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Chronic intestinal inflammation, as seen in inflammatory bowel disease (IBD), results from an aberrant and poorly understood mucosal immune response to the microbiota of the gastrointestinal tract in genetically susceptible individuals. Here we used serological expression cloning to identify commensal bacterial proteins that could contribute to the pathogenesis of IBD. The dominant antigens identified were flagellins, molecules known to activate innate immunity via Toll-like receptor 5 (TLR5), and critical targets of the acquired immune system in host defense. Multiple strains of colitic mice had elevated serum anti-flagellin IgG2a responses and Th1 T cell responses to flagellin. In addition, flagellin-specific CD4+ T cells induced severe colitis when adoptively transferred into naive SCID mice. Serum IgG to these flagellins, but not to the dissimilar Salmonella muenchen flagellin, was elevated in patients with Crohn disease, but not in patients with ulcerative colitis or in controls. These results identify flagellins as a class of immunodominant antigens that stimulate pathogenic intestinal immune reactions in genetically diverse hosts and suggest new avenues for the diagnosis and antigen-directed therapy of patients with IBD.
Crohn disease (CD) and ulcerative colitis (UC), collectively referred to as inflammatory bowel disease (IBD), are relatively common inflammatory diseases of the gastrointestinal (GI) tract. Histopathologically and anatomically, these two conditions are distinct, with CD characterized by transmural inflammation that can occur throughout the GI tract, and UC characterized by more superficial inflammation confined to the colon and rectum. Interestingly, both diseases are dependent upon factors present within the complex intestinal microbiota. Indeed, a unifying hypothesis has emerged that proposes that IBD results from a dysregulated mucosal immune response to the intestinal microbiota in genetically susceptible individuals (reviewed in refs. 1, 2).
While the dependence of IBD on intestinal microbes is increasingly clear, the molecular mechanisms underlying this dependence are not. The intestinal mucosa is exposed to the largest concentration of foreign bacterial antigens of any tissue in the body, estimated to be up to 1012 organisms per gram of stool in the normal colon. An emerging concept is that there is an active "dialogue" between the microbiota, intestinal epithelial cells, and mucosal immune cells, with each partner communicating with the others (3). In this context, "innate" immune responses, which recognize conserved microbial products such as lipopolysaccharide (LPS) and peptidoglycan (PG), are likely to be important in these microbial-host interactions and intestinal homeostasis. Critical to the host's "sensing" of microbes are members of the Toll-like receptor (TLR) family that, alone or in combination, recognize a wide array of microbe-associated molecular patterns on either pathogens or commensals (reviewed in refs. 4–6). Various TLRs are expressed on intestinal epithelial cells (7–10) and more broadly on macrophages and dendritic cells in the lamina propria. In addition, the identification of Nod2, an intracellular protein that recognizes muramyl dipeptide, as a susceptibility gene for CD highlights the potential role of pattern-recognition receptors and their ligands in diseases such as IBD (11).
Given the involvement of innate immune mechanisms in the modulation of T cell responses, the bacterial dependence of IBD is likely to involve both bacterial products such as LPS, PG, and other TLR ligands, and specific bacterial antigens capable of stimulating CD4+ T cell responses. CD4+ T lymphocytes have been identified as the crucial effector cells in experimental models of IBD (12–14), and these pathogenic CD4+ T cell responses are directed against the enteric microbiota. Enteric bacterial antigen–reactive CD4+ T cells are able to induce colitis when adoptively transferred into immunodeficient recipients (14). The in vitro data to date suggest that there is a relatively small number of immunodominant antigens that stimulate the pathogenic T cell responses (15), but the complexity of the intestinal microflora has posed a significant challenge to their identification. One notable successful example is the identification of "I2" by Braun and colleagues (16). This antigen, derived from a Pseudomonas species present within the intestinal microflora, was discovered using a molecular technique, representational difference analysis (RDA), to identify DNA sequences present in intestinal tissue from IBD patients but not in normal control (NC) tissue (17). These data highlight the utility of using unbiased molecular approaches to address this challenging problem.
Accordingly, we used a molecular technique known as serological expression cloning (SEC) to identify specific bacterial antigens driving experimental IBD. SEC involves the screening of DNA expression libraries in lambda phage with defined antisera. In clinical or experimental systems of infectious diseases, in which entire microbial genomes can be screened with defined reactive sera, SEC has proven to be extremely useful in the identification of antigens and vaccine targets (18, 19).
In this manuscript, we describe the molecular cloning of antigens by SEC using sera from colitic C3H/HeJBir mice. Interestingly, the dominant antigens identified were a family of related, novel flagellins. Strong reactivity against specific flagellins was seen in multiple models of experimental colitis across several distinct MHC haplotypes. There was a clear IgG2a predominance to the anti-flagellin response, suggesting a concurrent Th1-biased T cell response against flagellin. Indeed, marked reactivity against flagellin was seen in mesenteric and splenic T cell cultures from colitic animals, and flagellin-specific T cells were able to induce colitis when adoptively transferred into immunodeficient animals. Surprisingly, the reactivity against these flagellins (but not against the dissimilar Salmonella flagellin) was also seen in human IBD sera, with significant reactivity in patients with CD but not UC or control patients. Given the recognition that flagellin is the ligand for TLR5 (20), these data not only provide insight into antigens that may be involved in the pathogenesis of IBD but also highlight a link between IBD and innate immunity.
Serologic expression cloning resulted in 55 clones that were sequenced and identified. Using the basic local alignment search tool to search the GenBank databases demonstrated that 15 (26.8%) of these clones were flagellin-like sequences. None of the sequences directly matched any flagellin in the GenBank database, and all flagellin sequences identified represented unique clones. Given the average insert size of 0.8 kb in the library, no full-length flagellin clones were identified. However, all of the flagellin clones contained sequences derived from the conserved NH2 terminus, with varying amounts of the hypervariable central domain, and only two clones contained partial sequence from the conserved carboxy domain. Sequences from the 15 flagellin clones identified (CBir1–CBir15) were aligned at the protein level to flagellin sequences available in the public domain using the Clustal program in DNAStar. As shown in Figure Figure1B,1B, these flagellins are most closely related to flagellins from Butyrivibrio, Roseburia, Thermotoga, and Clostridium species and appear to align, by similarity, in the Clostridium subphylum cluster XIVa of Gram-positive bacteria. Sequences from the remaining 40 clones (see Table Table1)1) were also unique and were either related to known proteins (33 clones) or without significant homology to known proteins (7 clones).
Because of strong serum antibody reactivity to one particular flagellin clone, called CBir1, we cloned and expressed its full-length gene. During this effort, we also cloned a second, highly homologous and reactive flagellin (83.5% similarity to CBir1 at the NH2 conserved domain) and refer to it here as Fla-X (Figure (Figure2B).2B). Recombinant proteins representing full-length sequence and NH2 and carboxy fragments of both CBir1 and Fla-X were subsequently expressed in E. coli with a six-histidine tag to aid in protein purification (Figure (Figure2,2, C and D, respectively).
