression patterns upon phagocytosis by macrophages 2SO4, 1.7 g/l yeast nitrogen base (YNB) without amino acids and ammonium sulfate), incubated at 30°C while shaking (250 rpm), transferred once, and grown over night. In order to reduce variability in the cell suspension, the cells were washed twice with 0.9% NaCl solution and dissolved in 1× phosphate buffered saline. After adjusting the cell number to 2×103 cells/ml, 20 mM tert-Butyl hydroperoxide  were added and treated and control samples incubated for one hour at 30°C, while shaking. 100 µl cell suspensions from both samples were spread on YPD plates, incubated for 48 h at 30°C and colonies counted. Survival was calculated as the ratio of treated over untreated cells. Each strain was tested three times and results were clustered by origin and plotted in box-and-whisker plots. A one-way ANOVA with Bonferroni correction was carried out on experimental triplicates of each strain in every group to assess differences in variation for all groups with N≥10.Two-day old cultures from YPD plates were inoculated into liquid synthetic defined media Click here for additional data file.Figure S1UPGMA tree. The tree was generated from a pair-wise genetic distance matrix based on haplotype data of the 87 strains that were included in PCA. The strains are color-coded by origin (legend) and groups recognized in PCA indicated at internodes in the tree. Strains marked with * denote isolates that are not included in PCA groups A, B or C confidence envelopes. The numbered bar below the tree indicates total genetic distance observed in the data set.(1.34 MB TIF)Click here for additional data file.


Neuroscientists have observed that the human brain is comprised of neurons. We have observed that babies start speaking at an early age, yet no young animals, including pets, have so far been seen to speak, at least not in the articulated fashion of human babies. To understand this highly cognitive ability, many psycholinguistic data have been gathered, from behavioral, to neurolinguistic, to recent neuroimaging studies, each measuring macroscopic properties of the brain. Nevertheless, the challenging question remains unanswered of how such complicated behavior emerges from the microscopic (or mescoscopic) properties of individual neurons and of networks of neurons in the brain.We would like to tackle this question by developing and analyzing a Potts attractor neural network model, whose units hypothetically represent patches of the cortex. The network has the ability to spontaneously hop (or latch) across memory patterns , thus producing an infinite sequence of patterns, at least in some regimes . We woulBefore training the network on the corpus, the critical issues to be addressed, and the central ones here, are: how should the words be represented in a cognitively plausible manner in the network? how should the correlation between words, in terms of both meaning and statistical dependences, be reflected in their  representations? how should two main characteristics of a word, the meaning (semantic) and the syntactic properties, be represented in the network?We represent words in a distributed fashion on 900 units, 541 out of which express the semantic content and the rest, 359 units, are representative of the syntactic characteristics of a word. The distinction between the semantic and syntactic characteristics of a word has been loosely inspired by a vast number of neuropsychological studies [The preliminary analysis of the produced patterns indicates the resemblance between the statistics of the representation of words and the patterns that can generate the latching behavior of the network. This is a promising step towards building a neural network that can spontaneously generate a sequence of words (sentences) with desired syntactic and semantic relationships between words in sentences.


Boronic acid molecules form inversion-related hydrogen-bonded dimers in an R               2               2(8) motif. The structure is consolidated by intermolecular C—H⋯O bonds and C—H⋯π interactions.In the crystal structure of the title compound, C Å                           b = 12.339 (4) Å                           c = 16.983 (5) Å                           β = 95.651 (5)°V = 2548.0 (14) Å3                                                   Z = 4                           Kα radiationMo −1                        μ = 0.13 mmT = 297 K                           0.52 × 0.45 × 0.42 mm                                 Bruker SMART CCD area-detector diffractometerSADABS; Bruker, 2000T                           min = 0.934, T                           max = 0.947Absorption correction: multi-scan (23787 measured reflections4486 independent reflectionsI > 2σ(I)3727 reflections with R                           int = 0.052                                                            R[F                           2 > 2σ(F                           2)] = 0.083                           wR(F                           2) = 0.186                           S = 1.19                           4486 reflections294 parametersH-atom parameters constrainedmax = 0.40 e Å−3                        Δρmin = −0.32 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXS97  I, global. DOI: 10.1107/S1600536811051609/pk2368Isup2.hkl            Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811051609/pk2368Isup3.cml            Supplementary material file. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Arginine, an α-amino acid, has been reported to exert beneficial effects that ameliorate health problems and prevent excessive fat deposition. In this study, we investigated whether the activation of cell signaling by arginine can induce osteogenic differentiation and modulate excessive adipogenic differentiation in human mesenchymal stem cells (MSCs). Arginine potently induced the expression of type Iα1 collagen, osteocalcin, and ALP in a dose-dependent manner without causing cytotoxicity. Arginine significantly increased the mRNA expression of the osteogenic transcription factors runt-related transcription factor 2 (Runx2), DIx5, and osterix. Furthermore, arginine demonstrated its antiadipogenicity by decreasing adipocyte formation and triglyceride (TG) content in MSCs and inhibiting the mRNA expression of the adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and fatty acid binding protein 4 (Fabp4). This effect was associated with increased expression of Wnt5a, and nuclear factor of activated T-cells (NFATc), and was abrogated by antagonists of Wnt and NFATc, which indicated a role of Wnt and NFATc signaling in the switch from adipogenesis to osteoblastogenesis induced by arginine. In conclusion, this is the first report of the dual action of arginine in promoting osteogenesis and inhibiting adipocyte formation through involving Wnt5a and NFATc signaling pathway. Human bone marrow mesenchymal stem cells (MSCs) are pleiotropic cells that differentiate into either adipocytes or osteoblasts ,2. ChangOsteogenesis and adipogenesis, which share a reciprocal relationship in the bone marrow, are complex processes that include the proliferation of precursor cells and their commitment to a specific lineage and then terminal differentiation ,7. In MSN-terminal kinase (JNK) pathway and the Wnt/calcium (Ca2+) pathway [2+ pathway ultimately leads to the binding of the nuclear factor of activated T-cells (NFATc) to specific DNA-binding sites [Wnt signaling is a key pathway that controls bone formation and adipogenesis by regulating critical events such as cell-fate determination and cell proliferation and differentiation . Wnt sig pathway ,17. The ng sites ,18,19. TAstragalus membranaceus Bunge, and it is one of the 20 most common natural amino acids [Arginine is a major compound produced by no acids . Oral adno acids , and it no acids . Argininno acids . Argininno acids . HoweverIn this study, we investigated whether arginine enhances osteogenic differentiation and inhibits adipocyte formation in MSCs by modulating osteogenic and adipogenic transcription factors and the Wnt signaling pathway.To examine how arginine affects cell proliferation, we treated MSCs with 0, 0.1, 1, and 10 µM arginine for 1, 3, 5, 7, and 10 days. Arginine dose-dependently enhanced cell proliferation after treatment for 48 h and increased the proliferation of cells in a statistically significant manner, by nearly 36%, at a concentration of 10 µM A. HoweveTo determine whether arginine can stimulate osteogenic differentiation, we measured the effect of arginine on the levels of the bone-formation markers type Iα1 collagen, osteocalcin, and alkaline phosphatase (ALP). Our results showed that the treatment of MSCs with 1 μM arginine for 3, 7, 14, and 21 days increased the mRNA expression of type Iα1 collagen, osteocalcin, and ALP in a statistically significant manner, but did not enhance the expression of type IIα1 collagen relative to the control level at each time point A. The exIn vivo studies have demonstrated that in mice in which the PPARγ gene was deleted, the levels of TGs, free fatty