Activation of the hypoxia inducible transcription factor HIF and the NF-ĸB pathway promotes inflammation-mediated tumor progression. The cellular transcription factor ZNF395 has repeatedly been found overexpressed in various human cancers, particularly in response to hypoxia, implying a functional relevance. To understand the biological activity of ZNF395, we identified target genes of ZNF395 through a genome-wide expression screen. Induced ZNF395 expression led to the upregulation of genes known to play a role in cancer as well as a subset of interferon (IFN)-stimulated genes (ISG) involved in antiviral responses such as IFIT1/ISG56, IFI44 and IFI16. In cells that lack ZNF395, the IFN-α-mediated stimulation of these factors was impaired, demonstrating that ZNF395 is required for the full induction of these antiviral genes. Transient transfections revealed that ZNF395-mediated activation of the IFIT1/ISG56 promoter depends on the two IFN-stimulated response elements within the promoter and on the DNA-binding domain of ZNF395, a so-called C-clamp. We also show that IĸBα kinase (IKK)-signaling is necessary to allow ZNF395 to activate transcription and simultaneously enhances its proteolytic degradation. Thus, ZNF395 becomes activated at the level of protein modification by IKK. Moreover, we confirm that the expression of ZNF395 is induced by hypoxia. Our results characterize ZNF395 as a novel factor that contributes to the maximal stimulation of a subset of ISGs. This transcriptional activity depends on IKK signaling further supporting a role of ZNF395 in the innate immune response. Given these results it is possible that under hypoxic conditions, elevated levels of ZNF395 may support inflammation and cancer progression by activating the target genes involved in the innate immune response and cancer. Gene expression arrays repeatedly found the expression of the cellular factor ZNF395  significantly increased in various cancers such as renal cell carcinomas, osteosarcomas and Ewing sarcomas ,2,3,4. NHypoxia reflecting oxygen deficiency often occurs in developing tumors and is also a characteristic feature of acute foci of tissue inflammation. Prolyl hydroxylases (PHDs) hydroxylate HIF-α subunits and target them to ubiquitin-dependent degradation. Under conditions of hypoxia, PHDs are inhibited. HIF-α subunits then accumulate and translocate to the nucleus where they dimerize with their stable partner HIF-1β, and bind to their target sequence known as hypoxia response element (HRE). HIF induces the expression of more than 100 genes regulating glucose metabolism, cell proliferation and cell survival as well as genes that drive angiogenesis and induce immune tolerance . TheWe have isolated the open reading frame (ORF) for ZNF395 through its ability to bind to regulatory sequences within the control region of several papillomavirus types (PV) . It was 2 gas into the incubator to obtain an O2 concentration of 2%. Cells were then incubated for 12h and immediately harvested.The human skin cancer cell line RTS3b is described in  and was pcDNA3.1-FLAG-ZNF395, pcDNA3.1-FLAG-ZNF395ΔCR1 and pcDNA3.1-FLAG-ZNF395mtCR3 were described previously ,25. pcDNhttp://www.ncbi.nlm.nih.gov/geo) and assigned the accession number GSE44327.Total RNA was prepared by the RNeasy Mini Kit from Qiagen. cDNA synthesis and hybridization to Affymetrix Exon 1.0 ST array was performed by the group of Prof. Nürnberg . The raw data were processed using Affymetrix Power Tools, version 1.12.0 and the Robust Multiarray Average (RMA) algorithm . DownstrSmall interfering RNAs  were obtained as a pool of four annealed double-stranded RNA (dsRNA) oligonucleotides: IKKα (M-003473-02) and IKKβ M-003503-02) from Dharmacon, ZNF395 (sc-77820) from Santa Cruz. As a control, siRNA against the oncogene E6 of HPV8 (8E6) [03-02 froTGAGATCTGGCTATTCTGTCTTGTGG-3; reverse 5´-ATGGTTGCAGGTCTGCAGTTTATCTG-3´). Gel shifts were performed as described previously [5´-TTTAGTTTCACTTTCCCCTTTCGGTTTCCCTAGGT-3´; ISRE-mut114/101: 5´-TTTAGTGTCACTTTCCCCTGTCGGTTTCCCTAGGT-3´, which were labeled with 32P-γ-ATP or used as competitors.The rabbit polyclonal antibody against ZNF395 was described previously . The M2 eviously  with theWe have isolated the cDNA for ZNF395 from HaCat cells . For thiThe finding that six of the genes identified in the microarray are known regulated IFN genes implied a role of ZNF395 in the IFN-α-mediated stimulation of these target genes. In order to analyze the contribution of ZNF395 we transiently transfected normal RTS3b cells with control siRNA (siControl), which was directed against the HPV8E6 oncogene that is not expressed in these cells, or siRNA targeting ZNF395 (siZNF395) and incubated the cells 40h later with IFN-α for 6h. QRT-PCR revealed that the expression of ZNF395 was not altered in the siControl transfected cells due to IFN-α, indicating that ZNF395 is not regulated by this signal. The ZNF395-specific siRNA reduced the ZNF395 expression more than 90% . IFN-α iTo investigate the role of ZNF395 in the induction of ISG56, we analyzed recombinant ZNF395 in transient transfection assays with a reporter construct containing 654bp of the upstream control region of ISG56 that includes the promoter. Overexpression of ZNF395 induced the ISG56 promoter 4-fold, which is in the range observed in the microarray . We idenWe then determined the sequence requirements of ZNF395 in the ISG56 promoter. As shown in The results described so far show that ZNF395 is required for the full induction of ISG56, IFI44 and IFI16. These IFN-stimulated factors are known to be part of an antiviral innate immune response. In addition, ISG56 can be directly activated by IRF3 upon TLR3 signaling in response to dsRNA. TLR3 is expressed in cells of the immune system and epithelial cells, as are RTS3b (data not shown).We wanted to analyze the fate of ZNF395 within this pathway by transient transfections. Incubating transfected cells with IFN-α increased the ISG56 promoter in the ISG56-Luc construct by 6-fold, which was not affected by the co-expression of ZNF395 . When weWe confirmed the role of IKK in controlling the stability of ZNF395 by siRNA-mediated knockdown. Transfecting U87-MG cells with increasing amounts of siRNAs targeting either IKKα or IKKβ, resulted in the dose-dependent detection of endogenous ZNF395. This was not observed with the control siRNA, which was used in the highest amounts , lane 8 The impact of IKK signaling on ZNF395-mediated activation of the ISG56 promoter was analyzed by treating transiently transfected cells with BMS-345541. Although the concentration of the protein was increased in the presence of the inhibitor, as revealed in 2 atmosphere, and found a 5.3- and 1.7-fold increase of ZNF395 expression due to hypoxia, respectively. In correlation, endogenous ZNF395 became detectable by IB with protein extracts from these cells when they were incubated in 2% O2 atmosphere  that functions as an IKK phosphorylation motif and binding site for the ubiquitin ligase β-Trcp in some IKK substrates such as CBP, IĸBα, p105, p100, β-catenin and FOXO3a . We are Our data suggest that IKK-activity is required for ZNF395 to activate transcription and directs ZNF395 to ubiquitin-mediated degradation. The IKK-induced turnover of ZNF395 may contribute to promoter clearance to allow reinitiation of transcription. A similar regulation has been described for IRF3. IRF3 undergoes virus-dependent phosphorylation mediated by IKKε/TBK1, which results in the nuclear translocation, DNA binding and increased transcriptional activation. Phosphorylation of IRF3 in response to virus infections also provides a signal for proteasomal degradation . MoreoveOur results illustrate that the expression of ZNF395 is not affected in response to IFN-α, but is induced by hypoxia. Correlatively, ZNF395 was found to be upregulated as part of a response to hypoxia in glioblastomas and neuroblastomas  and in aThe results of the microarray also revealed an increased expression of four genes known to be associated with cancer upon induction of ZNF395. These include the transcription factor MEF2C, found to be activated in a subset of T-acute lymphoblastic leukemia cell lines , CALCOCOTaken together, we show that ZNF395 is required for the full induction of several ISGs and is thus part of the innate immune response. This is supported by the observation that the transcriptional activity of ZNF395 depends on IKK signaling. Furthermore, the finding that the target genes of ZNF395 are involved in the innate immune response and cancer, implies that ZNF395 may support inflammation and cancer progression under hypoxia, which deserves further investigation.