Western blot analyses using these purified recombinant flagellins and fragments with sera from the diseased C3H/HeJBir mice demonstrated the strong reactivity to flagellin, predominantly to the NH2-terminal fragments (Figure (Figure3A).3A). Little or no antibody reactivity was seen to the carboxy-terminal CBir1 or Fla-X recombinant fragments in the sera tested (Figure (Figure3).3). This selective reactivity to the NH2 domain is consistent with the presence of an NH2 domain in all flagellin clones identified in the initial serological screen (Figure (Figure1).1). In addition, strong reactivity to both flagellins was seen using sera from two additional experimental models of colitis: mdr1a–/– mice (Figure (Figure3B)3B) and B6.IL-10–/– mice (data not shown). These last two models are on strains with different haplotypes from each other (H-2s and H-2b, respectively) and from the C3H/HeJBir strain (H-2k), and the sera were obtained from mice from geographically different mouse facilities. In addition, these additional colitic strains have very different mechanisms underlying the genetic predisposition to develop IBD; that is, epithelial barrier dysfunction in the mdr1a–/– mice and a defect in regulatory T cells in the B6.IL-10–/– mice. Little or no reactivity was seen to CBir1 or Fla-X in noncolitic mouse serum from control MHC haplotype–matched noncolitic mice (Figure (Figure3,3, A and B). Interestingly, we also saw a similar pattern of reactivity to the NH2 termini of both CBir1 and Fla-X with a serum pool from patients with CD (Figure (Figure33C).
In order to generate more quantitative data with multiple IBD models at various time points in the course of disease, we developed an antibody subclass ELISA against full-length or fragments of CBir1 or Fla-X (Figure (Figure2,2, C and D). With this assay, we confirmed Western blot data showing that the antibody reactivity observed was predominantly to the NH2 terminus (data not shown) and of the IgG2a subclass. High titers of anti-flagellin antibody were seen in the four genetically distinct models of IBD tested (colitic mice: C3H/HeJBir [Figure 4], mdr1a–/– [Figure 4], BALB/c.IL-10–/– [not shown] and B6.IL-10–/– [not shown]), while minimal to no reactivity was seen in serum from the H-2–matched, control, noncolitic mouse strains. Given the nonuniform incidence of colitis in the mdr1a–/– colony at varying time points, we randomly chose 23 animals in the colony and assigned quantitative histopathological scores using a scale (from 0 to 60) that incorporates both the degree and extent of inflammation in the large intestine (21). We measured antibodies against Fla-X and CBir1 by ELISA in a "blinded" manner using sera from these animals and found that an increased titer of anti-flagellin IgG correlated positively with worsening IBD histopathology (r = +0.758 and +0.719, respectively; Figure Figure5).5). Weak correlations were found between antibody and mouse age (r = +0.325; data not shown) and between colitis score and mouse age (r = +0.372; data not shown).
Subsequently, we tested a large panel of sera from controls and patients with IBD for reactivity against CBir1 and Fla-X using antigen-specific ELISAs. We found a significantly higher level of serum anti–CBir1/Fla-X flagellin in CD patients than in NCs, disease controls (DCs), and UC patients (Figure (Figure6A).6A). It should be noted that more than 50% of the UC sera were from patients with a modified Truelove and Witts severity index greater than 7, indicating moderate to active disease. The observation of serum responses to flagellins CBir1 and Fla-X in a group of CD patients, but not UC patients (Figure (Figure6A),6A), highlights the possibility that anti-flagellin responses may be valuable in the diagnosis of IBD, in particular with regards to the more precise discrimination between UC and CD, and the definition of CD patient subsets.
Reactivity to the Salmonella muenchen flagellin (which is highly similar to the flagellin of Eschericia coli [84–91% at the conserved NH2 end]), however, showed no significant correlation to disease (Figure (Figure6B).6B). As shown in Figure Figure6B,6B, the mean values for the anti-Salmonella response were nearly identical in the control, CD, and UC populations. In all populations, there appeared to be a minority of samples without significant reactivity and a majority of samples that were "positive." While these data may reflect the random exposure to Salmonella in humans due to prior infection (possibly subclinical) or a cross-reactivity to an undefined but closely related flagellin (probably from the Enterobacteriaceae family), it is clear that the serological response to the Salmonella flagellin molecule does not correlate with IBD. Similarly, there was no correlation between reactivity to Salmonella flagellin and colitis in the C3H/HeJBir or mdr1a–/– strains compared with the MHC haplotype–matched controls (data not shown).
Marked reactivity against flagellin is seen at the T cell level, and flagellin-specific T cells are able to induce colitis when adoptively transferred. Given the strong IgG2a bias seen in the antibody response in the mouse IBD strains, we hypothesized that flagellin-specific Th1 T cells would be present in mice with IBD. To address this possibility, we purified CD4+ T cells from pooled spleens and mesenteric lymph nodes from colitic mdr1a–/–, C3H/HeJBir, and C3H/HeJ.IL-10–/– mice (and haplotype-matched, noncolitic FVB and C3H/HeJ mice) and tested the cells for reactivity against purified CBir1 and Fla-X in vitro in the presence of antigen-presenting cells (APCs). CD4+ T cells from the colitic mdr1a–/–, C3H/HeJBir, and C3H/HeJ.IL-10–/– mice, but not from age-matched control FVB or C3H/HeJ mice raised in the same mouse facility, responded to CBir1, as assessed by proliferation (Figure (Figure7).7). It was possible that the responses seen were due to the fact that the flagellin molecule was nonspecifically activating the cultured T cells via TLR5 or TLR4 activation (through endotoxin contamination of the recombinant protein). This possibility was excluded by the lack of stimulation in both the noncolitic T cell cultures (Figure (Figure7)7) and in an independent T cell culture system that showed no influence of Fla-X or CBir1 on the ovalbumin-specific proliferation of CD4+ T cells from DO11.10 ovalbumin-specific T cell receptor–transgenic animals (Table (Table22).
We have previously shown that a T cell line specific for cecal bacterial protein/antigen (CBA), but not CD4+ T cells polyclonally activated by anti-CD3, could induce mucosal inflammation when adoptively transferred into H-2–matched immunodeficient scid/scid mice (14). To address the potentially pathogenic role of flagellin-specific T cells in the initiation of mucosal inflammation, we generated a CD4+ T cell line reactive with CBir1 flagellin from C3H/HeJBir mice by repeated stimulation with antigen and APCs. This CD4+ T cell line strongly responded to CBir1 but not to Fla-X or a variety of other microbial, food, and epithelial antigens (Figure (Figure8).8). These CBir1-specific CD4+ T cells were adoptively transferred into C3H/HeJ-scid/scid recipients. Control SCID mice received anti-CD3–activated CD4+ T cells as a negative control or a CD4+ T cell line reactive to CBA as a positive control. Quantitative histopathological scores were assigned at 8 weeks after transfer (Figure (Figure9A).9A). The CBir1-specific CD4+ T cell line induced colitis in all recipients of an intensity that was similar to or greater than that induced by the CBA-specific CD4+ T cell line, whereas none of the recipients given anti-CD3–activated C3H/HeJBir CD4+ T cells developed disease (representative histology is shown in Figure Figure99B).
Data from both humans and experimental animals underscore the critical role of intestinal bacteria in the establishment and maintenance of IBD (2). The molecular mechanisms underlying this clear bacterial dependence on mucosal inflammation are likely to relate to the intrinsic ability of these organisms to trigger both acquired and innate immune responses in the gut. Using an unbiased molecular screen to search for bacterial antigens relevant to IBD, the dominant antigens identified were a family of related, novel flagellins. A strong, IgG2a-biased serological response to these specific flagellins was seen in multiple models of experimental colitis across several distinct MHC haplotypes. In addition, marked reactivity against these flagellins was seen at the T cell level, and flagellin-specific T cells were able to induce colitis when adoptively transferred into immunodeficient animals. Interestingly, while these flagellins were identified from mouse cecal bacteria, there was clear, specific reactivity against these molecules in patients with CD (but not in patients with UC or in NCs). Given the activity of flagellin as a specific ligand for TLR5, these data provide a potentially important link between adaptive and innate immune responses in mucosa in the pathogenesis of IBD.