acids, and cholesterol and the accumulation of hepatic TG were lower than those in control mice [To determine the effect of arginine on adipogenic differentiation in MSCs, 1 μM arginine was added to cells cultured in an adipogenic medium, and then adipocyte-specific gene expression was examined using quantitative real-time reverse-transcription PCR (qRT-PCR) during adipogenic induction. During the 14 days of differentiation, the induced adipocytes were stained with Oil red O. The staining-positive cells displayed substantial lipid accumulation at 3 days when compared with the uninduced cells A. The inrol mice . C/EBPα,rol mice . Lipid arol mice . Fabp4 brol mice . Our resTo investigate the roles of arginine in Wnt/β-catenin signaling during the osteoblastic and adipocytic differentiation of MSCs, cells cultured in an adipogenic medium were treated with arginine for 48 h and then Western-blotting analysis and RT-PCR were performed. Doses of arginine ranging from 0.1–10 μM did not affect the expression levels of phospho-β-catenin and β-catenin in MSCs A. SubseqNFATc1 represents a downstream of target of Wnt signaling and is a key regulator of adipogenic and osteogenic differentiation ,35. We tTaken together, our results suggest that arginine regulates Wnt/β-catenin independent signaling and also the mechanisms involving NFATc that control MSC fate and differentiation, and contributes to the increase in osteoblastogenesis and decrease in adipogenesis.l-Arginine, ALP activity assay kit, Alizarin red S staining kit, Oil red O staining kit, ascorbic acid, glycerophosphate, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), dimethyl sulfoxide (DMSO), and insulin (INS) were purchased from Sigma-Aldrich . Fetal bovine serum (FBS), fetal calf serum (FCS), antibiotics, Dulbecco’s modified Eagle’s medium (DMEM), Trizol R reagent, and SDS-polyacrylamide gels were purchased from Gibco-BRL . The RT-PCR system kit was purchased from TaKaRa Biotechnology . The real-time SYBR Green RT-PCR system kit was purchased from Bio-Rad . Hybond-C nitrocellulose membrane was purchased from Amersham Biosciences .2 flasks and incubated at 37 °C/5% CO2. After 48 h, non-adherent cells were removed from the flasks by changing the medium. Thereafter, the medium was changed once every 3 days. Typically, cultures reached 90% confluence by 14 days, at which point the cells were trypsinized using 0.25% trypsin-0.53 mM EDTA, counted, and then plated again. Cells from passages number of 4~6 were used in all experiments. To determine whether changes in gene expression depended on canonical or noncanonical Wnt signaling, MSCs were treated with arginine in the presence of DKK1 (50 ng/mL) or sFRP1 (250 ng/mL) (Sigma) and then gene expression was measured as described below . To investigate the role of NFATc in mediating the effect of arginine, MSCs were treated with arginine in the presence of CSA (100 ng/mL) or FK506 (10 ng/mL), and then gene expression was measured.Human bone-marrow MSCs were obtained from Lonza . Cells were seeded into 25-cm4 cells/well in 96-well plates and allowed to attach for 12 h in DMEM containing 10% FBS. To assess the effect of arginine on cell proliferation, MSCs were treated with culture medium containing various concentrations of arginine  for 48 h or with medium containing 0, 0.1, 1, and 10 μM arginine for 3, 5, 7, and 10 days. At each time point, the arginine containing medium was removed and the cells were incubated with fresh serum-free medium containing the Cell Counting Kit-8 (CCK-8) reagent WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5--2H-tetrazolium) ; cells were incubated for 2 h at 37 °C in a CO2 incubator and then the amount of formazan dye generated, which is proportional to the number of living cells, was determined by measuring the absorbance at 570 nm using a multi-well plate reader . The percentage of proliferating cells was calculated by defining the cell viability measured in the absence of treatment as 100%.MSCs were seeded at a density of 2 × 104 cells/cm2 in 6-well plates. After 2 days, the medium was replaced with an osteogenic medium , and then fresh differentiation medium was added every other day and the cells were maintained at 37 °C in a humidified 5% CO2 atmosphere throughout the experiments until the cells were harvested. The differentiating cells were treated for 3, 7, 14, and 21 days with the osteogenic differentiation medium containing arginine at a final concentration of 1 μM, or the cells were treated with 0.1, 1, and 10 μM arginine. To determine the effects of arginine on osteogenesis in MSCs, the expression of osteogenesis-related biochemical markers such as collagen type 1α, osteocalcin, and ALP and transcription factors such as Runx2, DIx5, and osterix were measured using qRT-PCR and matrix calcium deposition was evaluated by performing Alizarin red S staining.To induce osteogenic differentiation, MSCs were plated at a density of 2 × 10After treatment with arginine for 21 days, mineralization was measured using Alizarin red S (Sigma) staining and phase-contrast microscopy. Cells were incubated with 2% Alizarin red at pH 4.2 for 10 min and then washed with distilled water; the sub-cultured cells were observed using phase-contrast microscopy to examine cell morphology and verify the presence of mineralized nodules. After staining, the cells were washed and photographed, and the dye retained by the cells was eluted by incubation with isopropanol and then quantified by measuring the absorbance at 510 nm.4 cells/cm2 in 6-well plates and grown to post-confluence for 3 days. The culture medium was subsequently replaced with the adipogenesis-induction medium  in order to commit the cells to the adipogenic lineage. This medium was changed every other day and maintained up to 14 days to ensure complete development of the adipocyte phenotype. To assess the effect of arginine, cells were treated for 3, 7, and 14 days with the adipocytic differentiation medium containing arginine at a final concentration of 1 μM, or cells were treated with 0.1, 1, and 10 μM arginine for 14 days, and then the cells were used for qRT-PCR and RT-PCR assays, Oil red O staining, and TG-content measurement. The expression of Wnt3a, Wnt5a, and β-catenin was also evaluated during the adipocytic differentiation of MSCs.MSCs were plated at a density of 3 × 10Intracellular lipid accumulation was measured using Oil red O staining; the staining of lipid droplets by this dye was used as an indicator of the degree of adipogenesis. Cells were carefully washed twice with phosphate-buffered saline (PBS) and then fixed and dried with 10% formalin for 20 min. Next, the 10% formalin was removed and 60% isopropanol was added to each well for 3 min and then the cells were washed thoroughly with PBS to remove unbound dye. Cells were incubated with the Oil red O solution for 20 min, and the staining of lipid droplets in differentiated MSCs was examined after rinsing the cells 3 times with distilled water. Cells were photographed, and then the dye retained by the cells was eluted by incubating the cells with isopropanol, and quantified by measuring the absorbance at 510 nm.The cellular content of TG was measured using a TG-determination kit . Briefly, the cells were rinsed 3 times with PBS and then scraped off the plate by using a rubber policeman. The cells were extracted using 1 mL of lysis buffer  and then sonicated for 30 s at 4 °C. Next, 20 μL of the cellular lysate was mixed with 3 mL of the enzyme solution supplied with the kit, and the mixture was incubated for 10 min at 37 °C. The absorbance at 550 nm was measured within 60 min. As an internal control, the DNA concentration in MSCs was determined using spectrophotometry and then the μg/μg value was calculated.Ct) method. The cycle of threshold (Ct) for each sample was averaged and normalized relative to that of GAPDH. The results were then analyzed using the comparative ΔΔCt method (2Ct)(−ΔΔ) for the purpose of quantifying relative gene-expression levels. To perform RT-PCR, the diluted first-strand cDNA samples were amplified using TaqDNA polymerase (TaKaRa Biotechnology). The mRNA expression levels of Wnt5a, Wnt3a, and β-actin were evaluated by performing ethidium-bromide staining of PCR products. The signal intensity was quantified using Gel Doc EQ , and the relative expression of the mRNAs was normalized by dividing the signal of the target genes by that of the respective β-actin sample. The sequences of the primers used for qRT-PCR and RT-PCR assays are listed in Total cellular RNA was extracted from osteogenic cells or adipocytes by using the Trizol reagent and then centrifuged at 12,000 rpm for 10 min at 4 °C. Next, 1 μg of total RNA was reverse transcribed into cDNA for 60 min at 42 °C and then for 15 min at 72 °C by using an RT-PCR mixture (TaKaRa Biotechnology) that contained the RT buffer, oligo(dT) 12-mer, 10 mM dNTPs, 0.1 M dithiothreitol, reverse transcriptase, and RNase inhibitor. The qRT-PCR assay was performed in a 25-μL reaction mixture containing the SYBR Green PCR Master Mix (Roche Diagnostics). The