Resveratrol, extracted from Chinese herbal medicine Polygonum cuspidatum, is known to inhibit invasion and metastasis of human colorectal cancer (CRC), in which long non-coding Metastasis Associated Lung Adenocarcinoma Transcript 1 (RNA-MALAT1) also plays an important role. Using MALAT1 lentiviral shRNA and over-expression constructs in CRC derived cell lines, LoVo and HCT116, we demonstrated that the anti-tumor effects of resveratrol on CRC are through inhibiting Wnt/β-catenin signaling, thus the expression of its target genes such as c-Myc, MMP-7, as well as the expression of MALAT1. In detail, resveratrol down-regulates MALAT1, resulting in decreased nuclear localization of β-catenin thus attenuated Wnt/β-catenin signaling, which leads to the inhibition of CRC invasion and metastasis. This finding of ours surely provides important pre-clinical evidence supporting future use of resveratrol in prevention and treatment of CRC. Colorectal cancer is the results of the mutation of multiple genes including proto-oncogenes and tumor suppressor genes. As the oncogenes controlling cell proliferation remaining highly expressed, or the tumor suppressor genes being mutated, the resulting cancerous cells evade immune system, form tumors in distal locations/organs, i.e. metastasis and the terminal stage of cancer begins The emergence of new Chinese medicine monomer anticancer drugs has provided a new option to the reptoire of synthetic drugs for cancer treatment et al firstly identified long non-coding RNA - MALAT1. In 225 cases of stage I non-small cell lung cancer (NSCLC), it was found in 70 cases, metastasis correlates with MALAT1 over-expression, in a course and tissue specific manner, suggesting that MALAT1 expression can serve as a potential marker of survival in stage 1 NSCLC patients Early in 2003, Ji et al found that in oral squamous cell carcinoma cells, the accumulation of β-catenin in the cytoplasm induces TCF/LEF transcriptional activity, and increase the MMP-7 expression, thereby inducing the conversion of epithelial cells to mesenchymal cells as well as enhancing invasion and metastasis et al demonstrated in CRC HT29 cell line, NGX6 gene product inhibited transferring of the β-catenin from the nucleus and cytoplasm to the cell membrane, thereby inhibiting the transcriptional activity of TCF and down-regulating the expression of Wnt target genes c-Myc, cyclinD1 and COX-2, leading to decreased cancer cell invasion and metastasis Many studies have shown that Wnt/β-catenin signaling pathway regulates tumor cell invasion and metastasis. Soichi Our present studies interrogated the mechanisms by which resveratrol regulates MALAT1 and Wnt/β-catenin signal pathway, resulting in repressed cancer cell invasion and metastasis.et alParaffin-embedded tumor and adjacent normal tissue samples from 60 CRC patients who underwent tumor resection at Putuo Hospital, Shanghai University of Traditional Chinese Medicine (SUTCM)between 2010 and 2012 were selected for hybridization with digoxigenin (DIG)-labeled MALAT1 DNA probe . The experiment was performed according to the method described by Tanner 5-AGGCGTTGTGCGTAGAGGA-3), reverse (5-GGATTTTTACCAACCACTCGC-3), TaqMan probe (5-fam+CCTAGACCAGCATGCCAGTGTGCC+tamara-3). Real time PCR was performed in the Applied Biosystems 7300 System (Applied Biosystems Deutschland GmbH) using Premix Ex Taq . GAPDH was used as internal reference.LoVo cells  were cultured in F12K medium supplemented with 10% fetal bovine serum, 100 g/ml streptomycin, 100 U/ml penicillin, at 37°C, 5% CO2, and high humidity. All the Chinese medicine monomer anticancer drugs were diluted serially to detect the half inhibition concentration on proliferation of LoVo cells. Proliferation of LoVo cells was determined by Cell Counting Kit-8 (CCK-8) assay as described previously Human MALAT1 cDNA (Gene ID: 378938) was amplified by RT-PCR with the RNA extracted from LoVo cells, and then cloned into pLV4 vector. MALAT1 were searched for suitable siRNA target sequences, three siRNA sequences were designed, synthesized, and confirmed by sequencing respectively. Lentiviral particles were produced by co-transfecting expression vector pLV4-MALAT1 cDNA or pLV4-MALAT1 shRNA with viral particle packaging helper vector into 293T cells. Preliminary experiments picked out the most efficient siRNA sequences of knockdown from the above mentioned three candidate siRNA : sense 5GAGAATT-3′. Titer of viral particles containing MALAT1 shRNA and MALAT1 cDNA was determined by limited serial dilution. The efficiency of knockdown or over-expression was determined by real time PCR. LoVo cells or HCT116 cells were infected with the lentivirus with the pLV4-MALAT1 cDNA, pLV4-MALAT1 shRNA or pLV4-vector.To test MALAT1 promoter or LEF/TCF promoter activity, LoVo cells were co-transfected with either the recombinant plasmid pGL3-basic-MALAT1 promoter or pGL3-basic-LEF/TCF promoter with a control positive plasmid pRL-SV40 as previously described 5 LoVo cells cultured in 100 µl F12K with 0.5% FBS were seeded into the upper part of a transwell chamber . For migration analysis, in the lower part of the chamber, 600 µl F12K with 15% FBS and 10µg/ml fibronectin was added and the assay was performed for 24 hours at 37°C and 5% CO2. Migrated cells were analyzed by crystal violet staining, followed by observing under a DMI3000 B inverted microscope . For invasion analysis, 100 µl matrigel  was firstly added unto the bottom of the transwell chamber before LoVo cells were seeded, and the following procedures were the same as migration analysis, except for the invasive cells being analyzed after co-culture for 48 hours. As to HCT116 cells, the procedures were similar, the only difference was cells being cultured with RIPM 1640.A total of 5×10Whole cell, cytoplasm, and nuclear proteins from LoVo or HCT116 cells were prepared according to the manufacturer’s instructions of ProteoJET Cytoplasmic and Nuclear Protein Extraction Kit . Proteins were loaded onto the SDS-PAGE gels for electrophoresis, transferred to PVDF membranes and blocked in 5% milk prior to incubation with the indicated primary antibodies and the secondary antibodies. The resulting immunocomplexes were visualized by enhanced chemiluminescence. Protein loading was normalized by β-actin (whole protein or cytoplasm protein) or PCNA (nuclear protein).RNA-binding protein immunoprecipitation (RIP) was performed according to the manufacturer’s protocols from the EZ-Magna RIP Kit . Briefly, LoVo cells were rinsed with ice-cold phosphate buffered saline (PBS), lysed by RIP lysis buffer. Both β-catenin  and nonspecific control rabbit IgG antibodies were used for the immunoprecipitation. RIP lysates and magnetic beads-bound β-catenin antibody were incubated together with rotating for overnight at 4°C. Afterwards, proteins in the immunoprecipitate were digested with proteinase K and bound RNAs were purified from the supernatants, reverse-transcribed using PrimeScript RT reagent Kit followed by quantitative analysis.5) were grown on chamber slides  for 8 hours, starved for 12 hours. Cells were fixed for 40 minutes with 4% paraformaldehyde in PBS at room temperature, permealized and blocked with 5% non-fat dry milk, 1% BSA, 0.5% Triton X-100 and incubated overnight with rabbit monoclonal anti-β-catenin . After washing the slides, Cy3-conjugated anti-rabbit IgG antibody  was added in blocking solution for 1 hour. 4–6-diamidino-2-phenylindole was applied to stain nuclei. Immunofluorescence images were taken with a DMI3000B inverted microscope .LoVo cells  than the adjacent normal colorectal tissue  of all candidate drugs . Next weNext, we found that resveratrol attenuated the cellular localization of β-catenin, a protein regulating cancer cell invasion and metastasis. As shown in We next studied the expression of downstream β-catenin target genes in the presence of resveratrol, for the main role of β-catenin in nucleus is through its regulation on gene transcription. Unsurprisingly, we found the expression of β-catenin downstream target genes c-Myc and MMP-7 was significantly reduced by resveratrol, in a dose-dependent manner . These dFurthermore, we discovered that resveratrol down-regulated the expression of long non-coding RNA-MALAT1 in a dose-dependent manner . To veriNext, using lentivirus-mediated constructs, we knocked down or over expressed MALAT1 gene expression in LoVo cells. Our data clearly demonstrated that the proliferation and invasion/migration capability of LoVo cells were significantly decreased by MALAT1 knockdown, and increased by MALAT1 over-expression .When we knocked down, MALAT1 gene expression using shRNA construct in LoVo cells, we were surprised to find that β-catenin protein was retained in cytoplasm while its presence in nuclei was remarkedly increased . AdditioTo confirm our finding of resveratrol suppressing migration and invasion of LoVo cells through MALAT1, a MALAT1 rescue experiment was performed. Our results demonstrated that lentivirus-mediated overexpression of MALAT1 significantly attenuated resveratrol-induced inhibition on cell migration and invasion in LoVo cells . In otheWe hypothesized that resveratrol inhibited the Wnt/β-catenin signal pathway through MALAT1 regulation. To test this hypothesis, we used immunostaining and Western blot analyses to observe whether β-catenin trans-located from cytoplasm to nuclear with the presence of resveratrol and manipulation of MALAT1 expression levels. To make the effect more obvious, we used LiCl to inhibit the GSK3β from binding to β-catenin protein, to increase the active status of the Wnt/β-catenin signal pathway. Our results established, as LiCl indeed induced the translocation of β-catenin protein from cytoplasm to nucleus, resveratrol (50 µM) significantly impeded this process . SubsequMoreover, we tested the effect of resveratrol on Wnt/β-catenin signaling using a reporter LEF/TCF promoter dual-luciferase construct. Our results clearly indicated that, resveratrol inhibited the promoter activity of LEF/TCF induced by LiCl, and co-repression of MALAT1 led to the enhancement of this effect, as co-overexpression of MALAT1 opposing it . All theTo confirm our key findings in LoVo cells, we tested another CRC HCT116 cell line using similar approaches. Our results showed that resveratrol inhibited the proliferation of HCT116 cells by the IC50 of 100 µM . HCT 116In addition, resveratrol also significantly inhibited the migration and invasion ability of HCT116. As in LoVo cells, overexpression of MALAT1 rescued HCT116 cells from suppression of migration and invasion by resveratrol . Westernet al has found that resveratrol has anti-cancer characteristic because of its repressive effect on protein-tyrosine kinase activity Colorectal cancer is one of the most common malignant tumors in the world, with high rate of recurrence and metastasis. The mechanisms of recurrence and metastasis remain very complicated. Currently, the treatment of colorectal cancer is mainly surgical resection, combined with chemotherapy, immunotherapy, and other alternative cares such as traditional Chinese medicine. Traditional Chinese medicine and its more recently derived extracted monomer treatment can improve cancer symptoms, reduce cancer metastasis, as well as reduce the risk of recurrence of colorectal cancer. However, the therapeutic targets and mechanisms remain elusive. Among these extracted monomers, resveratrol is a perfect example. As early as in 1993, Jayafilake Being the key transcriptional activation factor of Wnt/β-catenin signal pathway, upon upstream activation, β-catenin often translocates to the nucleus from cytoplasm, in coordination with other transcription factors such as TCF/LEF, to activate its target genes c-Myc, cyclinD1, MMP-7, and CD44, which in turn play pivotal role in tumor initiation and development Long non-coding RNA-MALAT1 was firstly reported in the invasive non-small cell carcinoma, and lately being found over-expressing in many other cancer tissues, that indicated MALAT1 is associated with invasion and metastasis By using a LEF/TCF promoter dual-luciferase reporter construct and activating the Wnt/β-catenin signal pathway with LiCl, we further demonstrated that knockdown of MALAT1 enhanced the inhibitory effect of resveratrol on the promoter activity of LEF/TCF induced by LiCl, while over-expression of MALAT1 weakened it. All these results suggested that the underlining mechanism of resveratrol may be through a repression of Wnt/β-catenin signaling, via down-regulation of MALAT1 expression.et al, β-catenin : LoVo cells were infected with the lentivirus with three pLV4-MALAT1 shRNAs or pLV4-vector. Their efficiencies of knockdown were detected by real time PCR.*p<0.05 or **p<0.01, compared with vector control. (B): MALAT1 mRNA expression levels were measured by real time PCR, CRC tissues and adjacent normal tissues. **p<0.01, compared with normal tissues or CRC tissues with no metastasis. (C): Immunofluorescent staining of β-catenin in CRC tissues and adjacent normal tissues.(TIF)Click here for additional data file.