Among bacterial antigens, flagellin is an interesting candidate to play a role in mucosal immune responses. Specifically, flagellin is a common bacterial antigen present on most motile bacteria in the gut (22). Moreover, flagellin is highly antigenic; indeed, responses against flagellin are protective in Salmonella infections in mice (23, 24). Furthermore, flagellin has been identified as a pathogen-associated molecular pattern and has been identified as the specific ligand for TLR5. Monomeric, translocated flagellin is capable of stimulating cytokine and chemokine production by TLR5-positive intestinal epithelial cells (8), and flagellin has been demonstrated to induce the maturation of dendritic cells via TLR5 (25, 26). Indeed, we have observed that full-length flagellin Fla-X (endotoxin free) is capable of stimulating TNF-α production by human macrophages in vitro (M.J. Lodes and R.M. Hershberg, data not shown). It is tempting to speculate that the intrinsic "adjuvanticity" of flagellin is likely to contribute to its antigenicity. While flagellin molecules clearly have the capacity to stimulate the production of proinflammatory cytokines via TLR5, we favor the hypothesis that the B and T cell responses to flagellin contribute more directly to the chronic intestinal inflammation seen in IBD.
Our clinical data (Figure (Figure6)6) are consistent with the fact that the aberrant response in patients with CD is specific to the subgroup of flagellins identified in our molecular screen. Specifically, there was no correlation between IBD and a response to flagellin from Salmonella muenchen, which is very similar (84–91%) in the NH2 conserved region to the flagellin from the commensal organism Escherichia coli. It must be emphasized that the flagellins identified were from a source of material devoid of known bacterial pathogens. The bacteria with genes that "encode" the flagellins CBir1 and Fla-X (the two dominant flagellins tested) are unknown; however, preliminary phylogenetic data suggest that these flagellins are most closely related to the flagellins of bacteria in the genera Butyrivibrio, Rosburia, Thermotoga, and Clostridium and fall within the Clostridium subphylum XIVa cluster of Gram-positive bacteria (Figure (Figure1B).1B). This group of bacteria contains many of the butyrate-producing bacteria that are found in the human gut (27, 28). We speculate that the aberrant response to the flagellin molecule(s) from these organisms is related to a combination of the intrinsic property of the molecules themselves (as immunogens and adjuvants) and an underlying genetic susceptibility. The question of the basis for selective responses to an antigen present in the normal commensal flora of most (if not all) individuals is a challenging one. Using monoclonal antibodies directed against CBir1, we have demonstrated that this antigen is present in the stool of wild-type strains (FVB, C57BL/6, BALB/c, and C3H/HeJ) and colitic strains (mdr1a–/–, B6.IL-10–/–, and C3H/HeJBir) (data not shown). These data indicate that the presence of the antigen itself does not strictly correlate with colitis. Still, the widespread presence of these antigens does not preclude the possibility of enhanced colonization of organisms expressing these flagellins in CD lesions. This idea is certainly consistent with the recent observations by Swidsinski and colleagues (29). Alternatively, alterations in barrier function (30, 31) or intrinsic innate immune responses (32, 33) may enhance the likelihood of the response to these flagellin molecules in patients prone to develop IBD.
In general, the data are consistent with the hypothesis that IBD is associated with a defect in tolerance to commensal organisms (34). The IgG2a-biased antibody against Fla-X and CBir1 highlights the Th1 bias of the T cell responses seen. The broad recognition of these flagellins in several different mouse models and in humans with CD indicates that these flagellins are among the immunodominant antigens of the microbiota. However, the exact role of these flagellins in the pathogenesis of IBD (e.g., whether they are predominant or obligatory for disease) compared with that of other microbial antigens remains to be defined. We favor a model in which a T regulatory response to specific flagellins (and/or other bacterial antigens) may be selectively impaired in IBD. In this context, specific flagellin molecules may represent novel targets for antigen-directed therapy in IBD. Experiments are currently underway to address this possibility.
Clearly, flagellin is not the only bacterial antigen involved in the pathogenesis of IBD. It is likely that there will be specific subsets of patients with IBD (both CD and UC) that display patterns of seroreactivity against the molecular determinants that we have identified, similar to the patterns currently seen with responses to oligomannans from Saccharomyces cerevisiae, I2, and ompC for E. coli (35). Indeed, the identification of flagellin complements the efforts Braun and co-workers in discovering bacterial antigens relevant to the pathogenesis of IBD. One interesting example is I2, an antigen derived from Pseudomonas fluorescens that appears to have "superantigen-like" activity (16, 17). As we observed with the specific flagellins identified here, only a subset of patients with CD show specific seroreactivity against I2. It should be noted that an unbiased molecular approach, RDA, was also used to identify I2. In contrast to the technique of SEC, which depends upon the selective antibody responses to a given antigen, RDA depends upon the selective presence of bacteria in anatomic sections of IBD. Given the heterogenous nature of IBD and the complexity of the intestinal microflora, we are continuing our efforts to identify additional antigens in patients that do not respond to flagellin. It will be of considerable interest to determine how the reactivity against these antigens correlates with genetic determinants, clinical phenotypes, and responses to treatment.
Pelleted bacteria from C3H/HeJBir mouse ceca were inactivated at 80°C for 20 minutes and then were treated with 2 ml lysozyme (20 mg/ml in Tris-EDTA [TE] buffer) for 1 hour at 37°C. This solution was rocked at room temperature for 10 minutes with 40 l proteinase K (10 mg/ml) and 140 l 20% SDS (Sigma-Aldrich, St. Louis, Missouri, USA) and then incubated for 15 minutes at 65°C, then 0.4 ml of 5 M NaCl and 0.32 ml of a 10% cetyltrimethylammonium bromide (CTAB) solution (1 g CTAB [Sigma-Aldrich], 1.4 ml 5M NaCl, and 8.6 ml distilled H2O) was added, followed by incubation at 65°C for 10 minutes. DNA was then extracted twice with phenol, followed by extraction with phenol/chloroform/isoamyl alcohol (24:24:2), and then with chloroform. Finally the DNA was precipitated with 0.6 volumes of isopropanol and resuspended in TE buffer.
A detailed description of library construction can be found elsewhere (19). Briefly, 20 g of genomic DNA of mouse cecal bacterium was resuspended in 400 l of TE buffer and was sonicated for five seconds at 30% continuous power with a Sonic Dismembrator (Fisher Scientific, Pittsburgh, Pennsylvania, USA) to generate fragments of approximately 0.5–5.0 kb. DNA fragments were blunted with T4 DNA polymerase (Invitrogen, Carlsbad, California, USA) and were ligated to EcoRI adaptors (Stratagene, La Jolla, California, USA) with T4 DNA ligase (Stratagene). Adapted inserts were then phosphorylated with T4 polynucleotide kinase (Stratagene) and were selected by size with a Sephacryl 400-HR column (Sigma-Aldrich). Approximately 0.25 g of insert was ligated to 1.0 g Lambda ZAP Express Vector treated with EcoRI and calf intestinal alkaline phosphatase (Stratagene), and the ligation mix was packaged with Gigapack III Gold packaging extract (Stratagene) following the manufacturer's instructions.