template source was either 5 ng of cDNA or a purified DNA standard. Various primers were used for amplifying type IIα1 collagen, type Iα1 collagen, osteocalcin, ALP, Runx2, DIx5, osterix, PPARγ, C/EBPα, and Fabp4. To standardize mRNA levels, we amplified β-actin as an internal control. The relative expression of target genes in the examined samples was obtained using the difference in the comparative threshold , 5 min per wash, on a shaker. Next, the blots were incubated with goat anti-rabbit IgG-HRP  for 2 h at room temperature, and then washed 4 times in TTBS, 5 min each time. Immunoreactive proteins on the blots were visualized using ECL™ Western Blotting detection reagents and the signals were detected using Image Station 4000R . Staining with an anti-β-actin antibody was used to verify that equal amounts of proteins were loaded in all lanes.Following incubation with various reagents, cells were lysed using the lysis buffer. Total proteins (20 μg/lane) were separated on 4%–12% SDS-polyacrylamide gels by preforming electrophoresis under reducing conditions, and then transferred to Hybond-C nitrocellulose membranes (Amersham Biosciences) at 300 mA for 90 min. After blocking with 5% non-fat skim milk for 2 h, the membranes were incubated with rabbit primary antibodies against phospho-β-catenin, anti-β-catenin and NFATc1  for 12 h at 4 °C. The membranes were washed 4 times in 1× TTBS . In the case of two groups, Student’s In conclusion, this study has presented the first experimental evidence of the pro-bone and anti-fat effects of arginine, which counteracts the age-related switch in MSC osteoblast-to-adipocyte differentiation through the Wnt and NFATc1 signaling pathways. A schematic diagram of these observed effects of arginine is shown in


Reconstructing the past variability of Arctic sea ice provides an essential context for recent multi-year sea ice decline, although few quantitative reconstructions cover the Holocene period prior to the earliest historical records 1,200 years ago. Photochemical recycling of bromine is observed over first-year, or seasonal, sea ice in so-called “bromine explosions” and we employ a 1-D chemistry transport model to quantify processes of bromine enrichment over first-year sea ice and depositional transport over multi-year sea ice and land ice. We report bromine enrichment in the Northwest Greenland Eemian NEEM ice core since the end of the Eemian interglacial 120,000 years ago, finding the maximum extension of first-year sea ice occurred approximately 9,000 years ago during the Holocene climate optimum, when Greenland temperatures were 2 to 3 °C above present values. First-year sea ice extent was lowest during the glacial stadials suggesting complete coverage of the Arctic Ocean by multi-year sea ice. These findings demonstrate a clear relationship between temperature and first-year sea ice extent in the Arctic and suggest multi-year sea ice will continue to decline as polar amplification drives Arctic temperatures beyond the 2 °C global average warming target of the recent COP21 Paris climate agreement. A seasonal cycle in polar atmospheric bromine was first discovered in the 1980s by Sturges and Barrie24611An understanding of polar tropospheric halogen chemistry has developed as ground- and ice-core based measurements have established clearer links between FYSI extent and halogen variability recorded in polar ice cores. Interactions between halogens and FYSI are monitored by satellite81525) derived from sea ice diatoms26Due to the poor geographical and temporal resolution of sea ice proxies, few quantitative reconstructions of Holocene Arctic total sea ice extent are available. The few reconstructions available are based on dinoflagellate cyst assemblagesThe drivers and feedbacks between polar climate and total sea ice remain a topic of active research. The ice- albedo feedback mechanism has been studied in detailHere we present bromine and sodium concentrations in the NEEM ice core to investigate Canadian Arctic sea ice variability since the last interglacial period. We define the Canadian Arctic as the area from which aerosols are entrained and transported to the NEEM site, comprising the Canadian archipelago, Baffin Bay and Hudson Bay regions see . Bromine1418Bromine and sodium concentrations have been determined in the North Greenland Eemian NEEM ice core34enr as Br/(Na*0.006) using the mass ratios of Br and Na and the average sea water Br/Na mass ratio (0.00618O temperature proxy (R2 = 0.50), Brenr is positively correlated with δ18O (R2 = 0.54) supporting the connection between warm climate conditions, greater presence of FYSI and enhanced bromine photochemistry.Bromine concentrations in the NEEM ice core record vary through the Holocene as well as during the rapid interstadial warming events . The lowtio 0.006. Althoug−1, a bromine-enriched air mass would travel more than 1,000 km in 72 hours, easily encompassing the distance from the Canadian Arctic to the NEEM site. In the absence of recycling, atmospheric bromine concentrations quickly decrease to levels an order of magnitude lower than those found in the FYSI scenarios. On the basis of the model results, we proceed with the interpretation that bromine enhancement at the NEEM site is quantitatively linked to the presence of FYSI and that bromine concentrations found at NEEM are reasonably independent of transport variability. This interpretation assumes explicitly that no bromine recycling occurs over MYSI or land ice, and that bromine enrichment may be employed as a quantitative indicator of bromine recycling over FYSI.To demonstrate the quantitative link between FYSI extent and bromine enrichment in the NEEM ice core, we have applied a 1-dimensional setup of a state-of-the-art chemistry transport model featuring interactive halogen photochemistry. The model is spun up with a constant atmospheric BrO concentration over open ocean and then transports the airmass over two types of surface: FYSI over which bromine is photochemically recycled; and a stable surface over which bromine is not recycled. This stable non-reactive surface is theorised to comprise a combination of MYSI and/or Greenland ice sheet surface (land ice). Three scenarios have been investigated, in which the airmass experiences bromine recycling for 48 hours, 24 hours, or not at all . This fienr 15.6 ± 3.2 (1σ) 9.0–11.5 ky b2k), associated with the prevalence of FYSI, support existing evidence of a rapid loss of MYSI at the end of the glacial and a relatively ice-free Arctic Ocean. In comparison, bromine enrichment is lower during the last 3 ky (Brenr 10.6 ± 4.9 (1σ) 0.03–3.03 ky b2k) and displays greater variability, suggesting greater extension of MYSI in the Canadian Arctic during the late Holocene. Expansion of MYSI in recent millennia is supported by reconstructions of Arctic sea ice determined from Arctic Ocean marine sedimentsBromine enrichment in the NEEM ice core is greatest during the early Holocene, indicating a substantial increase of FYSI and corresponding decrease of MYSI in the Arctic immediately after the glacial termination . Ice-cor13enr closely following changes in δ18O temperature proxy. Bromine enrichment during GI-1 is similar to late Holocene values, indicating a mixture of MYSI and FYSI in the Canadian Arctic. Between 11.8 and 12.8 ky b2k, consistently low Brenr values indicate a return to extensive MYSI cover in the Canadian Arctic.Over the last glacial period, bromine enrichment shows variability on the scales of Dansgaard-Oeschger (DO) events. DO events are rapid climate changes involving reorganisation of atmospheric and oceanic circulation on hemispheric and interhemispheric scales. Greenland interstadials (GI) 3 to 12  demonstrenr values found for glacial and late Holocene climates  to represent late glacial conditions (see 18O-log(Brenr) relationship  for the whole NEEM dataset presented here and therefore also use log(Brenr) for our linear model of FYSI response to Holocene temperature variability. The model . The residual standard deviation (RSD) was low for both halogens and ranged between 2–10%.Concentrations of Br and Na were determined by Inductively Coupled Plasma Sector Field Mass Spectrometry  equipped with a cyclonic Peltier-cooled spray chamber TraceSELECT® NH4OH, Sigma Aldrich), then 2% HNO3 acid , with 30 s of MQ water between reagents. Between each analysis a single cleaning cycle was run to return the background to within 1% of the initial background level. Bromine was calibrated by external calibration using standards of 10 to 4,000 pg g−1, prepared by diluting a 1,000 mg L−1 standard solution  in MQ water. All calibration curves showed correlation coefficients greater than 0.99 .The analytical system was cleaned for 24 h prior to each analysis session, consisting of alternating 180 s washes of 5% ammonium solution (The Tropospheric HAlogen chemistry MOdel (THAMO)How to cite this article: Spolaor, A. et al. Canadian Arctic sea ice reconstructed from bromine in the Greenland NEEM ice core. Sci. Rep.6, 33925; doi: 10.1038/srep33925 (2016).