Aberrantly activated Wnt signaling can result in the abnormal stabilization of β-catenin, a key causative step in a broad spectrum of cancers. Here we report the modulation of lithium chloride-activated canonical Wnt/β-catenin signaling by phytochemicals that have antioxidant, anti-inflammatory or chemopreventive properties. The compounds were first screened with a cervical cancer-derived stable Wnt signaling reporter HeLa cell line. Positive hits were subsequently evaluated for β-catenin degradation, suppression of β-catenin nuclear localization and down-regulation of downstream oncogenic targets of Wnt/β-catenin pathway. Our study shows a novel degradation path of β-catenin protein in HeLa cells by Avenanthramide 2p (a polyphenol) and Triptolide (a diterpene triepoxide), respectively from oats and a Chinese medicinal plant. The findings present Avenanthramide 2p as a potential chemopreventive dietary compound that merits further study using  The β-catenin also accelerates human papilloma virus type-16 mediated cervical carcinogenesis in transgenic mice . Intriguingly β-catenin is thought to influence the metastatic potential of tumor cells by affecting chromatin remodeling Members of the Wnt family of secreted growth factors play important roles during embryogenesis by regulating proliferation, migration, tissue polarity, and organogenesis, and contribute to the development of the genitourinary system. In the canonical Wnt pathway, β-catenin acts as the central component Naturally occurring compounds represent attractive candidates for development as chemopreventive agents when backed by evidence-based findings and mechanistic research. In particular, dietary agents circumvent the need for additional introduction of foreign compounds into healthy asymptomatic individuals. Dietary agents are also in general less toxic, more readily bioavailable, more accessible, and less expensive as compared to synthetic agents. In the past few years, several laboratories have identified numerous phytochemicals that have potentially useful biological properties; however, only a few groups have directly evaluated the ability of these agents to disrupt β-catenin-mediated Wnt signaling in vitro colon cancer cell proliferation and NFκB activation in endothelial cells by these compounds has been demonstrated in vitro antiproliferative activity and cellular mechanisms of Avns in human cervical cancer cells.Avenanthramides (Avns) are a group of naturally occurring polyphenols found uniquely, among food crops, in oats, a popular healthful cereal Phenethylisothiocyanate (PEITC) is a well-studied isothiocyanate, occurring naturally in the form of its glucosinolate precursor, gluconasturtiin, in the Brassicaceae family of vegetables such as cabbage, cauliflower, watercress and broccoli. PEITC showed chemopreventive potential against various cancers Tripterygium wilfordii Hook F. Triptolide has multiple pharmacological activities including anti-inflammatory, immune modulation, antiproliferative and pro-apoptotic activity Triptolide, a diterpene triepoxide, is a major active component of extracts derived from the medicinal plant Overall objective for the current study was to establish mechanistic insights of the selected phytocompounds related to Wnt/β-catenin signaling that may be relevant to a broad range of cancers. All experiments were conducted using HeLa cells of human cervical cancer origin . HeLa cells are co-infected with Papilloma viruses that contribute to a persistent inflammatory response within the cells. Papilloma viruses are thought to be causative factors for human cervical cancers. The HeLa genome has been remarkably stable after years of continuous cultivation; therefore, the genetic alterations detected may have been present in the primary tumor and reflect events that are relevant to the development of cervical and other forms of human cancer Plasmids 7TFP (Addgene plasmid 24308), pCMVdR8.2 (Addgene plasmid 8455), and pCMV-VSV-G (Addgene plasmid 8454) were purchased from Addgene . The p7TFP vector p-coumaric, ferulic or caffeic acid) in pyridine was acetylated with excess acetic anhydride. After reaction, cold water was added and the precipitant was collected and dried overnight. To the acetylated phenylpropanoid in dimethyl formamide, with a small amount of triethylamine, was added, dropwise, an equimolar amount of benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium hexafluorophosphate dissolved in CH2Cl2. Next 5-hydroxy anthranilic acid (equimolar) in dimethyl formamide was added dropwise. The reaction was stopped with 0.5 M HCl. After extraction of the acetoxyavenanthramide into ethyl acetate and rotary evaporation to dryness, the protecting acetyl groups were removed by addition of 5% pyrollidine in CH2Cl2. The de-protection reaction was quenched with 1 M HCl and the Avn extracted into ethyl acetate. Purification of the Avn was effected by LH-20 column chromatography.Avns 2p, 2f and 2c were synthesized by methods reported previously The human cervical cancer cell line HeLa , and human embryonic kidney 293T cells  were maintained in Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum. HeLa 7TFP stable reporter cell line was maintained in Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 0.5 µg/ml puromycin and 10% heat-inactivated fetal bovine serum. The cells were kept in a 37°C incubator with 5% CO2. Cells were subcultured after trypsinization when 90% confluent with a 1∶5 splitting ratio. For cell proliferation assay only, cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 5% heat-inactivated fetal bovine serum, and 50 µM 2-mercaptoethanol. Cells were seeded at different densities as described in each experiment. Viable cell counts were carried out by trypan blue staining using a hemocytometer.et. al.Lentiviral production was carried out following a modified protocol from Steward 6 cells each dish). After 1d, for each dish, 1 µg of pGL4.75 was mixed with transfection solution containing 3 µl TransIT LT1  and added to the culture. At 24 h post transfection, cells were trypsinized, resuspended and seeded into wells of a 96-well plate (2×104 cells per well). Six h later, media was replaced with new media containing treatments or vehicle- dimethyl sulfoxide (DMSO). Following 4 h incubation, 50 mM LiCl was added to each well and incubated for an additional 8 h.A dual luciferase assay was used to determine the extent of Wnt signaling activation by measuring β-catenin mediated TCF transcription in HeLa 7TFP stable reporter cells in response to treatments with the compounds. First, transient transfection was performed as previously described The Wnt activation in the various samples was determined by measurement of Firefly luciferase activity using a Dual Glo luciferase assay kit . Firefly luciferase activity was measured as luminescence using a Synergy H4 hybrid Reader  in cell extracts, normalized for Renilla luciferase activity, and expressed as percent change as compared to a LiCl treated positive control. Concentrations of the test compounds and their treatment incubation periods were either pre-determined using separate optimization experiments (data not shown) or obtained from previous reports The ability of HeLa cells to proliferate in response to various treatments were measured using CellTiter 96® AQueous One Solution Cell Proliferation Assay , a colorimetric method for determining the number of viable cells in proliferation. The assay utilizes a tetrazolium compound  and an electron coupling reagent . Manufacturer’s instruction was followed and treatments were compared to vehicle control (DMSO-treated cells) by reading absorbance at 490 nm on a BioTek Synergy H4 multimode plate reader . All data shown are means±S.E. from four independent experiments.6 each well. The next day, media was removed and new media containing indicated concentrations of test compounds  or DMSO (vehicle control) were added back. After 4 h, LiCl at a final concentration of 50 mM was added to wells for an additional 8 h incubation. Immunoblotting was carried out following a protocol described by Gao et. al.HeLa cells  were seeded into 6 well plates at a density of 1×105 each well. The next day, culture media was removed and an indicated concentration of each compound  or DMSO was added back. The optimized concentration of each compound was pre-determined by the Wnt reporter assay. After 4 h incubation, 50 mM LiCl was added to wells for an additional 8 h incubation. Immunofluorescence was measured as described previously an EVOS FL epifluorescent microscope . The imaging data were expressed as Dylight 488 , Hoechst (indicating nucleus), and Merge (overlay of Dylight 488 and Hoechst staining for the same view). Nucleus localization of β-catenin was determined by Dylight 488 staining in the nucleus. The cells with presence of β-catenin in the nucleus were counted among 100 cells for each treatment.HeLa cells were seeded into a 12 well plate at a density of 2×10et. al. previously Total RNA extraction, purification and cDNA synthesis were performed as described by Dey et. al. Real time quantitative PCR was performed as previously described t test. Treatments were considered significantly different if p<0.05 *, highly significant if p<0.01 * *, or extremely significant if p<0.001 * * *.Experimental observations are expressed as the mean ±S.E. In all figures, the significance of any treatment over the untreated control DMEM for  and 6 waTo identify naturally occurring chemical modulators of Wnt signaling, we developed a high-content assay to measure activation, nuclear translocation and subsequent binding of β-catenin to the TCF transcriptional complex in HeLa cells. Inhibition of the GSK-3 complex in the cytoplasm leads to production of aberrant β-catenin which subsequently translocates to the nucleus to bind to the TCF-complex triggering downstream activation of Wnt pathway genes. We used a HeLa cell line stably carrying a lentiviral 7TFP reporter cassette containing a Firefly luciferase reporter gene controlled by the TCF gene promoter . To demoTo identify natural modulators of canonical Wnt signaling and further validate our reporter assay system, we performed pilot and focused screening with five compounds from our laboratory natural product collection. Using greater than 25% inhibition at the highest tested concentration combined with concentration dependent inhibition curve as compared to LiCl positive control, we identified three compounds that downregulated TCF mediated Wnt signaling activation . Avns 2pWnt signaling activation has been linked to uncontrolled cell proliferation during carcinogenesis Further mechanistic investigation into Wnt signaling perturbation by Triptolide and 2p were carried out. Since Wnt signaling activation that was suppressed by the compounds, requires cellular production of β-catenin and its binding to TCF transcription complex in the nucleus Wnt signaling response is dependent on the nuclear localization of β-catenin where it interacts with TCF/LEF family of transcription factors to activate the Wnt pathway. Therefore, we wanted to confirm if cellular degradation of β-catenin by 2p and Triptolide  logicallAs demonstrated in The Wnt signaling pathway leads to the dephosphorylation, stabilization, and nuclear translocation of β-catenin. The stabilized β-catenin binds with the TCF transcription factor complex, leading to the activation of Wnt-responsive genes such as c-Myc. As one of the principal downstream targets of the β-catenin-TCF complex, c-Myc plays a key role in altered cellular metabolism during tumorigenesis in vitro studies. Formation of the β-catenin–TCFs complex in the