Immunoreactive proteins were screened from approximately 6 ∞ 105 plaque-forming units (PFU) of the unamplified cecal bacterium expression lambda library. Briefly, twenty 150-mm petri dishes were plated with E. coli XL1-Blue MRF′ host cells (Stratagene) and approximately 3 ∞ 104 PFU of the unamplified library and were incubated at 42°C until plaques formed. Dry nitrocellulose filters (Schleicher and Schuell, Keene, New Hampshire, USA), pre-wet with 10 mM isopropyl β-thiogalactopyranoside (IPTG), were placed on the plates, which were then incubated overnight at 37°C. Filters were removed and washed three times with PBS containing 0.1% Tween 20 (PBST) (Sigma-Aldrich), blocked with 1.0% BSA (Sigma-Aldrich) in PBST, and washed three times with PBST. Filters were next incubated overnight with E. coli lysate–adsorbed C3H/HeJ Bir mouse serum (1:200 dilution in PBST), washed three times with PBST, and incubated with a goat anti-mouse IgG + IgA + IgM (heavy and light chain) alkaline phosphatase–conjugated secondary antibody (diluted 1:10,000 with PBST; Jackson Laboratories, West Grove, Pennsylvania, USA) for 1 hour. Filters were finally washed three times with PBST and two times with alkaline phosphatase buffer (pH 9.5) and were developed with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (Invitrogen). Reactive plaques were then isolated and a second or third plaque purification was performed. Excision of phagemid followed the Stratagene Lambda ZAP Express protocol, and the resulting plasmid DNA was sequenced with an automated sequencer (ABI, Foster City, California, USA) using M13 forward, reverse, and sequence-specific internal DNA sequencing primers. Nucleic acid and predicted protein sequences were used to search the GenBank nucleotide and translated databases. Protein analysis was performed with the PSORT program (National Institute for Basic Biology, Okazaki, Japan) and with the IDENTIFY program of EMOTIF (Department of Biochemistry, Stanford University). Sequence alignments were produced with the MegAlign program (Clustal) of DNAStar (Madison, Wisconsin, USA). Note that 20 random clones from the lambda library were picked and sequenced prior to serolological expression cloning. None of the 20 were found to be derived from mouse DNA and no flagellins were identified.
Clone CBir1 contains the conserved NH2 and variable regions of an unknown immunoreactive flagellin. The full-length sequence was obtained by first amplifying the unknown CBir1 carboxy terminus from total genomic cecal bacterium DNA with Expand polymerase (Roche, Indianapolis, Indiana, USA) and the primers CBir1var1 (designed from the variable region of CBir1; CACAATCACAACATCTACCCAG) and CBir1 Carb Z (designed from the carboxy terminus of the related flagellin B of Butyrivibrio fibrisolvens, GenBank accession number AF026812; 5′-TTACTGTAAGAGCTGAAGTACACCCTG-3′). This PCR product was cloned with a Zero Blunt TOPO PCR Cloning Kit (Invitrogen), digested with EcoRI, and gel-isolated (carboxy end of CBir1). Clone CBir1 plasmid DNA, which represents the NH2 terminus plus flagellin central variable region and overlaps with the cloned carboxy region, was digested with ScaI and then gel-isolated. Both overlapping (181-bp) DNA fragments (approximately 20 ng each) were added to a PCR reaction with the primers CBir1 HIS and CBir1 TERMX (see below), and the amplification product was cloned and expressed as described below.
Fla-X is an immunoreactive full-length flagellin sequence with no known identity in the public databases. Full-length flagellin Fla-X was cloned from total cecal bacterium genomic DNA by PCR amplification with the primers CBir Fla-X HIS (5′-CAATTACATATGCATCACCATCACCATCACGTAGTACAGCACAATC-3′) and CBir1 TERMX (5′-ATAGACTAAGCTTACTGTAAGAGCTGAAGTACACCCTG-3′), and was expressed as described below. The amplification product was cloned with a Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and several clones were sequenced.
Recombinant Salmonella muenchen flagellin (≥95% pure by SDS-PAGE) was obtained from Calbiochem (La Jolla, California, USA). Expression of other recombinant flagellin proteins and deletion constructs was accomplished by amplification from the cloned plasmid or genomic DNA (full length Fla-X) with Pfu polymerase (Stratagene) and the following primer pairs: for full-length CBir1, CBir1 HIS (5′-CAATTACATATGCATCACCATCACCATCACGTAGTACAGCACAATTTACAGGC-3′) and CBir1 TERMX (5′-ATAGACTAAGCTTACTGTAAGAGCTGAAGTACACCCTG-3′); for the CBir1 NH2 plus variable regions, CBir1 HIS and CBir1 AV TERM (5′-ATAGACTAAGCTTAAGAAACCTTCTTGATAGCGCCAG-3′); for the CBir1 NH2 terminus, CBir1 HIS and CBir1 A TERM (5′-TAGACTGAATTCTAGTCCATAGCGTCAACGTTCTTTGTGTC-3′); for the CBir1 carboxy terminus, CBir1 C HIS (5′-CAATTACATATGCATCACCATCACCATCACAAGATGAACTTCCATGTAGGTGC-3′) and CBir1 TERMX; for full-length Fla-X, CBir Fla-X HIS (5′-CAATTACATATGCATCACCATCACCATCACGTAGTACAGCACAATC-3′) and CBir1 TERMX (ATAGACTAAGCTTACTGTAAGAGCTGAAGTACACCCTG-3′); for the Fla-X NH2 plus variable regions, Fla-X HIS (5′-CAATTACATATGCATCACCATCACCATCACGTAGTACAGCACAATCTTAGAGC-3′) and Fla-X AV TERM (5′-ATAGACTAAGCTTAGAGGCTGAAATCAATGTCCTCG-3′); for the Fla-X NH2 terminus, Fla-X HIS and Fla-X A TERM (5′-ATAGACTAAGCTTAATGTGCTGAAAGATATCTTGTCAC-3′); and for the Fla-X carboxy terminus, Fla-X C HIS (5′-CAATTACATATGCATCACCATCACCATCACTTCAGCCTCCATGTAGGTGCAGATGC-3′) and CBir1 TERMX. Primers contained restriction sites for cloning (in bold) and a six-histidine tag (in italics) for protein purification (NH2 terminus). The amplification products were digested with the restriction enzymes NdeI and HindIII or EcoRI, depending on the primer set used, gel-isolated, and ligated to a pET 17b plasmid vector (Novagen, Madison, Wisconsin, USA) previously cut with NdeI and with HindIII or EcoRI and dephosphorylated with alkaline phosphatase (MB grade; Roche). The ligation mix was transformed into XL1 Blue competent cells (Stratagene) and plasmid DNA was prepared for sequencing (Qiagen, Valencia, California, USA). Recombinant protein was expressed by transformation of plasmid DNA into BL21 pLysS competent cells (Novagen) and induction of a single-colony cell culture with 2 mM IPTG (Sigma-Aldrich). Recombinant protein was recovered from cell lysate with nickel-nitrilotriacetic acid agarose beads (Qiagen), following the manufacturer's instructions, and was dialyzed in 10 mM Tris, pH 4–11 depending on predicted recombinant pI characteristics. Recombinant proteins were "quality-checked" for purity by SDS-PAGE followed by staining with Coomassie blue and by NH2-terminal protein sequencing, and were quantified with a Micro BCA assay (Pierce, Rockford, Illinois, USA). Recombinants were assayed for endotoxin contamination with the Limulus assay (Bio Whittaker, Walkersville, Maryland, USA). Production of the Mycobacterium tuberculosis 38-kDa protein has been described previously (36).
Ninety-six–well EIA/RIA microtiter plates (3369; Corning Costar, Cambridge, Massachusetts, USA) were coated overnight at 4°C with 100 ng/well of the recombinant proteins. Solutions were then aspirated from the plates, which were then blocked for 2 hours at room temperature with PBS containing 1% (weight/volume) BSA. This was followed by washing in PBST. Serum diluted in PBS containing 0.1% BSA was added to wells and incubated for 30 minutes at room temperature, followed by washing six times with PBST and then incubation with secondary antibody–HRP conjugate (1:10,000 dilution) for 30 minutes. Plates were then washed six times in PBST and then were incubated with tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry, Gaithersburg, Maryland, USA) for 15 minutes. The reaction was stopped by the addition of 1 N sulfuric acid, and plates were "read" at 450 nm using an ELISA plate reader (Biotek instrument EL311, Hyland Park VA). Background values were determined by reading of reactions that lacked the primary antibody step.
Recombinant antigens (50–200 ng/lane) were subjected to SDS-PAGE analysis using 15% polyacrylamide "minigels." The antigens were transferred to nitrocellulose BA-85 (Schleicher & Schuell, Keene, New Hampshire, USA) and were blocked for 1 hour at room temperature with PBS containing 1% Tween 20. Blots were then washed three times, 10 minutes each wash, in PBST. Next, blots were probed for 1 hour at room temperature with serum diluted 1:500 in PBST followed by washing three times, 10 minutes each wash, in PBST. Blots were then incubated for 30 minutes at room temperature with secondary antibody–HRP diluted 1:10,000 in wash buffer and were again washed three times for 10 minutes each wash in PBST containing 0.5 M sodium chloride. Finally, blots were incubated in chemiluminescent substrate for ECL (Amersham Plc, Little Charlton, UK) for about 1 minute and then were exposed to X-ray film (XAR5) for 10–60 seconds, as required.