The cornea is the transparent outermost surface of the eye, consisting of a stratified epithelium, a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea, harboring three distinct cell types with expression of key epithelial, stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions. The cornea is the outer protective layer of the eye that provides 2/3 of the refractive power required to focus light on the photosensitive retina2345in vitro modeling of the cornea has traditionally used individual constituent cell types of the epithelium, stroma or the endothelium7in vitro. In addition, studies of the ECM types they produce usually require long-duration cultures to elicit potential differences underlying disease. Therefore, we sought to develop three-dimensional (3D) corneal organoids comprising all the component cell types of the cornea in a stable system. This provides a unique opportunity to investigate cellular disease phenotypes for long periods of time, including developmental aspects, and incorporates influences of interactions between different cell types and the ECM.Despite its relative simplicity, in vitro12Recent efforts have made tremendous gains in developing self-organizing 3-dimensional organoids from embryonic stem cells, and subsequently later from induced pluripotent stem cells (iPSC)1112131415Methodological details of producing cornea organoids (C-ORG) from iPSCs are outlined in KRT)’ KRT3 and KRT14 and these appear as bands of expected sizes in C-ORG samples  and the ECM marker Keratocan (KERA), as well as putative endothelial markers   on total RNA extracted from individual (not pooled) C-ORGs, donor corneas, as well as cultured corneal fibroblasts as references . The tra2122 samples . KRT 12  samples , and itsa sample . The PAX S100A4) 2627. TheCryo-sections of C-ORGs revealed a multi-layered architecture approximately 100 μm thick. The sections were immunostained for epithelial, stromal and endothelial markers . The cen13032TEM of the organoids at low ,C and hi37Results presented here support the development of a corneal organoid structure from human iPSCs. These organoids express markers of the three cell types of the cornea - epithelium, stromal keratocytes and the endothelium. Moreover, key extracellular matrix collagens and proteoglycan core proteins that make up the stromal matrix were present in the organoids. In the central organoid collagen fibrils were present. The fibrils demonstrated the beginning of a hierarchal structure with fibrils becoming organized into small bundles (fibers) often with a ~90 degree displacement of adjacent structures.The emergence of the cornea organoids during the development of eye cups from iPSCs should be considered in the context of signals that regulate eye development. The vertebrate eye field develops under the regulation of the transcription factors TBX2/3/5As this study relies on cell mediated self-assembly and maturation of structures, the process of differentiation is orchestrated in a manner similar to that seen in development. In contrast, other tissue engineering approaches, such as culture of differentiated stromal fibroblasts in collagen matrix, hydrogels and other types of scaffolds attempt to recreate the local environment of the mature tissue. Alternatively, cell-based therapeutic approaches engage in transplanting limbal and mesenchymal stem cells and rely on their ability to populate and repair ocular surfaces. A recent study has published a 2-dimensional culture system whereby iPSCs demonstrated the ability to organize into a “self-formed ectodermal autonomous multi-zone” that could recreate different ocular epitheliaLUM, KERA) and COL1A1 and COLVA1. Our study on the other hand used iPSCs to develop cornea organoids, where the corneal status was more stringently tested at the transcript and protein level for the presence of epithelial, stromal and endothelial markers. The organoids show remarkably appropriate immunofluorescence staining for epithelial markers KRT3, KRT14 and p63. The outer layers also show some extracellular staining of perlecan and collagen type VIII. Perlecan, a heparan sulfate proteoglycan is a major component of the epithelial basement membrane, but it is also present in the Descemet’s membrane of young corneasOne previous study has reported the development of cell spheres termed “cornea orb” from a human embryonic stem cell line and from a second cell line derived from a parthenogenetic, unfertilized eggThe organoid field in general is moving towards using induced pluripotent stem cells rather than embryonic stem cells to develop various organoids such as intestinal, kidney, liver and retinal organoidsAll experiments were carried out in accordance with the relevant guidelines and regulations set out by the Johns Hopkins Stem Cell Research and Oversight committee. Normal donor anterior stromal caps in Optisol –GS  were obtained from endothelial keratoplasty from Tissue Banks International  and the Indiana Lions Eye and Tissue Bank  under established guidelines related to informed consent for research use of human donor corneas. These experiments are covered by the Johns Hopkins Medicine IRB approved protocol entitled “Phenotypic and Genotypic Analysis of Keratoconus (NA_00006544).Stromal cells from donor corneas were extracted by serial extraction with collagenase as described before432, 10% CO2) cells were transitioned to a Neural induction medium: consisting of DMEM  with 1% B27 vitamin A (-)  and 38.8 mg/L insulin , 128 mg/L L-ascorbic acid , 28 μg/l sodium selenium , 21.4 mg/L transferrin , 38.8 mg/L NaHCO3. Osmolarity was raised +30 mOsm to ~330–340 mOsm by adding 0.88 g/L NaCl. Aggregates were transferred at D10 to 15 ml tubes, rinsed 3× in HBSS, and treated with 100 nM Smoothened agonist  from D10–D18. Long term medium was a 3:1 mix of DMEM (Invitrogen #11965):F12 (#11765: Invitrogen) with 1% B27 (#17504044: Invitrogen), 10% heat inactivated FBS , 1 mM pyruvate , 1xNEAA , 1X Glutamax  and 1 mM taurine . Vesicles were excised from D10–12 (as late as D16) with tungsten needles and fed every 2–3 days in suspension in ultra-low binding T-75 flasks (Corning) or untreated 10 cm polystyrene petri dishes. When 10 cm dishes were used, sedentary aggregates were monitored to ensure that they didn’t stick to the surface. Cells were maintained at 37 °C in standard 20% O2/5% CO2. To increase survival and differentiation, 500 nM all-trans retinoic acid  was added to the medium from D20 until D120  as per manufacturer’s instructions. 1 μg of RNA was reverse transcribed using the Superscript III RT kit . Quantitative PCR analysis was performed using the Bio-Rad CFX384 real time PCR machine, utilizing SYBR green chemistry (Applied Biosystems) 14 μl reaction volumes. Briefly, 20 nM of oligonucleotides were loaded with 3 ng of cDNA per reaction. The primer list and PCR conditions are provided in The organoids were embedded in optimal cutting temperature compound (OCT) and flash frozen; then 10 μm sections were taken on a cryostat. These were allowed to air dry for 30 minutes before being fixed in 4% paraformaldehyde in PBS for 15 minutes at room temperature. The sections were treated with 0.1% Triton X-100 in PBS for 5 minutes at room temperature. The sections were then blocked in 3% BSA 2% normal goat serum for 1 hour. Primary antibodies were then added at appropriate concentrations overnight at 4 °C in a humidified chamber .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


To assess the magnitude of difference in antibiotic use between clinical teams in the acute setting and assess evidence for any adverse consequences to patient safety or healthcare delivery.Prospective cohort study (1 week) and analysis of linked electronic health records (3 years).UK tertiary care centre.All patients admitted sequentially to the acute medical service under an infectious diseases acute physician (IDP) and other medical teams during 1 week in 2013 (n=297), and 3 years 2012–2014 (n=47 585).Antibiotic use in days of therapy (DOT): raw group metrics and regression analysis adjusted for case mix.30-day all-cause mortality, treatment failure and length of stay.Antibiotic use was 173 vs 282 DOT/100 admissions in the IDP versus non-IDP group. Using case mix-adjusted zero-inflated Poisson regression, IDP patients were significantly less likely to receive an antibiotic (adjusted OR=0.25 (95% CI 0.07 to 0.84), p=0.03) and received shorter courses (adjusted rate ratio (RR)=0.71 (95% CI 0.54 to 0.93), p=0.01). Clinically stable IDP patients of uncertain diagnosis were more likely to have antibiotics held . There was no significant difference in treatment failure or mortality , but IDP patients were more likely to be admitted overnight (adjusted OR=3.53 (95% CI 1.24 to 10.03), p=0.03) and have longer length of stay (adjusted RR=1.19 (95% CI 1.05 to 1.36), p=0.007).The IDP-led group used 30% less antibiotic therapy with no adverse clinical outcome, suggesting antibiotic use can be reduced safely in the acute setting. This may be achieved in part by holding antibiotics and admitting the patient for observation rather than prescribing, which has implications for costs and hospital occupancy. More information is needed to indicate whether any such longer admission will increase or decrease risk of antibiotic-resistant infections. Clinical relevance of study population, conducted in ‘real-life’ hospital environment.Robust risk-adjusted multivariable analysis of prescribing to show differences in antibiotic use is not an artefact of case mix.Analysis of clinical outcome as well as prescribing outcome showing antibiotic reduction was achieved safely.Data collection by single physician unblinded to management team, possibility of recording bias, though objective criteria used where possible.Single-centre study; relevance