nucleus is a prerequisite for the transcription of Wnt target genes that has been associated with the development of many types of cancers β-catenin is a multifunctional protein that plays an important role in ontogenesis and oncogenesis. A major consequence of aberrant Wnt signaling is stabilization of β-catenin, and this can be triggered using LiCl-elicitation for in vitro  in vitro . Concentin vitro , which ain vitro  and subsin vitro . Since cTriptolide, the main active component of the traditional Chinese herbal medicine Tripterygium wilfordii Hook F, decreased accumulation of β-catenin in the cytoplasm and attenuated its translocation in to the nucleus, thereby inhibiting transcriptional activation of TCF. Significant pro-apoptotic effects of Triptolide were observed in HeLa cells at nanomolar concentrations as compared to the reference compound FH535 which was effective at micromolar quantities . Althougin vitro conditions (unpublished results). Whether Triptolide can be targeted to insulin resistant patients with higher risk for cancer development might be a potential line of research.Triptolide also downregulated c-Myc mRNA at nanomolar concentrations in a concentration dependent manner . The intIn conclusion, our study elucidates a novel molecular paradigm mediating previously known pro-apoptotic effects of Triptolide in human cervical cancer cells. This involved disruption of aberrant β-catenin signaling. Our study also demonstrates novel pro-apoptotic effects of Avn 2p, also mediated through abrogation of aberrant β-catenin signaling. Given the extremely low concentrations at which Triptolide is biologically targeting the molecular players discussed above, further investigations are warranted to explore potential use of this compound in clinics for treatment of aggressive cancers involving abnormal β-catenin signaling. Triptolide has been tested on animals and human for various health benefits, hence its therapeutic and toxic concentration windows are already established


To achieve tight regulation of the Wnt/β-catenin signaling, tissue- and cell-specific coactivators and repressors need to be recruited. The identification of such factors may help to elucidate mechanisms leading to enhanced cardiac differentiation efficiency in vitro as well as promote regeneration in vivo. Using a yeast-two-hybrid screen, we identified four-and-a-half-LIM-domain 2 (FHL2) as a cardiac-specific β-catenin interaction partner and activator of Wnt/β-catenin-dependent transcription. We analyzed the role of this interaction for early cardiogenesis in an in vitro model by making use of embryoid body cultures from mouse embryonic stem cells (ESCs). In this model, stable FHL2 gain-of-function promoted mesodermal cell formation and cell proliferation while arresting cardiac differentiation in an early cardiogenic mesodermal progenitor state. Mechanistically, FHL2 overexpression enhanced nuclear accumulation of β-catenin and activated Wnt/β-catenin-dependent transcription leading to sustained upregulation of the early cardiogenic gene Igfbp5. In an alternative P19 cell model, transient FHL2 overexpression led to early activation of Wnt/β-catenin-dependent transcription, but not sustained high-level of Igfbp5 expression. This resulted in enhanced cardiogenesis. We propose that early Wnt/β-catenin-dependent transcriptional activation mediated by FHL2 is important for the transition to and expansion of early cardiogenic mesodermal cells. Collectively, our findings offer mechanistic insight into the early cardiogenic code and may be further exploited to enhance cardiac progenitor cell activity in vitro and in vivo. S The extensively described canonical Wnt signaling pathway has as a key mediator, the ubiquitously expressed protein β-catenin, which plays a dual role in cell adhesion and signal transduction . In abseSeveral studies suggest that members of the LIM-only protein family may act as coregulators of tissue-specific gene expression by interacting with different transcription factors. Specifically, the four-and-a-half-LIM-domain (FHL) 2 was shown to interact with β-catenin in myoblast and tumor cell lines and to regulate its transcriptional activity , 9. FHL2Given the role of β-catenin in the context of the Wnt canonical signaling in early and adult cardiogenesis, we were interested in identifying novel cardiac-specific Wnt/β-catenin regulators. Using a yeast-two-hybrid (Y2H) screen, we found in this study an interaction between FHL2 and β-catenin, resulting in an activation of the β-catenin-dependent transcription in cardiac cells. To explore the role of this interaction in cardiogenesis, we used embryonic stem cell (ESCs) and embryonic carcinoma cell (P19) embryoid body (EB) cultures, which can consistently recapitulate the sequences of cardiac genes expression observed in early cardiac development in the mouse embryo . We obsehttp://www.invitrogen.com) was used as described previously [The ProQuest Two-Hybrid System ; digested with EcoRI, BamHI, and XhoI restriction enzymes, electrophoresed, and transferred onto a nitrocellular membrane . The riboprobes were generated by ClaI digestion of the cmyc-Fhl2 plasmid  and Digoxigenin (DIG)-labeling . Probes were hybridized overnight at 68°C; membranes were washed at 68°C in low- and high-stringency buffer /0.1% SDS; 2× SSC/0.1% SDS). Detection was performed with an anti-DIG antibody and CPC-star (Roche) [Genomic DNA was purified using the DNeasy Tissue Kit  .http://www.bdbiosciences.com) with the cmyc-Fhl2 expression plasmid and a puromycin-expressing vector using the Xfect-stem reagent . Transfected cells were selected under puromycin (1 μg/ml). For differentiation, cells were aggregated in hanging drops containing 500 cells each to form EBs and cultured for 5 days in Iscove medium supplemented with 20% fetal calf serum (FCS) and 0.1 mM ascorbic acid as previously described [http://www.acros.be) or dimethylsulfoxid (DMSO) at the indicated time points.Double transgenic αMHC-GFP/αMHC-Neomycin resistance cassette sequence (NeoR) murine ESCs were transfected on Matrigel , digested with collagenase type II , purified by discontinuous Percoll gradient centrifugation, plated on gelatin, and cultured. Transfection was performed using FuGene (Roche) according to manufacturer's instruction.Neonatal rat cardiomyocytes (NRCMs) were prepared as described previously . Brieflyhttp://www.sigmaaldrich.com); FHL2, cardiac troponin T (cTNT), Igfbp5, and Ki67 ; 1:200 dilutions. AlexaFluor488- and AlexaFluor594-conjugated secondary antibodies  were used for labeling. For nuclei visualization, cells were stained with 4′,6-diamidino-2-phenylindole and mounted with ProlongGold (Invitrogen). Microscopic images were captured with a confocal microscope (Zeiss LSM710/NLO). Semiquantitative analysis was performed using the Axiovision software .ESCs were differentiated on 0.1% gelatin-coated coverslips. Cells were fixed in 4% paraformaldehyd (PFA), permeabilized in 0.1% Triton X-100, and blocked in 1% bovine serum albumin (BSA). Cells were incubated with respective antibodies against β-catenin (BD Bioscience); α-sarcomeric actinin  48 hours after transfection, according to manufacturer's instructions.NRCMs were cotransfected with FHL2- and/or nondegradable β-catenin-expressing plasmids along with the pTOPflash luciferase reporter and http://www.scbt.com). Cells were stained with anti-rabbit IgG-APC or anti-mouse F(ab)2-FITC . Respective isotype controls were used. Fluorescence signals were detected with a Calibur flow cytometer (BD).Cells were fixed in 1% formaldehyde/phosphate buffered saline (PBS), permeabilized in flow cytometry buffer containing 0.5% Saponin (Sigma-Aldrich), and stained with antibodies directed against α-sarcomeric actinin  and Nkx2.5 . cDNA was synthesized and quantitative real-time PCR analyses were performed with SYBR Green (Qiagen) on an iCycler instrument . Copy numbers were calculated using the iCycler software with a relative standard curve obtained using the log dilutions of gene of interest cDNA. All reactions were run in triplicates and normalized to gapdh. Primers are listed in supporting information Total RNA was isolated from cells, embryonic, and postnatal tissue using the RNA II kit  antibodies were used. Densitometric analysis was performed using Adobe Photoshop software.Forty-eight hours after transfection, cells were harvested, lysed with Baeuerle buffer with protease inhibitors, and immunoprecipitated with an anti-β-catenin antibody (Santa Cruz). Detection was done by immunoblotting using a c-myc (Santa Cruz) antibody. Protein lysate transfected with empty vector served as control. Whole cell lysates were immunoblotted with respective antibodies to detected protein expression. For Western blot anti-FHL2 (Abcam), anti-β-catenin (Santa Cruz) and anti-GAPDH . Altogether these data indicate that FHL2 binds β-catenin in cardiac cells to exert a synergistic activity on β-catenin transcriptional activity.A Y2H system using a human cardiac expression library was used to screen cardiac β-catenin binding proteins . From 31 plasmid C. Endogen = 6/group, n = 3/group, Next, we tested the hypothesis that FHL2 and β-catenin interaction and regulation play a role during cardiogenesis in ESC-EB cultures A. Doublen = 12/group, n = 6/group). Similarly, α-sarcomeric actinin/GFP-positive cells as detected by flow cytometry  in FHL2- versus wild-type-ESC-EBs (n = 6/group, Since FHL2 was reported to have a role in cell cycle regulation in noncardiac cell types , we compn = 6/group, n = 12/group, n = 3/group, We found activation of the β-catenin-dependent transcription upon FHL2 overexpression, which is known to block differentiation of cardiomyogenic cells . Therefon = 6/group, n = 6/group; supporting information n = 6/group, n = 12/group; n = 6/group, n = 6/group, Differentiating ESC-EBs with stable FHL2 expression appeared to be locked in a mesodermal progenitor state with limited cardiomyocyte differentiation. This block could be reversed by β-catenin/TCF4 inhibition with quercetin. To confirm that transient overexpression of FHL2 during cell commitment would enhance mesodermal cell lineage commitment and subsequently allow for enhanced cardiomyocyte differentiation, we expressed FHL2 transiently in P19 cells A. P19 ceβ-Catenin is the key mediator of canonical Wnt signals, a major regulator of cardiogenesis in vivo and in vitro , 28-32. A detailed comparative mRNA expression analysis showed that Fhl2 is expressed in cardiac tissue as early as ED 7.5. This is in line with previous reports showing Fhl2 expression in the earliest myocardial progenitor cells of the embryonic cardiac crescent . Fhl2 exPrevious studies showed an interaction between FHL2 and β-catenin resulting in enhanced β-catenin-dependent transcriptional activation in different tumor cell lines as HEK293, SW480, and A375 . In lineWe previously demonstrated that modulation of the Wnt/β-catenin/TCF signaling affects resident cardiac progenitor cell biology , 32. SpeWe showed that FHL2 gain-of-function expands cardiac mesodermal cells by enabling an abnormally persistent expression of genes between undifferentiated mesodermal and early progenitor status. This led to an augmentation of cells with early cardiac identity, but resulted in abrogation of terminal cardiogenic differentiation. Sustained β-catenin/TCF transcriptional activation mediated by FHL2 might explain the described phenotype since inhibition of canonical Wnt signaling is critical for cardiomyocyte differentiation , 40. ThiActivation of the β-catenin-dependent transcription mediated by FHL2 may induce early cardiac overspecification and thereby block