CD4+ T cells were isolated from mesenteric lymph nodes (MLNs) of mice with BD IMAG anti-mouse CD4 beads according to the manufacturer's instructions (BD Biosciences Pharmingen, San Diego, California, USA). Briefly, MLN cells were labeled with anti-CD4 beads and then were placed within the magnetic field of the BD Imagnet. The unlabeled cells in suspension were removed and the cells binding to beads were washed and used in the CD4+ T cell culture. More than 99% of cells were CD4+, as shown by flow cytometry (data not shown). For the generation of a T cell line reactive to CBir1, CD4+ T cells were isolated from MLNs of C3H/HeJBir mice as described above and were cultured with splenic APCs that were pulsed with CBir1 (100 mg/ml) overnight. The cells were restimulated every 10–14 days.
Spleen and MLN CD4+ T cells, isolated as described above, or a CBir1 flagellin–specific T cell line (4 ∞ 105 cells/well) were incubated in triplicate in the presence of antigen-pulsed, irradiated APCs (4 ∞ 105 cells per well; treated with 1–100 g/ml antigen for 18 hours at 37°C) in 96-well flat-bottomed tissue culture plates (Falcon, Lincoln Park, New Jersey, USA) at 37°C in 5% CO2 humidified air. [3H]thymidine (0.5 Ci) (New England Nuclear, Boston, Massachusetts, USA) was added at day 3 of culture and the cells were harvested at 16 hours after the pulse. The cells were harvested on glass fiber filters on a PHD cell harvester (Cambridge Technology Inc., Watertown, Massachusetts, USA), washed with distilled water, and dried. Proliferation was assessed as the amount of incorporation of [3H]thymidine into cell DNA, as measured by beta scintillation counting (Beckman Instruments, Palo Alto, California, USA) of the harvested samples, and was expressed as cpm ± SD. The preparation of epithelial cell proteins and food antigens has been described previously (14). Ethical approval for animal studies was obtained from the Institutional Animal Care and Use Committee at the University of Alabama (Birmingham, Alabama) and from Corixa Corporation.
APCs were BALB/c spleen cells that were pulsed for 24 hours with nothing, OVA peptide at 2 g/ml, CBir1 at 100 g/ml, or Fla-X at 100 g/ml, alone or in combinations as shown in Table Table2.2. These APCs were washed and irradiated with 3,000 rads prior to culture. CD4+ T cells were isolated from DO11.10 mice and were cultured at a density of 1 ∞ 105 with 4 ∞ 105 prepulsed APCs. [3H]TdR was added at day 3 of culture and the cells were harvested after 16 hours.
CD4+ T cells were cultured with cecal bacterial antigen–pulsed and irradiated C3H/HeJ splenic cells in complete medium at 37°C for 4 days in 5% CO2 air before being transferred intravenously into C3H/HeSnJ scid/scid recipients. Three months later, the recipients were killed and then the cecum and the proximal, medial, and distal portions of the colon were fixed in formalin. Fixed tissues were embedded in paraffin, and sections were stained with hematoxylin and eosin for histological examination. All slides were "read" by an experienced pathologist (A. Lazenby, Department of Pathology, University of Alabama at Birmingham) without knowledge of their origin.
Serum samples from 212 subjects (50 UC patients, 100 CD patients, 22 DCs, and 40 NCs) were obtained from the serum archive of the Cedars-Sinai IBD Research Center. Sera were produced from standard phlebotomy blood specimens and were given an "anonymous" number code, divided into aliquots, and stored at –80°C until use. The UC and CD patient specimens were obtained from a genetic case-control study (37, 38). Each patient's diagnosis was confirmed by clinical history, endoscopic and radiologic examination, and histopathology findings. The NC group is a collection of environmental controls that contain sera from individuals with no symptoms/signs of disease (i.e., spouses). DC samples include sera from patients with presumed infectious enteritis (stool culture negative for specific pathogens), blastocystis, celiac disease, collagenous colitis, irritable bowel syndrome, radiation proctitis, and acute schistosomiasis. The UC group includes both pANCA-positive and -negative specimens, while the CD group contains samples that are marker-negative, ASCA+; I2+; OmpC+ (I2-positive); OmpC+, I2+, and ASCA+; and pANCA+. Ethical approval for human studies was obtained from the institution review board at Cedars-Sinai Medical Center.
The nucleotide sequence data for the flagellins CBir1 and Fla-X have been assigned GenBank accession numbers AY551005 and AY551006, respectively.
This work was supported by Corixa Corp., the Broad Medical Foundation, NIH PO1 DK44240 to C.O. Elson, and NIH PO1 DK46763 to S.R. Targan. We thank Audrey Lazenby for review and grading of the histopathology.
Michael J. Lodes and Yingzi Cong contributed equally to this work.
Michael J. Lodes' present address is: CombiMatrix Corp., Mukilteo, Washington, USA.
Nonstandard abbreviations used: cecal bacterial protein/antigen (CBA); cetyltrimethylammonium bromide (CTAB); Crohn disease (CD); disease control (DC); gastrointestinal (GI); inflammatory bowel disease (IBD); isopropyl β-D-thiogalacto-pyranoside (IPTG); mesenteric lymph node (MLN); normal control (NC); PBS containing 0.1% Tween 20 (PBST); peptidoglycan (PG); representational difference analysis (RDA); serological expression cloning (SEC); tetramethylbenzidine (TMB); Toll-like receptor (TLR); Tris-EDTA (TE); ulcerative colitis (UC).
Conflict of interest: M.J. Lodes, R. Mohamath, M. Fort, and R.M. Hershberg, were previously employed by Corixa Corp., a publicly traded biotechnology company, which has sponsored a portion of this work. The majority of this work was funded by a grant from the Eli Broad Foundation for Medical Research to R. Hershberg, C. Elson, and S. Targan. C. Elson, who funded a portion of this work, was previously a consultant for Corixa Corp. S. Targan is a founding scientist, director, and consultant for Prometheus Laboratories.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Last Christmas the star of the show was Kidorable! Jillian received a Lotus raincoat and umbrella, as well as the matching hat and mitts. Plus, Santa brought a Kidorable bath towel, which is much loved a year later, but still in great shape. Matthew had mittens and a hat in his stocking as well. Matthew was at the end of sizing for Kidorable, but, I think Santa wanted to give it a try regardless:)
Jillian has wore her Lotus raincoat throughout the spring and summer as needed. The poor umbrella is no longer with us...as you know kids and umbrellas. But, I will say it put up with a lot of abuse, until it could no longer. The raincoat is in perfect condition and will be a great hand-me-down to Jillian's little cousin.
Jillian was perfectly happy with her Lotus raincoat, people always made a comment as to how cute she was, and how beautiful the coat is. Until...along came Lucky Cat Raincoat and matching boots! Poor Lotus who was once so loved, was now abandoned. How could it compete with two of Jillian's favourites? Bright pink and cats.
Seriously! What could be sweeter than this coat? Kidorable is amazing and I wouldn't even consider looking elsewhere for kids outerwear and accessories (now, if they only would branch into snowsuits and winter boots for us northerners.) Everything is durable, high quality and made to impeccable standards.
I am a part of the Kidorable birthday club. About a month before my kids birthday I receive an email with a special offer - which I love! I am a huge fan of Kidorable, and a repeat customer, and I am thrilled that they offered a review opportunity here at my blog. Thank you!
Kidorable is an affordable line of kids apparel and gear...it is designed for kids to love, and parents to ooh and awe over!
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(based on availability)
Mandatory Entry
1. Must be a blog follower
[to follow find the follower widget and click to join]
2. Visit Kidorable and choose a favourite item...coat, boots, umbrella, backpack?
please ensure the mandatory entry is complete if you wish to qualify for additional entries
Additional Entries
Like Kidorable on facebook
Follow Kidorable on twitter
Subscribe to my blog emails
Post my blog button, let me know where I can find it
*New* post my holiday gift guide button, let me know where I can find it = +3 entries
Follow me on twitter
Follow me on facebook network blogs
Enter any other current giveaway @CMCM - one entry for each giveaway entered
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*tweet daily for extra entry*
@ccmouse ~ Ready for a rainy fall? Get prepared by winning an umbrella from @Kidorable http://tinyurl.com/355qjal
This giveaway is open for entries from Oct 25 - Nov 15, 2010
Please ensure your email is visible
Winner will be contacted by email and have 48 hours to respond
This blog bares no responsibility for the delivery of this giveaway prize
comment 177
Flower Child