to other institutions requires further exploration.Owing to the increasing profile of the threat of antimicrobial resistance, there has been an increased focus on measuring and reducing antibiotic use.Previous studies have shown that management input from an infectious diseases acute physician (IDP) can reduce antibiotic use in the acute hospital setting. Studies have demonstrated that IDP input for patients managed under other acute physicians is associated with shorter durations of treatment, with more patients having treatment stopped or shortened.11et alet al12A few studies have compared management of acute medical patients by infectious disease and non-infectious disease-trained hospitalists.Prescribing quality assessments in the acute medical setting have suggested that up to one-third of prescriptions may be inappropriate.The goal of our study was to identify whether general medical physicians could make significant, safe antibiotic reductions, and to assess the magnitude of this achievable reduction in an entire, mixed patient cohort from point of referral to the acute medical service, rather than in selected conditions.We also wished to investigate whether there was any evidence for adverse effects from lower antibiotic use on patient outcome. We were able to do this because in our institution, a tertiary centre, some acute medical unit teams are led by an IDP. Our primary objective was therefore to assess the difference in total amount of antibiotic used between IDP-led and non-IDP-led teams. Comparison at a consultant team level reflected working practices, and previous studies supporting the commonly held view among physicians that it is the ‘senior doctor who ultimately decides what antimicrobial is prescribed’.10.1136/bmjopen-2015-010969.supp2Supplementary figuresAntibiotic prescribing data were collected prospectively on all acute admissions to the medical service in a UK tertiary care centre  by the lead author, an active physician in the department, but not working during this period. Data were collected as a benchmarking audit of antimicrobial prescribing. In this service, patients are admitted from primary care and Emergency Department referrals, and managed under an on-call consultant acute physician and their team, who share an on-call team rota with three shifts per day . The team makes initial management decisions (including antibiotic prescribing) before or during a consultant ward round of all newly admitted patients. After initial care, a small proportion of patients are transferred for operational reasons to the nearest available bed under any team or to other non-acute specialities (<5%); the majority are managed under admitting team , readmission, escalation/restarting antibiotic treatment and disease-related complications  diagnosis codes, length of stay, mortality and readmissions. Patient-level prescribing and clinical response data were not available in this data set. During this period, the IDP performed clinical duties in the acute medicine service, and other departments together with periods of leave, thus admitting ∼1% of patients in this data set.Antibiotic use was calculated using several metrics using previously published definitions,We hypothesised, based on small pilot data, that IDP management would lead to 30% less total antibiotic use, with no difference in clinical adverse composite outcome. In total, 367 patients provided at least 80% power to detect a reduction in overall antibiotic use from 300 to 210 DOT/100 admissions (30% reduction) based on 10 000 simulations from a Poisson distribution, assuming that 20% patients were admitted under IDP during audit period, overall 30% patients received antibiotics and two-sided α=0.05. Acute medicine admits 300–400 patients per week; 1 week was therefore chosen as a representative audit sample including out-of-hours and weekend prescribing.2 or Fisher's exact tests for categorical factors. Antibiotic prescribing was modelled as a two-step process , representing clinical experience and national prescribing guidance,Baseline characteristics between IDP and non-IDP groups were compared using Wilcoxon Rank Sum tests for continuous and Pearson χThe baseline antibiotic prescribing audit was approved by the Oxford University Hospitals Clinical Audit Service. Mortality and admission comparisons over 2012–2014 were an approved analysis within the Infections in Oxfordshire Research Database, which has Research Ethics Committee (14/SC/1069) and UK Health Research Authority (5-07(a)/2009) approval as an anonymised database without individual informed consent. The IDP gave consent to be identified in these data, and for the prescribing outcome analysis to be performed.During a 1-week period, the IDP-led team admitted 56 patients, and non-IDP Acute Physician teams admitted 241 patients. Teams admitted similar patients, except IDP patients had slightly lower white cell counts (WCC) and were more likely to be admitted with a frailty syndrome . Group mIn a patient-level risk-adjusted model , broad-spectrum antibiotic DOT (p=0.08), number of patients started on an intravenous antibiotic (p=0.12) or intravenous DOT (p=0.23). The reduction in total antibiotic use was not due to reduced use of antibiotic combinations; analysis by total length of therapy (which counts days on single or combination therapy as a single day) also showed a 39% unadjusted total reduction (129 vs 210 LOT/100 admissions) . AntibioThere was no difference in antibiotic prescription rates between IDP versus non-IDP groups in perceived high-risk patients , or in patients with low likelihood of infection. However, there was a difference in antibiotic use in cases of uncertain infective diagnosis without adverse features . In the There was no evidence that management under IDP was associated with risk-adjusted 30-day all-cause mortality or the composite ‘treatment failure’ outcome . Similar to our 1-week data set, this comprised patients admitted to the acute, unselected medical take, during which time the same consultant teams were operating with broadly the same antibiotic policy. Patient characteristics available in the 1 week and 3-year data sets were broadly similar in terms of age, gender, Charlson score  and admission at a weekend  an attitude in the IDP team of ‘you must justify why you gave antibiotics’ compared with ‘give antibiotics, just in case’ (as previously described by others),Study strengths include the ‘real life’ clinical relevance of the study population and robust risk-adjusted multivariable analysis of patient-level data to study prescribing and clinical outcome. Limitations include the restricted audit size, which was also smaller than planned. While we were able to subsequently examine a larger administrative data set with similar patient characteristics to assess outcome, individual-level prescribing and clinical data were not available, requiring extrapolation that IDP and non-IDP antibiotic use would have been similar over the longer period of time. During the audit, data collection was by a single clinician unblinded to management team, with possibility of recording bias for clinical factors, although objective criteria were used wherever possible. Although acute physicians were made aware of the audit and the exact date were not announced, the IDP may have been more aware of the monitoring due to closer professional relationships with the lead author, and larger differences in prescribing found due to extra attention on antibiotic use in the IDP team. The study focused on one IDP; while IDPs with similar training and integration to acute medicine exist around the UK, replicating the findings with more IDPs at different locations is required before these findings can be generalised to IDPs as a group. The study was single centre; understanding generalisability to other centres remains important. Cost impact has not been assessed, though we note that as overnight stays currently cost over £250,Our findings confirm those of other studies of the potential for antibiotic use reduction in the acute mixed-case setting. A study of 129 adult patients admitted to a US tertiary hospital found 30% of total antimicrobial days were judged unnecessary by study investigators,29et al who found IDP management was associated with shorter lengths of stay.imilar to previous studies, we found IDP input associated with a reduction in antibiotic use.Our study adds to this body of work by demonstrating reduced antibiotic prescribing using patient-level data to adjust for case mix and conducting a robust analysis of clinical outcome data to assess patient safety, providing further evidence of the potential for safe antibiotic reduction. It also examines for the first time the effects of IDP management in patients at presentation, rather than after a period of assessment and provisional diagnosis of infection being made. It reports an association with reduced antibiotic use and increased admissions in the acute setting, contrary to previous studies.13In conclusion, our study provides further support that antibiotic reduction can be achieved safely in the acute setting, but at the expense of more and longer admissions. This could be done by replacing a perceived ‘antibiotic safety net’ (prescribe and discharge) with one based on observation and greater degree of diagnostic certainty (hold and observe). While the findings require replication in other healthcare settings, they suggest that attempts to reduce antibiotic use may come at the expense of increasing admissions and exposure to the hospital environment; this may increase costs to healthcare services and the burden on already-stretched acute services. Given that admission to hospital is a risk factor for resistant infection, it may also conversely increase the spread of resistance.