further cardiac differentiation at the cardiomesoderm progenitor stage. This hypothesis was substantiated by the observation of persistent elevated expression of Igfbp5 at 7 and 16 days of differentiation in FHL2-ESC-EBs. Igfbp5 has been identified to mark cells in the precardiac region and early cardiac crescent in mouse embryos . FailureFHL2 gain-of-function mediated increased cell proliferation in ESC-EBs as indicated by enhanced Cyclin-D1 and KI67 expression. This may subsequently prevent differentiation and further explain the reduced and delayed cardiomyocyte differentiation in ESCs. Whether this process is mediated by β-catenin transcriptional activation, which is known to activate proliferation and the target gene Cyclin-D1 , 40, or Collectively, our observations indicate that FHL2 functions as a cardiac-specific coactivator of Wnt/β-catenin transcriptional activity to control early cell lineage commitment during cardiac differentiation. Previous studies in skeletal muscle cells reported that FHL2 promotes myogenic differentiation via repressing LEF/TCF-dependent transcription , 25. TheFhl2-KO mice do not present obvious defects in cardiogenesis. Whether Fhl2 deficiency in these mice was compensated by other FHL-protein family members or whether the genetic defect would render them more susceptible to cardiac damage under myocardial stress remains to be studied in more detail. Despite this uncertainty, we provide novel evidence that Wnt/β-catenin modulation with FHL2 may be instrumental in optimization of cardiomyocyte derivation from stem cells for applications in studies of fundamental heart muscle biology, drug discovery, and potentially also cell-based heart repair.Our findings suggest that FHL2 is an element of the putative cardiogenic differentiation code. In particular, cardio-mesoderm specification induced via Wnt/β-catenin-dependent transcription was enhanced by FHL2. This may in part be mediated by Igfbp5 activation. However, sustained FHL2 overexpression arrested cardiac differentiation at the cardio-mesoderm progenitor stage. This is in agreement with the biphasic role of Wnt/β-catenin in cardiac differentiation. While our study seems to have identified a novel regulator of cardiogenesis in vitro, it has to be acknowledged that


The inconsistent implementation of evidence-based practice has become a significant concern in the traumatic stress field. The European Society for Traumatic Stress Studies (ESTSS) has played a major role in highlighting this issue and has contributed to a number of European initiatives to improve dissemination and implementation. Key initiatives include the introduction of the ESTSS General Certificate in Psychotrauma Psychotraumatology and the European Network for Traumatic Stress (TENTS); these are discussed in this paper. Just before I left the British Army in 1993, I attended my first European Society for Traumatic Stress Studies (ESTSS) conference in Bergen, Norway. I had never quite experienced anything like it before but have on numerous occasions since. I remember Colin Murray Parkes giving a keynote presentation on bereavement, paying more money for a beer than I had imagined possible and meeting people for the first time who have subsequently become great friends and collaborators. Ironically, I even met Jonathan Shepherd in Bergen who I have subsequently worked closely with in Cardiff.Psychological Debriefing  time in my life.When I became President in 2007, Berthold Gersons had already undertaken a lot of work to transform the ESTSS and I was able to continue with that work. I remember several tense meetings with the presidents of different societies but by positively working together, we managed to find a way forward. It was great to see the creation of new societies in the Netherlands, United Kingdom, Poland, and Georgia and a large expansion of the ESTSS membership.In addition to driving forward the transition of ESTSS, I was keen to develop the Society's work in the dissemination and implementation of evidence-based practice during my Presidency. We introduced the ESTSS General Certificate in Psychotraumatology; an initiative that I continue to believe has the potential to be developed further.The ESTSS Board approved the introduction of the ESTSS General Certificate in Psychotraumatology in December 2007. All members of the ESTSS are eligible to register for the certificate program subject to a one-off fee to cover the administration of the certification process. To complete the Certificate, individuals must attend a 1-day ESTSS-approved introductory workshop and then 4 days of other approved workshops. Satisfactory completion of a test for each workshop attended and completion of all workshops within 3 years of attendance at the first qualifying workshop are the final requirements. A wide range of workshops are eligible for the Certificate allowing those undertaking it to develop a strong grounding in psychotraumatology.There are now an increasing number of individuals from across Europe who have been awarded the Certificate, and it has attracted people to the Society who may not have become involved otherwise. I consider the Certificate an excellent way to disseminate knowledge in an evidence-based manner.In parallel with the development of the Certificate, members of the ESTSS Board along with some other interested parties created a team, led by Miranda Olff, to apply for European Union (EU) funding to develop European guidance on psychosocial responses following traumatic events. The European Network for Traumatic Stress (TENTS) was formed . The TENTS guidelines together with guidance produced by NATO  facilitaI continue to feel very attached to ESTSS and remain committed to helping it to fully exploit its key role in disseminating and implementing evidence-based practice in the traumatic stress field across Europe and beyond.


Kengyilia is a group of allohexaploid species with the StYP genomic constitutions in the wheat tribe. To investigate the evolutionary dynamics of the Pgk1 gene in Kengyilia and its diploid relatives, three copies of Pgk1 homoeologues were isolated from all sampled hexaploid Kengyilia species and analyzed with the Pgk1 sequences from 47 diploid taxa representing 18 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) Kengyilia species from the Central Asia and the Qinghai-Tibetan plateau have independent origins with geographically differentiated P genome donors and diverged levels of nucleotide diversity at Pgk1 locus; (2) a relatively long-time sweep event has allowed the Pgk1 gene within Agropyron to adapt to cold climate triggered by the recent uplifts of the Qinghai-Tibetan Plateau; (3) sweep event and population expansion might result in the difference in the dN/dS value of the Pgk1 gene in allopatric Agropyron populations, and this difference may be genetically transmitted to Kengyilia lineages via independent polyploidization events; (4) an 83 bp MITE element insertion has shaped the Pgk1 loci in the P genome lineage with different geographical regions; (5) the St and P genomes in Kengyilia were donated by Pseudoroegneria and Agropyron, respectively, and the Y genome is closely related to the Xp genome of Peridictyon sanctum. The interplay of evolutionary forces involving diverged natural selection, population expansion, and transposable events in geographically differentiated P genome donors could attribute to geographical differentiation of Kengyilia species via independent origins.The level and pattern of nucleotide variation in duplicate gene provide important information on the evolutionary history of polyploids and divergent process between homoeologous loci within lineages.  Duplication is a prominent feature of plant genomic architecture. Genome duplication or polyploidy provides a reservoir of duplicate genes as substrates for potential evolutionary innovation Kengyilia Yen et J. L. Yang, a polyploid perennial genus in the wheat tribe (Poaceae: Triticeae), includes about 22 perennial species distributed in a different range of natural habitats over the upper and middle mountain ranges of Central Asia and the Qinghai-Tibetan Plateau Kengyilia species arose from two hybridization events followed by genome doubling of three ancestral diploid species with different genomes St, Y and P Pseudoroegneria (Nevski) Á Löve and Agropyron Gaertn., respectively Kengyilia genome Kengyilia species associated with geographical origin from Central Asia and the Qinghai-Tibetan plateau. Zhou et al. Kengyilia species might genetically arise from its parental lineages with two different geographical origins . Based on the cytogenetic and geographic data, Yen et al. Agropyron (P genome) species might play an important role in influencing the genetic differentiation of Kengyilia species. While these studies add to our understanding of phylogeny and genetic differentiation of Kengyilia, little is known about the evolutionary forces acting on the geographical differentiation of Kengyilia, and further information on whether the biological factors from the P genome influences the patterns of genetic diversity of Kengyilia species is still outstanding.Previous studies based on RAPD (Random amplified polymorphic DNA polymorphism) Pgk1 gene showed that it is present as a single copy per diploid chromosome in grass Pgk1 gene has been successfully used to study the phylogeny and evolutionary history of Triticum/Aegilops complex Pgk1 gene were isolated from each the fifteen sampled Kengyilia species and analyzed with those from 47 diploid taxa representing 18 basic genomes in Triticeae. The objectives were to: (1) document the patterns of molecular evolutionary divergence among homoeologues of the Pgk1 gene in hexaploid StYP Kengyilia and between polyploidy and its diploid genome donor; (2) determine whether the patterns of Pgk1 sequence variation within the P genome lineages reflects the geographical differentiation of Kengyilia species; (3) explore evolutionary forces acting on the Kengyilia species with different geographical region; (4) identify the possible origin of the Y genome.Phosphoglycerate kinase (Pgk1), a key ATP-generating enzyme in the glycolytic pathway, catalyzes the conversion of 1, 3-diphosphoglycerate to 3-phosphoglycerate. Analysis of the Kengyilia species were included in this study and were analyzed together with 47 diploid taxa representing 18 basic genomes in the tribe Triticeae , while the seed materials with ZY and Y numbers were collected by ourselves, which no specific permit is required. The plants and voucher specimens are deposited at Herbarium of Triticeae Research Institute, Sichuan Agricultural University, China (SAUTI).Fifteen riticeae . Pgk1 sePgk1 gene was amplified with the Pgk1-specific primers PgkF1 (5′-TCGTCCTAAGGGTG TTACTCCTAA-3′) and PgkF2 (5′-AAGCTCGCGCCACCACCAGTTGAG-3′). PCR was conducted under cycling conditions reported previously DNA extraction followed a standard CTAB protocol Pgk1 gene from each Kengyilia accession, we performed the following process. Firstly, approximately 30 positive clones from each accession were screened by direct PCR using primer PgkF1 and M13R (on the side of the cloning site in the plasmid). Secondly, St-type (5′-GGTATTCTTGTGTTCCACACCA-3′) and P-type (5′-ATCZAGACYTCTAATCAAGCA-3′) Pgk1-specific primers were designed and used each together with the reverse primer PgkF2 to screen the St- and P-type Pgk1 sequences from above 30 positive clones with Pgk1 inserts, respectively. The positive clones containing the Y-type Pgk1 sequences were also obtained. The cloned PCR products were commercially sequenced in both directions by TaKaRa Biotechnology Co. Ltd. , and