subset: commoncrawl


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MATLAB Newsgroup
I need to implement an algorithm for automatic modulation recognition using matlab. I am planning to do this by using pattern recognition, I have read about the decision tree approach, it says to "ideniify the constant envelope signals (CW, FM, FSK). PSK sigals are
not considered as constant envelope signals, since in practice
they are band-limited, which leads to a non-constant
envelope. The feature used to identify the envelope variations
is the maximum of the squared Fourier transform of the
normalized signal amplitudes "
How can i implement this technique in matlab, i need help with the code. I also need to calculate the instantaneous freq/phase, etc. I am using a .wav file.
hey are u still working on it?
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Mr. Morrison, LJA high school science teacher was our guest speaker today. He came to help us answer our inquiries about the Elements and the Periodic Table. He even brought in some real elements to show us. He taught us some songs about the elements and then took us outside where he showed us what happens when elements react to each other. The students were ready to join his 10th grade science class. Ask your child to tell you about his visit!
Guest Speaker/ Science Expert
Julie Larson Hicks
03/14/2013 3:43pm
Sounds like a great time! Keaton was telling me about elephant toothpaste!
Catie Ferandin
04/24/2013 10:29pm
That was a cool project can we do it again?
Leave a Reply


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Jams, Jellies & Marmalades
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text:
There are many good reasons
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text:
- Reported To: FHC
- Reported At: Fastheld
- Reported When: Wed Jun 22 20:19:43 2005
FASTHELD - The body of a man was found dumped in an alley near Shadow Gate in the Shadow District. A prostitute directed soldiers of the Emperor's Blades to the corpse, believed to be of a man in his late 20s or possibly 30s, with long dark blond hair. The corpse had been stripped of all clothing and valuables. His identity is unknown at this time.


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Vallejo Mayor Osby Davis said Wednesday that he will seek an independent investigation into the recent controversial officer-involved shooting death of Mario Romero.
Davis said he will ask the City Council to request that Attorney Gen. Kamala Harris' office look into concerns raised since police officers killed Romero, 23, while he sat with another man in his car outside his North Vallejo home early Sept. 2.
Police contend that when officers approached Romero's car, he reached for a weapon that later turned out to be a pellet gun, and that is why they fired at him. Family members, however, have vehemently denied that he had such a weapon, or police assertions that he had 50 ecstasy tablets in his car.
The shooting, which also wounded the other man, has sparked an ongoing community debate, as well as several public protests and thousands of comments posted on social media sites, including the Times-Herald's.
"It must be understood," Davis said. "that this request should not be taken as an indication of a lack of confidence in the established Solano County Officer Involved Shooting Protocol, or any lack of confidence in the Vallejo Police Department or the ability of the District Attorney's office to conduct fair investigations.
"However, it is my belief that under the current circumstances a secondary review will help to establish trust in the process."
Family, chief response
Romero's sister, Cynquita Martin, told the Times-Herald that, "I'm very pleased" by Davis' move.
"I see this as a positive step for the City council and mayor to earn the trust back from the community, toward the victory and justice for Mario," Martin said. She said she will watch developments and "attend (City Council meetings) until justice is served."
The mayor also said he wants police officers and all patrol cars to be equipped with video cameras.
Vallejo Police Chief Joseph Kreins said that he welcomed an independent review if it will "restore or enhance the public's trust and the department's credibility."
"The more sets of eyes you can put on something is a good thing," Kreins told the Times-Herald.
Kreins added that he doubts that any attorney general's review will produce a different outcome than the D.A.'s office investigation.
"The (D.A's investigation process) has worked for many years and continues to work," Kreins said. "But when folks are suggesting that they don't have the confidence in that process, then I have no problem with an independent review."
City Manager Dan Keen said Kreins was informed in advance of the mayor's desire to seek an outside investigation.
"It was a decision that the mayor made but he did discuss it with he (Kreins) and I," Keen said.
Current protocol
Under the current protocol, officer-involved shootings are investigated not only by the Vallejo Police Department, but also by the Solano County District Attorney's office. None of those recent shooting investigations have been completed, but Romero supporters last week called for an independent probe either by an outside police agency or the attorney general.
Martin said that while the outside investigation was a key demand that family members had made, a rally will be held Thursday at 2 p.m. at the district attorney's office. Several others have been held since the shooting.
Both Keen and Davis said it's their understanding that the state would step in after the department and the District Attorney complete their investigations. When those two reports will be done is not known, Davis said.
State Attorney General spokeswoman Lynda Gledhill said that the agency would not comment on the Davis press release.
Gledhill added that state investigations of officer-involved shootings have likely been done, but are not done on a regular basis.
Davis hopes the City Council will consider his proposals next Tuesday.Meanwhile, Keen wants to ensure that the council has all the data on how much everything would cost.
Interrupted meetings
Davis' proposals were issued a day after the Vallejo City Council abruptly ended an economic study session after protesters suddenly filled the council chambers and demanded to speak about the officer-involved shootings. The week before, dozens spoke to the council decrying not only the Sept. 2 incident but also four other fatal officer-involved shootings this year.
Keen said the intention now is to encourage the state to look at just the Sept. 2 shooting, based on the public outcry surrounding it, but not the other incidents. Davis also said his request involves the Romero shooting, not the other officer-involved shootings.
Meanwhile, referring to Tuesday night's halted meeting, both Davis and Keen said the city is exploring ways to allow residents to voice concerns and still conduct city business "in an orderly fashion."
In addition to asking that Attorney General Harris intervene, Davis said he will ask that Keen move to buy body and in-car cameras to be integrated with the vehicle's mobile data computers and emergency systems.
Currently, only some officers wear body cameras, but no cars are equipped with in-car versions.
Kreins said his goal is that all officers be issued body cameras, but he would not require them to be worn.
"My issue with not keeping it discretionary is that it might interfere with the officer's safety and the critical incident at hand," Kreins said. "The last thing I want is my officers worried about the cameras at a critical scene. In our job things happen very quickly."
Kreins favors in-car cameras because they will turn on and off automatically. However, it might take months before all 45 vehicles in the Vallejo police fleet are equipped with them.
"I'm hoping it's something that we can do in the next few months. But there's the issue of money and technology," Kreins said. They cost about $10,000 per car for cameras and officer training.
Keen said the body cameras could probably be bought without triggering "meet and confer" meetings with the police union.
Davis also asked Keen to:
• Conduct research to select and implement a decision-based firearms/use of force training simulator system.
• Reaffirm the Police Department's protocol and procedure for communicating with the public and families in incidents concerning loss of life or great bodily injury as a result of violence. The Romero family, in particular, has angrily contended that police have not communicated properly with them, or in a timely way about the shooting.
The Romero family, in particular, has angrily contended that police have not communicated with them about the shooting. Martin said Thursday that her brother's body is still at the coroner's office, that police still have not contacted family members, and that no funeral arrangements have been made.


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tag:blogger.com,1999:blog-7210858986261575108.post7477820299860728662..comments2012-11-24T00:36:06.677-06:00Comments on Veronica Roth: DIVERGENT Cover and SummaryVeronica Rothhttp://firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-60532090424641138732012-11-10T14:20:32.624-06:002012-11-10T14:20:32.624-06:00Read it in class for a Literature circle and then ...Read it in class for a Literature circle and then I couldn't put it down... I Loved it!!! Thank you for your hard work and i can't wait to read email@example.com:blogger.com,1999:blog-7210858986261575108.post-87927430522791121602012-11-08T16:51:35.070-06:002012-11-08T16:51:35.070-06:00I fell in love with this book on page 1! Great job...I fell in love with this book on page 1! Great job Veronica and keep up the great firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-42327202621298578202012-11-06T22:24:33.567-06:002012-11-06T22:24:33.567-06:00Awesome book! LOVED IT! I have also read Insurgent...Awesome book! LOVED IT! I have also read Insurgent, which I might add was equally as awesome. Great email@example.com:blogger.com,1999:blog-7210858986261575108.post-58356191543010589132012-10-17T17:10:23.770-05:002012-10-17T17:10:23.770-05:00 This was an AMAZING book and I wouold recremend i... This was an AMAZING book and I wouold recremend it to ANYONE!!!!!!!April Millerhttps://firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-54378198537486569452012-10-03T16:07:09.575-05:002012-10-03T16:07:09.575-05:00CONGRATULATIONS!You have a reader from England! I...CONGRATULATIONS!You have a reader from England!

I've just finished reading Insurgent and I can't wait for the next book! When I heard rumours about the movie coming out in 2014 I was like: NO IT HAS TO BE SOONER ! xD

I keep reading the books, too, even though I've already read them!