Mitosis is a complex and dynamic process that is tightly regulated by a large number of mitotic proteins. Dysregulation of these proteins can generate daughter cells that exhibit genomic instability and aneuploidy, and such cells can transform into tumorigenic cells. Thus, it is important for faithful mitotic progression to regulate mitotic proteins at specific locations in the cells at a given time in each phase of mitosis. Ubiquitin-dependent modifications play critical roles in this process by regulating the degradation, translocation, or signal transduction of mitotic proteins. Here, we review how ubiquitination and deubiquitination regulate the progression of mitosis. In addition, we summarize the substrates and roles of some deubiquitinating enzymes (DUBs) crucial for mitosis and describe how they contribute error correction during mitosis and control the transition between the mitotic phases. In general, a tumor is caused by abnormal cells that undergo unrestricted divisions and proliferation. These events are the same as those that manifest upon the failure to control mitosis, and such a failure eventually results in cell death or tumorigenesis. Mitotic defects can occur at any phase of mitosis. Specifically, dysregulation of the spindle assembly checkpoint (SAC) leads to prolonged mitotic arrest and constitutes the major cause of several mitotic defects. Hence, the SAC is an important target for the development of antiproliferative chemotherapeutic strategies . Moreove(1)Prophase: Mitosis begins with the nuclear envelope breakdown (NEBD), an essential step for spindle assembly, followed by condensation of replicated DNA in the chromosome. During prophase, the two duplicated centrioles move to the opposite poles, where each pair forms a centrosome. The two centrosomes then nucleate the polymerization of microtubules from the opposite ends, forming the spindle.(2)Prometaphase: This phase is a dynamic part of mitotic progression. Microtubules rapidly assemble and disassemble by growing out from the duplicated centrosomes to find the accurate attachment site at the kinetochores of the chromosomes. The attached microtubules pull each chromosome from the opposite sites until all the chromosomes are bi-oriented and aligned.(3)Metaphase: The assembly of the mitotic spindle and its correct attachment to the kinetochore of sister chromatids are stabilized, completing the alignment of sister chromatids at the equator of the spindle for proper segregation of chromosomes toward the opposite poles of the spindle ,4,5. How(4)Anaphase: After the requirements of the SAC are satisfied, the cell enters anaphase. During this phase, microtubules attached to the duplicated chromosomes shorten from the opposite sites, separating the chromosome pairs and pulling each chromosome of a pair toward opposite spindle poles . Followi(5)Telophase: Once all the chromosomes reach their poles, the final phase of mitosis, termed telophase, begins. During telophase, the nuclear envelope reforms around the nuclei of daughter cells and chromosomes decondense .(6)Cytokinesis: This step refers to the division of the cytoplasm into two daughter cells. A cytokinetic furrow formed by the contraction of the actomyosin ring splits the cytoplasm into two domains. At this stage, two daughter cells remain connected by a narrow intracellular bridge containing antiparallel bundles of microtubules that overlap at the mid-body. The physical separation of the two daughter cells is finally accomplished by the fission of the plasma membrane via the process called abscission ,10.The cell cycle refers to a series of processes including DNA synthesis (S phase), cell growth (G1 phase), evaluation of the accuracy of the genomic materials (G2 phase), and cell division (M phase). Of these phases, mitosis (M phase), which occurs only in eukaryotic cells, is an important step in ensuring the stability of the entire genome by duplicating the genetic information and equally segregating it into two daughter cells . MitosisUbiquitination is a reversible PTM, which involves the covalent attachment of the small conserved protein ubiquitin (Ub) to a target protein, almost exclusively at a lysine residue . It requDeubiquitination is the reverse process of ubiquitination, that is, excise the conjugated monoubiquitin or polyubiquitin chains from the modified proteins . This prAlthough DUB paralogs are highly conserved in function, they can be divided into the following six families based on the architecture of their catalytic domains: ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado-Josephin proteases (MJDs), JAB1/MPN/MOV34 (JAMMs), and MIU-containing novel DUB family (MINDY). The first four families are cysteine proteases, whereas JAMMs are metalloprotease . MINDY iSeveral E3 ligases participate in mitotic control at almost every phase. Of these, the anaphase-promoting complex/cyclosome (APC/C) E3 ligase plays an important part, as it controls the metaphase-to-anaphase transition by ensuring accurate chromosome segregation and regulates the mitotic exit by mediating the degradation of key mitotic cyclins ,20,21. AModulation of the localization of Aurora B affects its functions during mitosis. During metaphase, Aurora B participates in the destabilization of erroneous microtubule attachments at the kinetochores ,33. DuriSeveral DUBs have been reported involved in mitotic progression . For exaThe SAC plays an important role against chromosomal missegregation and generation of aneuploidy progeny, which is a remarkable common characteristic of tumorigenesis, by delaying sister chromatid separation until all chromosomes achieve bipolar kinetochore–microtubule attachment. Once the last chromosome bi-orients on the mitotic spindle, APC/C activated by the binding of CDC20 ubiquitinates securin and cyclin B1 to induce their proteasomal degradation and then initiates chromosome segregation .−/− mouse embryonic fibroblast (MEF) cells [−/− MEF cells, abnormal spindle geometry and incomplete centrosome separation are increased, indicating that defects in these processes due to USP44 loss of function result in mitotic chromosome missegregation and aneuploidy. USP44 directly interacts with the centrosome component centrin and localizes to the centrosome during interphase. The DUB activity of USP44 and its ability to interact with centrin are critical for preventing chromosome lagging. Aneuploidy is very common in human lung adenocarcinoma, and reduced expression of USP44 is frequently observed in patients with lung adenocarcinoma [USP44 is a critical regulator of the SAC. Stegmeier et al. identified that USP44 is required for efficient SAC signaling and anaphase onset. They observed that the depletion of USP44 abolishes the checkpoint function of the SAC, but overexpression of a siRNA-resistant USP44 rescues the function of the SAC in USP44-depleted cells . In addiF) cells . An incrF) cells . SeveralF) cells . In USP4arcinoma . In otheUSP9X has been implicated in several disorders, including X-linked intellectual disability , ParkinsUSP9X has previously been reported to deubiquitinate and consequently stabilize the prosurvival BCL2 family member MCL1 by preventing its proteasomal degradation, thereby promoting cell survival and contributing to chemoresistance in B-cell lymphoma . HoweverRecently, Skowyra et al. showed that USP9X inhibits the degradation of SAC-controlled APC/C substrates, such as cyclin B1, cyclin A, and NIMA-related kinase 2A (NEK2A) during a mitotic arrest b. SpecifCYLD is a deubiquitinating enzyme that cleaves Lys63-linked polyubiquitin chains off its target proteins . OriginaUSP35 functions as a mitotic regulator by deubiquitinating Aurora B kinase and maintaining its stability during mitosis . Aurora While USP35 counteracts the ubiquitination effect of APC/C on Aurora B, Cezanne antagonizes the degradation of other APC/C substrates without the involvement of Aurora B. Cezanne, which is also called OTUD7B, belongs to the OTU family that has ubiquitin linkage specificity , and thiAs reviewed here, ubiquitination and deubiquitination catalyzed by E3 ubiquitin ligases and DUBs, respectively, regulate mitotic progression at all stages of mitosis. Ubiquitin-dependent modifications during mitosis have been shown to play important roles in the regulation of the key components of several events, including chromosome condensation, alignment, and segregation, for a faithful mitotic progression. Additionally, the misregulated expression of DUBs involved in the modifications supports aberrant mitotic progression, leading to the development of many types of cancers . As show


The feasibility of improving the extraction rate of common bean antioxidants by ultrasonic treatment was investigated. Scanning electron microscopy (SEM) and spectrum Fourier transform infrared spectrophotometer (FT-IR) analysis revealed that ultrasonic treatment substantially altered the cellular structure of common bean seed, resulting in increased surface area, eroded cell walls, and greater exposure of cellulose and hemicellulose. The highest antioxidant activity was obtained at optimal extraction conditions  which were optimized by response surface methodology. In terms of the extraction rate of common bean antioxidants, ultrasound-assisted extraction (UAE) exhibits about seven-fold higher extraction efficiency than conventional solvent extraction (CSE). In addition, 10 phenolic compounds in the common bean extracts were detected and quantified by high performance liquid chromatography (HPLC), including protocatechuic acid, catechin, chlorogenic acid, epicatechin, ferulic acid, coumarin, rutin, myricetin, cinnamic acid, and genistein. In summary, ultrasonic treatment is an ideal candidate methodology for improving the extraction rate of common bean antioxidants. Phaseolus vulgaris L.) are the second most important bean species in the world after soybean [In recognition of the great importance of pulses for sustainable agriculture and human nutrition, Food and Agriculture Organization of the United Nations (FAO) officially proclaimed 2016 as “the International Year of Pulses”, aiming to draw attention to these important crops. Common beans  berries [Ultrasound-assisted extraction of antioxidants in common beans is influenced by several factors such as extraction solvent, extraction time, ultrasonic power, and solvent-to-solid ratio, which can be optimized to reduce process costs . Respons berries . In addiDried speckled kidney bean, a type of common bean  was purchased from Wal-Mart Stores in Kunming, Yunnan, China, and was produced in 2017. Prior to extraction, the dried beans were ground to fine powder using a laboratory-scale dry grinder . The powder was sifted through a 40-mesh sieve to obtain kidney bean powder of