an additional internal primer (5′-GATGGAGCTGTTTCAAACC-3′) was used to sequence the internal portion of the cloned PCR products. All the sequences from Kengyilia species were determined based on at least five independent St-, Y- and P-type clones, respectively.PCR amplicons of single-copy nuclear genes from allopolyploid species will produce a heterogeneous mix of homoeologues. To separate the homoeologues of the Multiple sequences were aligned using ClustalX F) and the numbers of shared polymorphisms (SS) To assess the divergence and genetic relationships between allopolyploids and its diploid progenitors, nucleotide diversity was estimated by Tajima's π Pgk1 gene, the ratio of nonsynonymous to synonymous substitution (dN/dS) were computed using the modified Nei-Gojobori method in MEGA 4.0 dN and dS was estimated using the Z statistics, with standard errors based on 1000 bootstrap replicates using MEGA 4.0 dN to dS was estimated using profile likelihood Pgk1 gene using DnaSP 4.10.9 To detect selective constraints on the coding portions (the introns were excluded) of the homoeologous http://www.sinauer.com). ML heuristic searches were performed with 100 random addition sequence replications and TBR branch swapping algorithm. The robustness of the trees was estimated by bootstrap support (BS) Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI). The evolutionary model used for the phylogenetic analysis was determined using ModelTest v3.0 with Akaike information criterion (AIC) Pgk1 sequences within Kengyilia and its putative diploid species was evaluated with a likelihood ratio test comparing the likelihood scores from the unconstrained and clock-constrained analyses, implemented in PAUP*4.0b10. Substitution rates were significantly heterogeneous , implying a very poor fit to the molecular clock. Therefore, divergence times with 95% confidence intervals were estimated using Bayesian relaxed molecular clock method, implemented in BEAST v1.4.6 Pgk1 gene clock of 0.0051 substitutions per site per MY (million year) http://tree.bio.ed.ac.uk/).Clock-like evolution of Pgk1 homoeologues, three distinct types of Pgk1 sequences  were obtained from all 15 Kengyilia species. At least 15 positive clones  were sequenced from each accession. In cases when multiple identical sequences resulted from cloned PCR products of one accession, only one sequence was included in the data set. Consequently, 45 unique sequences were obtained and analyzed together with those from 47 diploid taxa representing 18 basic genomes in Triticeae.Following the screen of Pgk1 gene includes 5 exons and 4 introns, which was in agreement with previous studies Pgk1 sequence matrix including both exons and introns contains 1522 characters, of which 20.04% (305/1522) were variable, and 8.74% (133/1522) were parsimony informative. Sequence alignment showed that an 83-bp insertion was detected for the P-type sequences at position 1295–1377 in the intron region from eight Kengyilia species , two Agropyron mongolicum accessions (PI 531543 and PI 499392) and eight Agropyron cristatum accessions  (MITE stowaway element.The DNA sequence of the ZY09005) . SecondaZY09005) . BLAST sKengyilia, the Pgk1 sequences of all the polyploid species were included in the phylogenetic analyses (ML and BI), together with 47 diploid taxa representing 18 genomes in Triticeae. ML analysis yielded a single phylogenetic tree (−Lnlikelihood = 9139.1240), with the following estimated ML parameters: the assumed nucleotide frequencies A: 0.2682, C: 0.1918, G: 0.2332, T: 0.3068, the proportion of invariable sites = 0.2519, gamma shape parameter = 0.6693. ML and Bayesian analyses recovered the same topology. The tree illustrated in To reveal the putative genome donors of Kengyilia species were split into three well supported clades (Kengyilia and the sequences of Agropyron (91% PP and 71% BS). Three subclades  with high statistical support were recognized in this clade. Subclade A included all the Kengyilia species and Agropyron cristatum accessions from the Qinghai-Tibetan Plateau and Agropyron mongolicum from the Alashan (margin of the Qinghai-Tibetan Plateau)(100% PP and 87% BS). It was worth mentioning that the sequence in subclade A had an 83-bp MITE stowaway insertion at position 1295–1377. Subclade B contained all the Kengyilia species from Central Asia and one A. cristatum accession from Central Asia (PI 486160) (99% PP and 78% BS). Subclade C consisted of four A. cristatum accessions from Central Asia and three A. cristatum accessions from East Asia (100% PP and 99% BS). The Clade II comprised all the St-type sequences of Kengyilia and the sequences from Pseudoroegneria species and Lophopyrum elongatum (100% PP and 98% BS). In this Clade, Pseudoroegneria spicata and Lo. elongatum were grouped with six Kengyilia species from the Qinghai-Tibetan Plateau (100% PP and 83% BS). Kengyilia longiglumis from the Qinghai-Tibetan Plateau was clustered with all the sampled Kengyilia species from Central Asia (92% PP and 74% BS). Three Pseudoroegneria species formed paraphyletic, and Kengyilia stenachyra was placed at the base of the Clade II. The Clade III included all the Y-type sequences of Kengyilia and the sequence from Peridictyon sanctum (100% PP and 86% BS). In Clade III, Kengyilia kaschgarica was grouped with Kengyilia gobicola with 100% PP and 61% BS, while the remaining Kengyilia species formed one subclade (100% PP and 64% BS).The phylogenetic tree showed that the St-, P- and Y-type sequences from d clades . The Claπ and θw, were separately calculated for the St, Y and P genomes of Kengyilia, and for Agropyron , the estimates of nucleotide diversity were π = 0.0064, wθ = 0.0093. The estimates of nucleotide diversity in the P genome of Kengyilia from Central Asia were π = 0.0088, wθ = 0.0120, while in the P genome of Agropyron from Central Asia, the estimates of nucleotide diversity were π = 0.0142, wθ = 0.0170. π was also separately calculated for synonymous and nonsynonymous sites. The overall number of polymorphic sites at homoeologous loci of Pgk1 sequence from Kengyilia St, Y and P genomes was 51, 85, and 82, respectively. The overall number of polymorphic sites in the P genome of Kengyilia was lower than that in the P genome of diploid Agropyron. The Tajima Kengyilia, and −1.5089 (P<0. 05) and −1.9190 (P<0. 05) for Agropyron, respectively. The same parameters in the P genome lineage from Central Asia were −1.5244 (P<0. 05) and −1.5709 (P<0. 05) for Kengyilia, and −1.2570 (P<0. 05) and −1.2570 (P<0. 05) for Agropyron, respectively.Two overall measures of nucleotide diversity, gropyron . The estPgk1 locus between Kengyilia and its putative diploid donor were shown in Kengyilia and that of Pseudoroegneria. Thirty-four shared polymorphisms and no fixed difference were found between the all the sampled P-type sequence of Kengyilia and that of Agropyron. For the P genome lineage of sympatric origin, the number of shared polymorphisms was higher than the number of fixed difference, while for the P genome lineage of allopatric origin, the number of shared polymorphisms was lower than the number of fixed difference.Speciation genetics suggested that hybridization or differentiation between two species can be inferred through comparisons of shared nucleotide polymorphisms with fixed differences N/dS is indicative of the change of selective pressures. The dN/dS ratios of >1,  = 1 and <1 indicate positive selection, neutral evolution and purifying selection on the coding portions, respectively. Prior to the estimation of selective constraints on the coding portions of the Pgk1 gene in Kengyilia St, Y and P genome and its putative diploid genome donor, the average non-synonymous (dN) and synonymous (dS) distances with standard errors were calculated using the modified Nei-Gojobori method  below 1 and nearly 3-fold higher than that in the Central Asia Agropyron. The McDonald–Kreitman (MK) test of selective pressures was performed to compare the Qinghai-Tibetan Plateau P genome lineage with Central Asia P genome lineage. Total 19 mutations were found in the Qinghai-Tibetan Plateau Agropyron, of which 11 were nonsynonymous and eight were synonymous. Among the 19 mutations found in Central Asia Agropyron, six were nonsynonymous and 13 were synonymous. Significant departure from neutrality was detected for Agropyron (P = 0.037) from the Qinghai-Tibetan Plateau.The non-synonymous to synonymous rate ratio di method . Both Z-Pgk1 sequences from Kengyilia and its putative diploid species generated a time-calibrated tree . This is in agreement with recent genealogical analysis of single-copy nuclear gene Acc1 sequence in the species with Y genome in Triticeae , where the Y-type Acc1 homoeologues were clustered with the sequences from Dasypyrum (V genome) species, Heteranthelium piliferum (Q genome) and Peridictyon sanctum (Xp genome). Therefore, it suggested that the Y genome may be closely related to the Xp genome in Peridictyon sanctum.The Y genome is represented in all the tic tree , the Y-tAgropyron is the P haplome donor to Kengyilia and contains approximately eight diploid (PP) or tetraploid (PPPP) or hexaploid (PPPPPP) species. Agropyron cristatum and A. mongolicum are the only two diploid species within AgropyronA. cristatum is widely distributed in Eurasian temperate region, while A. mongolicum is restricted in some regions of northern China. The present Pgk1 gene data showed that the sampled A. cristatum from East Asia, the Qinghai-Tibetan plateau, and Central Asia did not form monophyletic group but were scattered into three distinct subclades . Genetic differentiation among A. cristatum population based on pairwise FST estimates was relatively high, ranging from 14.93% to 65.74% . These results indicated that A. cristatum populations from different geographical origins were genetically heterogeneity. High level of genetic differentiation and divergent population structure of A. cristatum could be attributed to restricted gene flow caused by geographical isolation.Genus Kengyilia species were geographical differentiated. Our phylogenetic analysis of Pgk1 sequences demonstrated that the separation of the P genomes in Kengyilia species is in good agreement with their geographical origins – Central Asia and the Qinghai-Tibetan plateau distinction. The accessions of Kengyilia species and A. cristatum from the Qinghai-Tibetan Plateau, and A. mongolicum from Alashan is differentiated from the accessions of Kengyilia species and one A. cristatum accession from Central Asia. Second, more shared polymorphisms than fixed differences (Kengyilia (CA) – Agropyron (CA): 9 vs. 2; Kengyilia (QTP) – Agropyron (QTP): 6 vs. 0) were observed between the sympatric P genome lineage, while less shared polymorphisms than fixed differences (Kengyilia (CA) – Agropyron (QTP): 3 vs. 14; Kengyilia (QTP) – Agropyron (CA): 5 vs. 7) were found between the allopatric P genome lineage. Third, an 83 bp MITE element insertion in the Pgk1 gene were found in the P genome of the Qinghai-Tibetan Plateau Kengyilia species and their sympatric diploid donors, while this element was absent in the same position of the sequence alignment in the P genome lineages from Central Asia. Finally, time-calibrated phylogeny suggested that speciation event of Central Asia Kengyilia species (about 3.68 MYA) may be prior to that of the Qinghai-Tibetan Plateau Kengyilia species (about 2.25 MYA). These different lines indicated that the P genome in Kengyilia species is highly differentiated according to their geographical origin. The Central Asia and the Qinghai-Tibetan Plateau Kengyilia species thus have independent origins.Data from RAPD