-LoreleiLoreleihttps://email@example.com:blogger.com,1999:blog-7210858986261575108.post-35306894602589122352012-10-01T18:58:55.514-05:002012-10-01T18:58:55.514-05:00I loved this book and I need the second oneI loved this book and I need the second email@example.com:blogger.com,1999:blog-7210858986261575108.post-10673620907039075652012-09-17T19:01:13.727-05:002012-09-17T19:01:13.727-05:00Loved the book!!!!! So exciting and romancy! !!!Loved the book!!!!! So exciting and romancy! !!!firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-46678450752844985282012-09-10T18:54:19.500-05:002012-09-10T18:54:19.500-05:00Roth, I really enjoyed your book. It was an amazin...Roth, I really enjoyed your book. It was an amazing adventure, and it had an amazing cast of characters. Tris, especially was a particular favorite not of characters of this book series, but a great many. I have never read about a character with so similar a personality as myself, and for this small fact alone this book has become a fast favorite. I'm currently waiting to get my hands on the next books in the series once I have the available funds and leisure time.
I understand that I may be simply seeing so much of a connect because this was written in the first person, and that is one reason I generally avoid first person books, because I never wanted to be "that girl", well, "that girl who identifies with everything written in the first person", and the fact that First person books in general are just not my cup of tea.
I was in the store when I saw Divergent on the shelf, and I picked it up because the cover caught my eye. I realized it was first person, but I figured I would give it a try sense I had just finished The Hunger Games, and it was written First Person, and I rather enjoyed it, so I figured, why not? give it a go.
I am unbelievably happy to have picked up this novel. It's amazing, and I found I could relate to the characters with ease.

It is wonderfully written, it has a very enticing plot, and the characters have very intriguing personalities and reactions.

I'm proud to have read it, and since I finished it, I have been skimming through the internet everyday to learn about when this will be made in to a movie and when and where the auditions will take place. I would be thrilled to have any part in this film once it begins to be underway, if there is anyway I possibly can.

Honestly, this is one of the first books I've ever felt such a drive to be potentially involved in when it became a movie. I love all the characters in this book, I love their thoughts, their reasoning, their reactions, and their actions. I hope I will be able to be involved in this film in someway, anyway, just because it's one of those rare books that "hit home" with me.

It could just be because of how pleased I am to have a main character with such similar reasoning and reactions as myself, it could be because it's an amazing novel, or it could just be because I feel this pull towards it.

Though, I will not say me and Tris perfectly identify, I think that would be nearly impossible, and feel I should clarify that, I find the main difference, is that of all the Factions, the 3 I identify most with are Candor, Dauntless, and Erudite. But I think differences between myself and Tris would only be natural considering we were raised very differently, but I would say above anything else, If I were to put my family into a faction, we would fall Heavily under Candor, Honesty is very important to us, but second to that is Being Informed (Erudite). I relate to As far as Dauntless goes, I have a great dislike for "leaning on " others, simply because they wont always be there, it's a fact. and because I think it is important to stand up for yourself and others.

In fact, the main thing I loved about this book, was how all of the Factions could be inter connected.
It takes Bravery to stand up for someone, to take care of someone, to be honest with someone, to be kind to a complete stranger, and to be curious.

well, I think I have rambled enough, I hope I can have some large, small or any part in this film, and I wish the best for it even if I am unable to be involved, at least I tried. I can't wait to finish the series! Thank you so much for writing. You are an amazing author of an amazing novel, and I admire you for it. May all be well!
~ MelanieMiyanahttps://email@example.com:blogger.com,1999:blog-7210858986261575108.post-75822597922168562872012-09-06T20:09:14.891-05:002012-09-06T20:09:14.891-05:00OMG I LOVE THESE BOOKS!!! I READ THEM IN DAYS!!! ...OMG I LOVE THESE BOOKS!!! I READ THEM IN DAYS!!! im trying out for Tris in the movie :D :D <3pinkeypie213https://email@example.com:blogger.com,1999:blog-7210858986261575108.post-13986766791278092582012-09-05T19:51:56.682-05:002012-09-05T19:51:56.682-05:00I first picked up divergent for the cover, once I ...I first picked up divergent for the cover, once I began reading I couldn't stop and it only took me 4 days to read the 2. Can't wait for the email@example.com:blogger.com,1999:blog-7210858986261575108.post-90180096495527018632012-07-29T13:47:53.658-05:002012-07-29T13:47:53.658-05:00thank you for you :0thank you for you :0nalgenehttps://firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-53049445636540481562012-06-21T20:28:59.907-05:002012-06-21T20:28:59.907-05:00I read this book. Very good!!!I read this book.
Very good!!!Jessica Klinghttps://firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-26644428662955945352012-06-21T19:42:25.079-05:002012-06-21T19:42:25.079-05:00I saw this after I read the book and I have to say...I saw this after I read the book and I have to say i love it! I am ten years old (soon to be 11), and that just shows that everyone can read it and enjoy it! :)email@example.com:blogger.com,1999:blog-7210858986261575108.post-31455089911388367572012-06-17T20:52:02.221-05:002012-06-17T20:52:02.221-05:00We have to read this book as Summer assignment and...We have to read this book as Summer assignment and I love this book.I want to read this one again and again.Nawid email@example.com:blogger.com,1999:blog-7210858986261575108.post-82623802422718838322012-06-14T02:51:31.452-05:002012-06-14T02:51:31.452-05:00Hello, my name is Simona and I am from Roumania. I...Hello, my name is Simona and I am from Roumania. It came to my knowledge in an interview, that you stayed a while in Cluj-Napoca in the fall of 2011. I have no words to describe the book. Is amaizing!Absolutely amaizing!
When I read the book you have climbed the charts of my favorite authors.
You have done an amaizing deed with this story. Let me introduce you my humble congratulations.
And if you want to give me some advise about writing a book...I have 15 years old, and I want to write a book, but some advice from you would be more than a dream:). If you still agree, let me know and I will give you my mail.

A lot of hugs from youre fan no. 1 in Romania>:D<>:D<>:D<, Simona.weareholdingangelshttp://email@example.com:blogger.com,1999:blog-7210858986261575108.post-76363081031870739862012-05-12T23:44:05.298-05:002012-05-12T23:44:05.298-05:00willysbookreviews.blogspot.com I feel the urge to...willysbookreviews.blogspot.com

I feel the urge to type in caps, but I will resist.

This book was one of the best books I have ever read. The only reason it is one of them is because I have not finished the sequel "Insurgent" and the third has not come out yet.

This book was-by far-better than the hunger games. It is also a dystopia. And the main character is awesome. She is a total bad ass. I love the way she develops as a person throughout her experience in her faction. Also resisting giving off spoilers.

The entire story hooks you in. It makes you want to be as awesome as she is. I want to go read Insurgent right now, but I am writing some reviews.

You will not want to stop reading it. And I am so glad I waited to read Divergent until the release of Insurgent because I would have died. Sadly, I will die when I finish Insurgent because I will have to wait for the third for about a year and a half at the least.

POTENTIAL SPOILER:

The whole star crossed lovers was amazing, and greatly portrayed. I was in shock when I read over the scene where she and Four kissed.
And them doing so while doing completely dangerous, adrenaline pumping, dauntless acts is remarkable.

In comparison to Tris, MY LIFE SUCKS!