particle size less than 500 μm and was stored at 4 ℃ and used within 1 month.g, 22 ± 1 ℃) and the supernatant was collected and stored at 4 ℃, and analyzed within 24 h.The ultrasonic treatment of common beans was performed in a tunable ultrasonic bath. The sample (0.5 g) and certain volume of extraction solvent were added to a 50 mL tube. Ultrasonic power, extraction time, and extraction temperature were controlled by the equipment panel. Here, the extraction solvent consisted of water and acetone in different ratios, and the detailed ratios, extraction time, extraction temperature, and ultrasonic power were based on experiment design charts. After extraction, the sample was centrifuged  and the supernatant was collected and stored at 4 ℃.In order to compare with UAE, CSE was carried out under the optimal extraction conditions obtained by response surface methodology. Samples (0.5 g) were extracted at 30 ℃ for 68 min, using 18 mL 50% acetone as solvents in an electric hot water bath . After extraction, the sample was centrifuged , μmol Fe (II)/g dry weight (DW)) and Y2 , μmol Trolox/g DW), respectively. The Pareto Chart of Standardized Effects was used to illustrate the main effect or interactive effect, which is considered to be statistically significant when its magnitude is larger than the other contrast column effects [A two-level factorial (default generators) design was applied to build the distribution matrix, which was generated by Minitab 17 software . This design comprehensively examined the effects of ultrasonic time, extraction temperature, ultrasonic power, liquid-to-solid ratio, and acetone concentration on the common bean antioxidants and screened the key factors that have significant effects on the extraction process. The low and high levels were assigned as “−1” and “+1”, respectively,  and the  effects .1, 40-60-80 min), acetone concentration , and liquid to solid ratio  were selected as the major influential independent variables, and their related coded levels of independent variables were presented in 3 central composite design was used to evaluate the effects of the three independent variables on the extraction efficiency of antioxidants, which were reflected by two dependent responses Y1 and Y2.Based on the DOE results, the extraction time  was used for data regression analysis and estimation of regression equation coefficients. An empirical model was obtained by correlating the measured responses with independent variables using multiple regression analysis. The following equation was used to predict the second−order response function for the experiments.The antioxidant activities of common bean extracts were evaluated by DPPH free radical scavenging activity (DPPH) assay and ferric-reducing antioxidant power (FRAP) assay according to our previously published work , and thev/v) formic acid in water) and solvent B (0.1% (v/v) formic acid in methanol). The analysis was performed by a gradient elution program: 0 min, 5% B (v/v); 3 min, 10% B (v/v); 13 min, 28% B (v/v); 23 min, 45% B (v/v); 38 min, 70% B (v/v); 43 min, 90% B (v/v); 48–63 min, 95% B (v/v). The sample injection volume was 20 μL, and the flow rate was set at 1.0 mL/min. Phenolic compounds were identified by comparison with the retention time and UV spectra of standards, and their contents were calculated based on the peak area under the maximum absorbance and calibration curve of the corresponding standard at 10–100 μg/mL. The results were expressed as mg/100g DW.The supernatant obtained under optimal extraction conditions was collected and lyophilized and then dissolved in methanol for HPLC analysis. Major phenolic compounds in the common bean extracts were identified and quantified using Shimadzu LC-20AR HPLC system  equipped with a Shim-pack GIS C18 reversed phase column , a binary pump, and a diode array detector . The mobile phase consists of solvent A  was used to observe the surface structures of raw and treated samples. A spectrum Fourier transform infrared spectrophotometer  was applied to analyze the infrared signatures of the raw and treated common beans.p value less than 0.05 defined as statistical significance. The statistical software Design Expert 8.06 , IBM SPSS Statistics 19 , Minitab 17 software , and Origin 8.5  were used for statistical analysis.All the measurements were performed in triplicate, and the results were expressed as mean standard deviation (SD), with DOE technique offers a structured approach for generating a significance test of factor levels and providing a prediction model for response. Here, a two-level factorial (default generators) design was used to build the distribution matrix and the results are shown in p < 0.0001) for FRAP and DPPH values. The lack of fit for both models was not significant (p > 0.05), which indicated that the established models fully explain the relationship between independent variables and responses. The R2 and adjusted R2 (Adj. R2) were close to 1, indicating a high degree of correlation between the predicted values and experimental values. Moreover, the predicted R2 (Pred R2) of each model was in reasonable agreement with the Adj. R2, indicating that the model predicts the response value very well. The Adeq Precision of each model is larger than 4, indicating that the model can be navigated in the design space. Furthermore, the coefficient of variance (CV) of each model was rather low (CV < 3%), which further supported the good adaptability of the model, thereby assuring better reproducibility. The resulting response surface 3D graph corresponding to each response is shown in Experimental modeling results for antioxidant activity  show tha1, X2, X3), quadratic (X2X3), and interactive , acetone concentration (X2), and liquid-to-solid ratio (X3) showed a highly significant (p < 0.0001) positive effect on FRAP; moreover, their quadratic terms (p < 0.0001) negative effect on FRAP. The interaction between the acetone concentration and liquid-to-solid ratio (X2X3) revealed a significant (0.0024) negative effect on FRAP  and liquid-to-solid ratio (X3) showed a highly significant (p < 0.0001) positive effect on DPPH, and their quadratic terms (p < 0.0001) negative effect on DPPH; moreover, p = 0.0210) negative effect on DPPH. The interaction between the extraction time and acetone concentration (X1X2) had a significant (p = 0.0004) negative effect on DPPH. The interaction between the extraction time and liquid-to-solid ratio (X1X3) revealed a significant (p = 0.0421) positive effect on DPPH  were estimated by RSM, taking into account only the significant terms, which were shown in Equations (2) and (3):R2 value  fairly matched the adjusted R2 value   .2) and liquid-to-solid ratio (X3) revealed a positive effect for both responses. The interactive effects exhibited quite different patterns for the two responses (Y1 and Y2). Similar results were also obtained by others [In brief, acetone concentration /g DW and 50.9 μmol Trolox/g DW, which were well matched with the predicted values of 77.2 μmol Fe(Ⅱ)/g DW and 51.8 μmol Trolox/g DW with the coefficient of variance values of 2.68% and 1.84%, respectively.Two extraction methods (UAE and CSE) were compared with regard to their extraction efficiency. These two extraction methods were performed under the same optimum extraction conditions obtained by central composite design (CCD) .In terms of extraction efficiency, UAE gave FRAP and DPPH values of 75.2 μmol Fe(Ⅱ)/g DW and 50.9 μmol Trolox/g DW, respectively, while CSE offered 9.87 μmol Fe(Ⅱ)/g DW (FRAP) and 7.76 μmol Trolox/g DW (DPPH), indicating that the UAE method yielded about eight-fold higher FRAP and about seven-fold higher DPPH than CSE method. Producing higher antioxidant activities through UAE can be attributed to the ultrasonic wave facilitating solvent penetration into the sample matrix and increasing the rates of mass transfer of antioxidants to the extraction solvent . SimilarPhaseolus vulgaris L.) [Ten polyphenolic antioxidant compounds in the extracts, including protocatechuic acid, catechin, chlorogenic acid, epicatechin, ferulic acid, coumarin, rutin, myricetin, cinnamic acid, and genistein, were detected and quantified by HPLC with standard curves. The chromatography of standards is shown in aris L.) . In addiaris L.) . In addiaris L.) . Interesaris L.) . Flavonoaris L.) . Phenoliaris L.) . TherefoDifferences of the common bean slurries before and after ultrasonic treatment a show en2O molecules and produces hydroxyl and hydrogen radicals, which then excite a chain reaction. The chain reaction produces more free radicals or oxidizing species such as hydrogen peroxide, oxygen, and ozone [−1 was enhanced, and it represented the stretching vibration of O–H and hydrogen bonds in phenolic and aliphatic structures [−1 represented the stretch vibrations of the C–H bond in the methylene of cellulose [−1 change, which may be related to the demethylation of the syringyl structure of lignin [−1 is the C–H signal in planar deformation of glucosyl group in lignin [−1, which is assigned to C–O stretching and is likely the C–O bond of cellulose and hemicellulose [In addition to the physical effects of ultrasound, the sonochemical effect is another important factor in the change of the physicochemical properties of common beans. The sonochemical effects originate from a series of complex free radical reactions in water. Initially, ultrasonication cleaves the O–H bonds of Hnd ozone . The oxiructures . The enhellulose ,33. The f lignin . The enhn lignin . These rellulose . This inIn this study, ultrasound-assisted extraction of common bean antioxidants was optimized by RSM. The optimal extraction parameters were determined, and the predicted values were close to the experimental values. The optimal UAE conditions for common bean antioxidants included an extraction time of 68 min, extraction solvent concentration of 55%, liquid-to-solid ratio of 36 mL/g, extraction temperature of 30 ℃, and ultrasonic power of 480 W. Under these optimal conditions, the maximum FRAP and DPPH values of common beans were 75.2 μmol Fe(Ⅱ)/g DW and 50.9 μmol Trolox/g DW, respectively.Compared to conventional extraction methods, ultrasonic treatment significantly increases the extraction rate of antioxidants, which were further analyzed using HPLC, and 10 phenolic compounds were detected, including protocatechuic acid, catechin, chlorogenic acid, epicatechin, ferulic acid, coumarin, rutin, myricetin, cinnamic acid, and genistein. In addition, the effects of ultrasound on common bean structures were analyzed using SEM and FT-IR, finding that ultrasonic treatment led to a loose structure and erosion of the surface of common beans, which could result in more rapid and greater penetration of the solvent into the plant material.