Pgk1 gene genealogical structure and patterns of shared/fixed polymorphisms indicated the occurrence of independent origins of Kengyilia species. This offers an opportunity to address the potential evolutionary outcomes of independent origins within Kengyilia. On the basis of Pgk1 sequences of the P genome lineage from the Qinghai-Tibetan Plateau, the level of nucleotide diversity in Kengyilia  was higher than that in diploid Agropyron , and Tajima's and Fu and Li's D statistic suggests a departure from the equilibrium neutral model at this locus, with an excess of rare sequence variants in Kengyilia species. Greater diversity could reflect gene flow from diploid Agropyron population. For the P genome lineage from Central Asia, the level of nucleotide diversity in Kengyilia  was lower than that in diploid Agropyron , and the values of Tajima's and Fu and Li's D statistic in Kengyilia were significantly negative and lower than the values of same parameter calculated from Agropyron. This is compatible with a genetic bottleneck created by recent polyploidization. Diverged levels of nucleotide suggested that the P genome lineages of Kengyilia with independent origin have distinct evolutionary potentials.Recent studies using genetic markers in many genera suggested that multiple origins (including independent origin) of polyploid species are the rule rather than the exception Pgk1 homoeologues from all the hexaploid Kengyilia species studied here suggested the retention of triplicated Pgk1 homoeologues in Kengyilia species. The dN/dS ratio of three divergent Pgk1 homoeologues was significantly below 1 , suggesting that the Pgk1 sequences are selectively constrained as most mutations in functional genes are expected to be disadvantageous.Since gene duplication results in functional redundancy, divergent selective pressure may act on the duplicated copies that are critical for the subsequent variation, retention or loss of the duplicated genes N/dS value of the Qinghai-Tibetan Plateau Agropyron was nearly 3-fold higher than that in Central Asia Agropyron. Significant MK test (P = 0.037) between them suggested an excess of nonsynonymous substitutions, which is traditional viewed as an outcome of positive selection. This appears paradoxical in light of the strong signature of purifying selection in Pgk1 sequences. Relaxed purifying selection, selective sweep, and population expansion may explain this paradox. The relaxed purifying selection hypothesis is very unlikely because it did not explain our result of less significant Tajima's D values in the high dN/dS group (P = 0.044) than in the low dN/dS group (P = 0.017). The deficiency of synonymous polymorphisms in the high dN/dS group in comparison with the low dN/dS group was also not the result of relaxed purifying selection. The following evidences support the hypothesis of selective sweep. First, the dN/dS value of the Qinghai-Tibetan Plateau Agropyron was significantly higher than that in Central Asia Agropyron. Significant difference in dN/dS ratio was considered to be a result of selective sweep N/dS group than in the low dN/dS group, which is consistent with the expectation that sweep results in reduced polymorphism in the high dN/dS group N/dS group than in the low dN/dS group. Corresponding to this suggestion, the present result showed that the Tajima's D value in the high dN/dS group (−1.5089) is more negative than that in the low dN/dS group (−1.2570), and the levels of silent diversity in the high dN/dS group (π = 0.0089) is less than that in the low dN/dS group (π = 0.0236). Finally, given that the Pgk1 gene in the Qinghai-Tibetan Plateau Agropyron population has undergone selective sweep, it is suggested that the sweep event for the Pgk1 gene in Plateau may be associated with an evolutionary adaptation to local cold climate conditions. Recent study focusing on the responses of plant to cold stress has revealed that Phosphoglycerate kinase (Pgk1) is an up-regulated response protein to cold stress, a feature of adaptive evolution N/dSAgropyron population from the Qinghai-Tibetan Plateau was dated to 2.25 MYA, and the divergence time of the P genome lineage including the low dN/dSAgropyron population from Central Asia was dated to 3.38 MYA. Considering the conversion of low to high dN/dS value resulting from selective sweep, the age of selective sweep might have happened ≈2.3–3.4 MYA. Therefore, it is possible that a relatively long-time sweep event allow the Pgk1 gene within Agropyron to adapt to cold climate triggered by the recent uplifts of the Qinghai-Tibetan Plateau. Because demographic processes such as range expansion can have a similar impact on DNA variation to that caused by a selective sweep in a population N/dS ratio. Analysis of diversity showed that the levels of diversity in the Qinghai-Tibetan Plateau Agropyron population  is significant lower than that in Central Asia population , indicating a recent expansion in the Qinghai-Tibetan Plateau Agropyron population. This suggestion is further determinative of the more significant negative Tajima's and Fu and Li's D values. Population expansion in associated with Pleistocene glacial cycles might have accelerated the fixation of mildly deleterious replacement mutations that became effectively neutral in the Qinghai-Tibetan Plateau Agropyron populations.The dN/dS value in the P genome of the Qinghai-Tibetan Plateau Kengyilia species was lower than that in their sympatric diploid relatives, indicating that more strongly purifying selection acts to conserve the function of the Pgk1 gene in Kengyilia. However, a slightly elevated dN/dS value in the P genome of the Central Asia Kengyilia species compared to their sympatric diploid relatives might suffer from polyploidization bottleneck as suggested by the present estimate of diversity, because population bottleneck may result in reduced selection Duplicate genes in polyploid lineages are often preserved in function by more strongly purifying selection N/dS value of the P genome of Kengyilia lineage from the Qinghai-Tibetan Plateau (dN/dS = 0.2645) was higher than that from Central Asia (dN/dS = 0.2018). Non-significantly negative Tajima's D value coupled with greater diversity in the high dN/dS group excluded the possibility that selective sweep could resulted in the difference in the dN/dS value of the P genome of Kengyilia. A highly non-significant difference between this two dN/dS ratios (P = 0.499 by Fisher's exact test) based on the McDonald-Kreitman test was found, rejecting the hypothesis of relaxed purifying selection. Considering the difference in the dN/dS value of two allopatric Agropyron lineages, it is possible that the difference in the dN/dS value of the P genome of two allopatric Kengyilia lineages may be genetically from geographically differentiated P genome donors via independent origins. This difference is not completely erased, although polyploidization bottleneck might occur in Central Asia Kengyilia lineages and strong purifying selection might act on the Pgk1 gene in the Qinghai-Tibetan Plateau Kengyilia lineages.It is worth mentioning that the dTable S1Kengyilia species and other related genera in Triticeae used in this study.(DOC)Click here for additional data file.Table S2Estimates of nucleotide diversity and test statistics at Pgk1 locus in Kengyilia St, Y and P genome and its putative diploid genome donor.(DOC)Click here for additional data file.Table S3Detection of selection pressure on the coding portions of the Pgk1 gene in Kengyilia St, Y and P genome and its putative diploid genome donor.(DOC)Click here for additional data file.


I) of GOX, approximately pH 4.0. In addition, the molecular weight of chitosan also evidently influenced the encapsulation efficiency. Storage stabilities of GOX samples were investigated continuously over two months and the retained activity of CACM-GOX was 70.4%, markedly higher than the 7.5% of free enzyme. The results reveal the great potential of CACM-GOX as a flour improver.In order to improve its stability and catalytic rate in flour, the immobilization of glucose oxidase (GOX) was investigated in this work. The enzyme was encapsulated in calcium alginate-chitosan microspheres (CACM) using an emulsification-internal gelation-GOX adsorption-chitosan coating method. The interaction between alginate and chitosan was confirmed by infrared spectroscopy (IR). The resultant CACM in wet state, whose morphology was investigated by scanning electron microscopy (SEM), was spherical with a mean diameter of about 26 μm. The GOX load, encapsulation efficiency and activity of the CACM-GOX were influenced by concentration of chitosan, encapsulation time and encapsulation pH. The highest total enzymatic activity and encapsulation efficiency was achieved when the pH of the adsorption medium was near the isoelectric point (p The gluconolactone is then slowly hydrolyzed to gluconic acid via a nonenzymatic mechanism. The generated H2O2 is able to oxidize thiol groups into disulfide bonds and catalyze the water-soluble pentosans in the dough to gel in the presence of peroxidase, which explains its improving effect on wheat flour [Applying enzymes instead of chemical oxidants is a very interesting option to improve the bread-making performance of dough since they are considered as natural and non-toxic food components. Currently, the baking industry is deeply involved in research on alternatives to potassium bromate due to its potential hazards. Glucose oxidase (GOX) EC 1.1.3.4) is the preferred enzyme alternative to chemical oxidizing agents for bread improvement and has been cited for commercial use. It is a dimeric protein with a molecular weight of 160 kDa, containing one tightly but noncovalently bound flavin adenine dinucleotide (FAD) per monomer as cofactor and glycosylated with a carbohydrate content of 16% (w/w). In the presence of oxygen, GOX catalyzes the oxidation of β-.1.3.4 isat flour .2O2 generated during that period, which accounts for the dry and strong dough as well as bread with low volume and poor crumb [et al. [There are still some deficiencies of GOX despite its good effects on flour, and its encapsulation and immobilization has being studied due to its high oxidization rate and low stability in flour. As a fast-acting oxidant, GOX reduces the elasticity of dough immediately after mixing because of the abundant Hor crumb –10. Mean [et al. , 25% of 2O2 in dough.The basic idea behind enzyme immobilization is to entrap the protein in a semi-permeable support material that prevents the enzyme from leaving while allowing substrates, products and co-factors to pass through. The catalytic rate of the enzyme inside the microcapsules could be adjusted by the thickness of its wall material. Compared with free enzymes in solution, the immobilized ones are more robust and resistant to environmental changes. The immobilized GOX in the microspheres can not only prevent enzyme from denaturing but can also delay the generation of Hl-guluronic acid (G) and β-d-mannuronic acid (M) residues. Alginate supports are usually made by cross-linking the carboxyl group of the α-l-guluronic acid with a solution of a cationic cross linker such as calcium chloride. In the experiment, an internal gelation method was selected [Alginate is by far the most widely used polymer in immobilization and micro-encapsulation technologies ,13. Algiselected ,15. Insoselected –18.Chitosan is another natural polymer that has gained tremendous interests in immobilization technology. Chitosan is used either as a means of coating alginate beads in order to alter the diffusion rate of the GOX  or as anThe purpose of the present