Please visit my book blog,
willysbookreviews.blogspot.comWillyhttps://firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-39114437302389813332012-04-06T15:20:48.541-05:002012-04-06T15:20:48.541-05:00I havent been able to get my hands on the book >...I havent been able to get my hands on the book >:(email@example.com:blogger.com,1999:blog-7210858986261575108.post-91765137742163840682012-03-09T20:22:10.281-06:002012-03-09T20:22:10.281-06:00I loved the book! It was one of the best books I h...I loved the book! It was one of the best books I have ever read. I couldn't put it down! The imagery was amazing, and it made me feel like I was in the book! I love exciting stories like this, and your stories inspire me every day to be a great author just like you! Again, the book was AMAZING, and I loved it!Brynnan Holstinehttps://firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-83394694603668803822012-02-12T09:48:14.969-06:002012-02-12T09:48:14.969-06:00Omg just finished reading the book! It is amazing!...Omg just finished reading the book! It is amazing! The cover really says a lot. I have been reading non stop for two days and i was truly sad after reading the last page, not because it was sad but because it was the last page! I can't wait until Insurgent releases, by the way that cover looks even better! I really fell in love with the characters, except forPeter who I well and truly hate now! Hahaha I never thought I could connect with a Character so much, it was like I was right there with firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-24971521315055671712012-01-19T09:01:39.949-06:002012-01-19T09:01:39.949-06:00I love the cover! I haven't started to read it...I love the cover! I haven't started to read it, but I firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-80161148952464748942011-11-03T22:33:28.213-05:002011-11-03T22:33:28.213-05:00Divergent is an amazing book!! I have recently sta...Divergent is an amazing book!! I have recently started a new fan site devoted to all Divergent. Visit us at www.factionbeforeblood.com we are working hard in getting it up and runing, we will also be bringing a forum and lots more!!mandylorhttps://email@example.com:blogger.com,1999:blog-7210858986261575108.post-81375886843627969752011-11-01T08:30:21.114-05:002011-11-01T08:30:21.114-05:00I just wanted to let you know your book was one of...I just wanted to let you know your book was one of the best I've ever read, and I can't wait for the sequel!
Don't ever stop writing, friend,
Sam.Caddarenhttps://firstname.lastname@example.com:blogger.com,1999:blog-7210858986261575108.post-91192610622613609552011-10-22T16:18:55.416-05:002011-10-22T16:18:55.416-05:00I love divergent!! cant wait fo the next one!!I love divergent!! cant wait fo the next email@example.com:blogger.com,1999:blog-7210858986261575108.post-72973796974376833712011-10-14T15:39:08.739-05:002011-10-14T15:39:08.739-05:00MY FRIEND CHARLOTTE TOLD ME ABOUT THIS BOOK , AT T...MY FRIEND CHARLOTTE TOLD ME ABOUT THIS BOOK , AT THE TIME I DIDNT THINK I WOULD BE INTERESTED IN IT BUT WHEN I READ IT ............. OMG IT WAS THE BEST BOOK I EVER READ THE DANGER , THE EPICNESS, AND THE ROMANCE ITS THE PERFECT COMBO!!!!! .......... BTW I HAVE DECIDED TO MARRY TOBIAS<3 .......... PLEASE KEEP ON WRITING BECAUSE U R REALLY GOOD AT IT !!!!!!!!!!!!email@example.com:blogger.com,1999:blog-7210858986261575108.post-7193799588604004002011-06-29T18:29:02.555-05:002011-06-29T18:29:02.555-05:00"SO FRICKIN EPIC" does not begin to desc..."SO FRICKIN EPIC" does not begin to describe this book. Completely amazing. I have a small library on the upstairs landing, and this is the only book I have EVER read cover to cover 15 times consecutively. I love this book. 5000% AMAZING. I am eagerly awaiting the sequel and tri-quel [what time do they come out in Australia?]. Totally worth every email@example.com


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The Croatian scientific journal for different topics from Earth sciences, different engineering and similar fields. It is entirely open access (green model, CC-BY, http://www.sherpa.ac.uk/romeo/pub/2374/), without any hidden author's fees. Please, use OJS / log in / new submission for sending manuscript into journal. Published by the Faculty of Mining, Geology and Petroleum Engineering (https://www.rgn.unizg.hr/en/) as part of the University of Zagreb (http://www.unizg.hr/homepage/).
|Important: all authors are completely responsible for maintain metadata of their paper in OJS! The published PDF/print need to be the same as abstract/title/author list in OJS. The editors do not check this before publishing!
|Posted: 2017-07-06
No. 37 is published online
|No. 37 is published online as current issue (with online publication date 21th June). Print will be available about 10th July.
|Posted: 2017-06-21
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text:
Swing Dress - Simply Be, Leggings - Newlook Inspire, Socks - Primark, Brogues - Clarks, Clutch - Primark.
Now overly patterned things usually wouldn't be my first choice however this is glorious. The black and white monochrome mixed with a 60's inspired geo print makes me so happy! Anything 60's inspired and I am all over! The best way I can describe this material is 'mole skin'. Now, of course, it isn't mole skin, but it's soft, supple and slightly furry which in turn makes it ridiculously comfortable and allows the dress to fall beautifully. It also has two front pockets which, although a tad unnecessary for me personally as I'm not a pocket girl, they do add a cute twist to the standard swing dress. I felt glam but also comfortable and retro in this gorgeous dress so I cannot recommend it enough to ya'll. The sizing is also wonderful, no sizing up required, totally true to size!
Now let's get on to these brogues. THE BROGUES!!! Honestly? I am a sucker for a cute, CHEAP pair of shoes that will ultimately destroy my feet or last no longer than a few weeks. I had been telling myself for months I needed to invest in a GOOD pair of shoes so when I saw these, it was love at first sight. They are Clarks, so you know the quality would never questionable! But they are also totally stylish, on trend, comfortable and supportive. They have a tiny heel (no more than half an inch) which adds a feminine touch and the fit is impeccable. I have pretty giant feet, I'm a size 8 and sometimes I wear wide fit, but sometimes not - it all depends on the shoe. These are standard fit and my trotters were in heaven! They are a little pricey, but seriously worth every penny! Invest in a statement brogue and you are good to go! (The socks are also adorable, £1 from Primark! You have to be super careful putting them on as the main sock is a very thin tight-esque material but they are just so cute).
I also want ya'll to take a minute to appreciate this pop art inspired clutch. £6!! Six pounds!! Look at it! It's fun, ridiculous and a little scary. When I bought it, I thought, I don't care if I never use it, I just love it and have to have it however since buying it, I have used it more than any other clutch I own. So thanks Primark for this absolute beast.
I went with a little 60's inspired face to go with the dress - adding lash flicks under my bottom eyelashes in the style of the ever iconic Twiggy. I also busted out my classic, go to MAC lipstick of choice - Lady Danger. It's verging on an orange red but I think the final colour of it really depends on your own colouring and because I'm whiter than white, it swings more to the red end of the scale on me.
And finally, here is a little treat for ya'll. my new man friend. He is a handsome devil, dresses better than I do and he buys me flowers - for now that's all you're getting hehe.
It's so nice to be back in the blogging saddle! I won't be leaving it anywhere near as long this time!
Lots Of Love,
KLJ x


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Pizza Hut is the leading pizza restaurant with 111 shops and over 5000 staff in Hong Kong and Macau. Our key mission is to ensure every pizza centred food service occasion is an enjoyable experience to our customers. To support the mission and expansion in other countries, we are now inviting high calibre, passionate professional who strives for executional excellence to join our Team.
Training Officer
|Job level
|Work exp
|Minimum 2 Years
|Bachelor Degree
|Hong Kong
|Employment type
|Full Time
|5-day week, Competitive pay, Discretionary bonus, Education subsidies, General holidays, MPF, Maternity leave, Medical plan, Paternity leave, Social & recreation facility, Special / extra leave, Staff care centre, Statutory holidays
|Catering / Food & Beverage
|Job function
Human Resources > Generalist HR
Human Resources > Training & Development
|Published On
The incumbent will be responsible for designing, organizing and delivering training programs in the areas of customer service, supervisory training and soft skills for Operations staff; coordinating customer service related training events such as Mystery Shopper Program and HKRMA Service & Courtesy Award; providing assistance in ad hoc projects related to shop operations, training and people development and working closely with other internal and external organizations for various training or customer service related activities.
- Degree in Human Resources, Business or related discipline
- Minimum 2 years hands-on experience in designing and delivering training programs, preferably in catering or retail fields
- Excellent presentation, communication and interpersonal skills
- Strong sense of ownership, responsible and self-initiated
- Good command of both written and spoken English and Chinese
- Proficient in PC applications especially MS Word, Excel, Powerpoint and Chinese Word processing
cpjobs.com - Training Officer - is a part of Human Resources, Catering / Food & Beverage, in Hong Kong, placed by PIZZA HUT HONG KONG MANAGEMENT LIMITED