Platycarya strobilacea is a rich source of ellagic acid (EA) which has shown antioxidant, anticancer and antimutagen properties. Response surface methodology (RSM) was used to optimize the conditions for ultrasonic extraction of EA from infructescence of P. strobilacea. A central composite design (CCD) was used for experimental design and analysis of the results to obtain the optimal processing parameters. The content of EA in the extracts was determined by HPLC with UV detection. Three independent variables such as ultrasonic extraction temperature (°C), liquid:solid ratio (mL/g), and ultrasonic extraction time (min) were investigated. The experimental data obtained were fitted to a quadratic equation using multiple regression analysis and also analyzed by appropriate statistical methods. The 3-D response surface and the contour plots derived from the mathematical models were applied to determine the optimal conditions. The optimum ultrasonic extraction conditions were as follows: ultrasonic extraction temperature 70 °C, liquid:solid ratio 22.5, and ultrasonic extraction time 40 min. Under these conditions, the experimental percentage value was 1.961%, which is in close agreement with the value predicted by the model.The infructescence of  Extracts from red raspberry leaves or seeds, pomegranates, or other sources are said to contain high levels of EA, and are available as dietary supplements in capsule, powder, or liquid forms. A recent profusion of pomegranate nutraceutical products, ‘‘standardized to 40% EA” has appeared in the marketplace [Platycarya strobilacea Sieb. et Zucc, EA is present in the heartwood, as reported by Tanaka et al. [P. strobilacea. Polyphenols are widely distributed in the plant kingdom and are important components of common foods, including tea, red wine, fruits, beverages and various medicinal plants. The importance of polyphenols arises from their effects on sensory properties, including astringency and colour, and possible health effects that they may have. One of the polyphenols is ellagic acid (EA), a dimeric derivative of gallic acid, which mainly exists in higher plants, including fruits and flowers, combined with its precursor, hexahydroxydiphenic acid or bound in the form of ellagitannins . EA was ketplace . EA was ketplace . Plants ketplace . In the a et al. , but theP. strobilacea can be affected by many factors including ultrasonic extraction temperature, liquid:solid ratio, and ultrasonic extraction time. In such situations, where multiple variables may influence the extraction yield, response surface methodology (RSM) is an effective technique for optimizing the process [The efficiency of the extraction of EA from the infructescence of  process ,11. The  process . As a po process .P. strobilacea using response surface methodology (RSM) employing a CCD (three factors and five levels) to study the effects of ultrasonic extraction temperature, liquid:solid ratio, and ultrasonic extraction time on the extraction yield of EA.The purpose of the present study was to optimize the process for production of EA from the infructescence of P. strobilacea, as shown by a typical HPLC chromatogram of the extracts ; x2, ultrasonic extraction time (min); x3, ultrasonic extraction temperature (°C). ANOVA was used to evaluate the significance of the coefficients of the models. The regression coefficient values of equation are listed in P-values were used as a tool to check the significance of each coefficient, which in turn may indicate the pattern of the interactions between the variables. For any of the terms in the model, a large regression coefficient and a small P-value would indicate a more significant effect on the respective response variables [P, the more significant was the corresponding coefficient. It can be seen from this table that the linear coefficients  and the quadratic term coefficients  were significant, with very small P-values (P < 0.05). The other term coefficients were not significant (P > 0.05). Response surface optimization is more advantageous than the traditional single parameter optimization in that it saves time, space and raw materials. A total of 20 runs were needed for optimizing the three individual parameters in the current CCD. ariables . Thus, tP > 0.05) are eliminated from the model through a “backward elimination” process. The statistical parameters obtained from the ANOVA for the reduced models are given in P < 0.05 is obtained, implying that these models are significant. The adequate precision value is a measure of the “signal (response) to noise (deviation) ratio”. A ratio greater than four is desirable [To obtain a simple and yet realistic model, the insignificant terms . In the figures, the maximum predicted value indicated by the surface was confined to the smallest ellipse in the contour diagram. Elliptical contours are obtained when there is a perfect interaction between the independent variables ,17. The In P. strobilacea were ultrasonic extraction temperature 70 °C, liquid:solid ratio 22.5, and ultrasonic extraction time 40 min. Among the three extraction parameters studied, ultrasonic extraction temperature was the most significant factor affecting the EA extraction yield, followed by liquid:solid ratio, and ultrasonic extraction time according to the regression coefficients significance of the quadratic model  was collected from the ground in Liuan, Anhui Province, China. The infructescence was ground in a knife mill and the powdered sample was sieved to select particles smaller than 1 mm. EA was purchased from Sigma Chemical Co. . The solvents methanol and trifluoroacetic acid (TFA) were of analytical reagent (AR) purity grade. The CH3CN used for the analysis were of HPLC grade. Deionized water was used throughout.Dried infructescence of Y) was as follows:Y represents the response variable, a0 is a constant, ia, iia and ija are the linear, quadratic and interactive coefficients, respectively. iX and jX are the levels of the independent variables. The software uses this quadratic model to build the response surface. The adequacy of the model was determined by evaluating the lack of fit, coefficient of determination  and the Fisher test value  obtained from the analysis of variance (ANOVA) that was generated by the software. Statistical significance of the model and model parameters was determined at the 5% probability level (α = 0.05). Three-dimensional surface response plots were generated by varying two variables within the experimental range and holding the other constant at the central point. RSM was applied to evaluate the effects of ultrasonic extraction temperature, liquid:solid ratio, and ultrasonic extraction time on the yields of EA. RSM was performed using the Design-Expert software program version 7.0. The coded and uncoded independent variables used in the RSM design are listed in et al. [The EA was extracted according to the method described by Bianco et al. . An amou3CN with the solvent programmed as follows: 0-8th minutes 7% B, 8th-25th minutes 7%-32% B, 25th-30th minutes 32%-35% B, 30th-35th minutes 35% B. EA peak was identified by comparing its retention time with those of standards and the concentration was calculated from the calibration curves. The analysis was carried out in triplicate.The polyphenol fraction was isolated by methanolic extraction and EA was determined in the extracts by HPLC with UV detection. The detector was operated at 357 nm wavelength which corresponded to the experimentally found maximum absorption of the EA standard. An aliquot of the extract were filtered through a 0.45 µm syringe filter prior to HPLC-UV analysis. EA was separated using an Inertex C18 column . The solvent flow rate was 1 mL/min and the mobile phase was composed of solvent (A) water  and solvent (B) CHExperimental data was analyzed by multiple regressions to fit the quadratic equation to all independent variables. Analysis of variance (ANOVA) was performed to evaluate significant differences between independent variables. To visualize the relationships between the responses and the independent variables, surface response and contour plots of the fitted quadratic regression equations were generated using Design-Expert software version 7.0.The extraction conditions have a significant effect on the EA extraction yield. The use of the contour and surface plots in RSM was effective for estimating the effect of three independent variables . The optimum set of the independent variables was obtained graphically in order to obtain the desired levels of EA extraction. An optimal experimental extraction yield of 1.961% was obtained when the optimum conditions of EA extraction  were used. Under these optimized conditions the experimental EA extraction yield agreed closely with the predicted yield of 2.028%.