work was to encapsulate GOX into CACM with high encapsulation efficiency and residual activity. Immobilization conditions including the concentration and molecular weight of chitosan, encapsulation pH and absorption time were optimized. Thermal and storage stabilities of CACM-GOX compared with that of free enzyme were also studied in the paper.2.2.1.All chemicals and reagents used in this work were food-grade or reagent-grade. Glucose oxidase (Gluzyme Mono) containing 10,000 glucose oxidase units/g was kindly supplied by Novo Nordisk . Alginate  was purchased from Sinopharm Chemistry Reagent . Chitosans with different molecular weights and 91% deacetylation degree were purchased from Yuhuan Ocean Biochemistry .2.2.A modified emulsification-internal gelatin method ,17,21 waOne milliliter GOX of 1 mg/mL in a 0.2 M buffer solution of pH 4 (sodium acetic-acetic acid) was added into 2 mL CaAlg gel beads (dry weight of 7.7 mg/mL). The adsorption experiments were carried out for 60 min at 4 °C, followed by incubating the GOX CaAlg gel beads in 1 mL 1% (w/v) chitosan acetic acid solution  for 10 min at 4 °C, shaking quickly to ensure the reaction uniform. The CACM beads  were washed twice with 10 mL deionized water and blended with 0.26 mL 0.2 M acetic buffer solution of pH 6 for activity assay.2.3.d-glucose solution (1 mg/mL) and 0.1 mL free enzyme or appropriate volume of other enzyme preparation followed by adding acetic buffer (pH 6.0) to 4 mL. The mixture was incubated at 30 °C for 30 min under a vibrating state in a rocking bed. The reaction was stopped by holding the tube in boiling water. To measure residual sugar, 2 mL DNSA reagent was added into the above tube and the mixture was boiled for 5 min followed by cooling to room temperature and diluted to 25 mL. The latter was quantified by measuring absorbance at 520 nm using a spectrophotometer . A unit of glucose oxidase activity is defined as the amount of enzyme which oxidizes 1.0 μmol of β-d-glucose to gluconic and hydrogen peroxide per minute at 30 °C.The activities of free GOX and CACM-GOX were measured by the DNSA method ,22. The 2.4.Microsphere features such as shape and existence of aggregates were examined after isolation by an optical microscope Olympus CKX41 equipped with a digital camera . CACM was stained by neutral fuchsin. Scanning electron microscopy  was utilized to observe the morphology of coated microspheres. The particle size and size distribution of the CaAlg gel beads and the CACM were determined by a particle size analyzer .−1 at a resolution of 4 cm−1.Infrared (IR) spectra of alginate, chitosan, and CACM were recorded with a Fourier transform infrared spectrometer . Freeze-dried samples were scanned from 600 to 4000 cm2.5.2.5.1.To investigate optimum encapsulation pH and the total activity of CACM-GOX, 2 mL CaAlg gel beads (dry weight of 7.7 mg/mL) were added into 1 mL GOX of 1 mg/mL prepared in 0.2 M acetate buffer at pH 3.0, 3.5, 4.0, 4.5, 5.0 and 0.2 M phosphate buffer at pH 6.0 and 7.0. Encapsulation mixture was kept at 4 °C for 80 min. The experiment revealed that GOX was stable under pH 4.5 and 5.0 and no deactivation or precipitation was observed. The encapsulation efficiency was calculated in terms of the change of GOX activity in the supernatant before and after incubation of CaAlg gel beads in enzyme solutions.2.5.2.One percent (w/v) chitosan solutions were prepared by dissolving chitosans, which had the same degree of deacetylation (91%) but different molecular weight of 20,000 (LMW), 100,000 (MMWa), 200,000 (MMWb) and 450,000 (HMW), into 1% acetic acid solution. The GOX adsorption experiments were carried out as above followed by incubating the GOX CaAlg gel beads in each chitosan solution for 10 min at 4 °C.2.5.3.Chitosan acetic acid solutions of 0.5%, 1% and 2% with a predetermined molecular weight were prepared. 1 mL GOX solution was added into 2 mL CaAlg gel beads. Encapsulations were carried out for 10, 20, 40, 60 and 80 min separately, followed by incubation in each chitosan acetic acid solution (pH 4.0) for 10 min. The amounts of GOX loaded and activities of CACM-GOX were determined based on dry weight of the particles. The amount of GOX loaded was calculated by the following equation. Protein values were determined by the method of Lowry.2.6.Free GOX solution containing 1 mg/mL enzyme protein in 0.2 M buffer (pH 6.0) and CACM-GOX  with an activity of 8.86 U/mL were kept at 4 °C and 25 °C, and the residual activities of these samples were measured periodically for 60 days and 60 hours, respectively.2.7.Each experiment was repeated three times. The results were expressed as means ± S.D. and analyzed by SAS  analysis of variance method.3.3.1.The morphology of microspheres was determined in terms of their final shape and granulometry. Wet CaAlg beads presented as spherical by optical micrographs , CACM coThe micrographs of calcium-alginate microspheres (MMWb chitosans of 1.0%) of about 30 μm and approximate to regular spherical shape with a smooth external surface are shown in The particle size and size distribution of a representative preparation of CaAlg beads and CACM are presented in −1 and the absorption band of the N-H of non-acylated 2-aminoglucose primary amines bending vibration at 1570 cm−1. The peaks at 1419 and 1384 cm−1 belonged to the N-H stretching of the amide and ether bonds and N-H stretching (amide III band), respectively [−1 were observed as well. The carbonyl (C=O) stretching of the secondary amide (amide I band) at 1655 cm−1 and the bending vibrations of the N–H  at 1558 cm−1 [−1 due to carboxyl anions (asymmetric and symmetric stretching vibrations).In order to confirm alginate-chitosan interactions, samples were analyzed by IR spectroscopy. ectively . The bri558 cm−1  were ver−1 became broader, which illustrated hydrogen bonding was enhanced [−1) and asymmetric and symmetric –C–O stretching at 1636 and 1384 cm−1, respectively, disappeared, indicating that the –NH2+ of the chitosan had reacted with the –COO− of the alginate. A new peak at 1561 cm−1 was assigned to the amide bond of CACM [For the alginate-chitosan beads, the band around 3500–3100 cmenhanced . Moreove of CACM .3.2.3.2.1.Ka values of β-d-mannuronic acid (M) and α-l-guluronic acid (G) of alginate were 3.38 and 3.20, respectively. Therefore, both alginate and GOX were positively charged at pH 3, which was lower than the pI of alginate (pH 3.3), leading to low encapsulation efficiency due to electric repelling. A similar phenomenon occurs at pH values higher than 4.5 due to repulsive electrostatic interaction between negative charges. The total activity of CACM-GOX displayed the same trend with the immobilization efficiency, which suggested that GOX had good stability and low loss in the 80 min absorption process within the pH range of 3.0 to 7.0.To establish the optimum pH value for the encapsulation of GOX into CaAlg, the pH of encapsulation medium was changed between 3.0 and 7.0. As seen in 3.2.2.Four chitosan solutions with different molecular weights were selected to investigate the effects of molecular weight on the activity of GOX in the supernatant. The latter was calculated by measuring the difference between the total activity of GOX added in the preparation medium and the residual activity remaining in the aqueous supernatant suspension after the absorption process. As shown in 2+ and alginate resulted in many channels, beneficial to entrance of GOX into CaAlg beads [The encapsulation efficiency increased rapidly in the initial 20 min of absorption time and then slightly rose up to 80 min for the chitosans with high molecular weight. Chitosans of MMWb and HMW had similar variation law of encapsulation efficiency with high efficiencies of 93.1% and 91.1%, respectively, at 60 min. The egg-box structure formed by Calg beads ,27. The lg beads . Therefo3.2.3.p < 0.01) affected by the concentration of chitosan. It increased as the reaction proceeded for up to 80 min and was at the greatest rate within the first 20 min for all the three concentrations and almost remained constant for periods longer than 60 min, which indicated that the enzyme absorption to the CaAlg beads happened mainly at the beginning followed by lower speed as the concentration of free enzyme decreased. The encapsulation efficiency was also influenced significantly by chitosan concentration (p < 0.05) and achieved the minimum in the presence of 0.5% chitosan. Through measuring the protein concentration in the supernatant of water before and after washing CACM-GOX, it was found that GOX could not be effectively mobilized in microcapsules and was prone to being washed out in the presence of low-concentration chitosan, leading to lower GOX load and activity of CACM-GOX.The effects of chitosan concentration and encapsulation time on the encapsulation efficiency, the amount of GOX loaded and the total activity of CACM-GOX were evaluated. As seen in The amount of GOX loaded and the total activity of CACM-GOX also increased gradually with extending encapsulation time due to more and more protein absorption onto CaAlg beads. They were also influenced by the concentration of chitosan as the total weight of CACM increased due to more chitosan coating on the surface of CaAlg beads with increasing concentration. The phenomenon that chitosan competed with GOX for absorption  did not The specific CACM-GOX activity, to measure the catalytic rate of the immobilized enzyme, was taken as the ratio of total CACM-GOX activity to the encapsulation efficiency. It was maximum in the presence of 0.5% chitosan, the next was 1.0% followed by 2% , which m3.3.Free GOX solution containing 1 mg/mL enzyme protein in 0.2 M sodium phosphate buffer (pH 6.0) and CACM-GOX  with an activity of 8.86 U/mL were kept at 4 °C and 25 °C, and residual activities of these samples were measured periodically for 60 days and 60 hours, respectively.The immobilized GOX was much more thermally stable than the free. After 30 h incubation at 25 °C, free and immobilized GOX retained activities of 15.5% and 83.0%, respectively. Subsequently, the activity of CACM-GOX clearly decreased as seen in The storage stabilities of the CACM-GOX and free GOX were investigated at 4 °C and the results are shown in 4.The necessary conditions for successful encapsulation of GOX into CACM microspheres using an emulsification-internal gelation-enzyme adsorption-coating by chitosan method with high residual activity had been established.I of GOX. The high porous structure of CaAlg gel beads made by carbon dioxide release during the course of CaCO3 dissolution contributed to the high absorption efficiency of protein. The molecular weight of chitosan was a very important factor for the whole experiment. Chitosans with low molecular weight, 20,000 (LMW) and 100,000 (MMWa), can enter the interior of CACM with less space resistance, resulting in low encapsulation efficiency. Enzyme absorption occured mainly within the first 20 min and then the speed became slower. The encapsulation efficiency and GOX load, coating for 60 min under the condition of 1% chitosan, were 91.1% and 4.15 (mg/g), respectively. Low GOX load was beneficial for CACM-GOX application in flour and could make the extremely low dosage of enzyme disperse in flour matrix homogenously in favor of the release and catalysis of GOX. The specific CACM-GOX activity was remarkably influenced by the concentration of chitisan and the maximum was achieved in the presence of 1% chitosan in the experiment.Total activity of CACM-GOX and encapsulation efficiency were dependent on the adsorption medium pH with much higher activity and encapsulation efficiency at pH values near the p