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Molecular Bases of Disease| Volume 291, ISSUE 36, P18753-18764, September 02, 2016
Gingipains of Porphyromonas gingivalis Affect the Stability and Function of Serine Protease Inhibitor of Kazal-type 6 (SPINK6), a Tissue Inhibitor of Human Kallikreins*
Karolina Plaza
Departments of Microbiology
Magdalena Kalinska
Oliwia Bochenska
Departments of Analytical Biochemistry, Faculty of Biochemistry, Biophysics, and Biotechnology
Ulf Meyer-Hoffert
Department of Dermatology, University Clinic Schleswig-Holstein, 24105 Kiel, Germany
Zhihong Wu
Katherine Falkowski
Laura Sasiadek
Ewa Bielecka
Barbara Potempa
Department of Oral Immunology and Infectious Diseases, School of Dentistry, University of Louisville, Louisville, Kentucky 40202
Andrzej Kozik
Jan Potempa
Supported by National Institutes of Health Grants DE 09761 and DE 022597 and European Commission Grant FP7-HEALTH-F3-2012-306029"TRIGGER"). To whom correspondence should be addressed: Tel.: 48-12-6646343; Fax: 48-12-6646902
Tomasz Kantyka
Malopolska Centre of Biotechnology, Jagiellonian University, 30-387 Krakow, Poland
* This project was supported by Polish National Centre of Science Research Grants OPUS 2012/05/B/NZ6/00581 (to T. K.), Preludium UMO-2012/07/N/NZ1/00006 (to K. P.), and SYMFONIA UMO-2013/08/W/NZ1/00696 (to J. P.); Polish Ministry of Science and Higher Education Iuventus+ IP2011 022171 (to T. K.) and 2975/7.PR/13/2014/2 (to J. P.); German Research Foundation Grant ME2037/3-3 (to U. M. H.); and Young Scientist Grant for Faculty of Biochemistry, Biophysics, and Biotechnology K/DSC/003271 (to M. K.). The Faculty of Biochemistry, Biophysics, and Biotechnology of the Jagiellonian University in Krakow is a beneficiary of structural funds from the European Union (Grant POIG.02.01.00-12-064/08, "Molecular Biotechnology for Health") and a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education, Poland. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
4 K. Plaza, M. Kalinska, O. Bochenska, U. Meyer-Hoffert, Z. Wu, J. Fischer, K. Falkowski, L. Sasiadek, E. Bielecka, B. Potempa, A, Kozik, J. Potempa, and T. Kantyka, unpublished results.
1 Both authors contributed equally to this work.
Open AccessPublished:June 27, 2016DOI:https://doi.org/10.1074/jbc.M116.722942
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Periodontitis, a chronic inflammation driven by dysbiotic subgingival bacterial flora, is linked on clinical levels to the development of a number of systemic diseases and to the development of oral and gastric tract tumors. A key pathogen, Porphyromonas gingivalis, secretes gingipains, cysteine proteases implicated as the main factors in the development of periodontitis. Here we hypothesize that gingipains may be linked to systemic pathologies through the deregulation of kallikrein-like proteinase (KLK) family members. KLKs are implicated in cancer development and are clinically utilized as tumor progression markers. In tissues, KLK activity is strictly controlled by a limited number of tissue-specific inhibitors, including SPINK6, an inhibitor of these proteases in skin and oral epithelium. Here we identify gingipains as the only P. gingivalis proteases responsible for SPINK6 degradation. We further show that gingipains, even at low nanomolar concentrations, cleaved SPINK6 in concentration- and time-dependent manner. The proteolysis was accompanied by loss of inhibition against KLK13. We also mapped the cleavage by Arg-specific gingipains to the reactive site loop of the SPINK6 inhibitor. Moreover, we identified a significant fraction of SPINK6-sensitive proteases in healthy saliva and confirmed the ability of gingipains to inactivate SPINK6 under ex vivo conditions. Finally, we demonstrate the double-edge action of gingipains, which, in addition, can activate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Altogether, the results indicate the potential of P. gingivalis to disrupt the control system of KLKs, providing a possible mechanistic link between periodontal disease and tumor development.
bacterial pathogenesis
enzyme inhibitor
kallikrein
protein degradation
SPINK6
gingipain
The family of kallikrein proteases (KLKs)
The abbreviations used are:
kallikrein-like proteinase
serine protease inhibitor of Kazal-type
7-amino-4-methylcoumarin
Tricine
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
contains 15 enzymes that are expressed in many different tissues, suggesting a range of physiological and pathophysiological functions. This diversity of functions is exemplified by the involvement of kallikreins in such processes as normal skin desquamation, psoriatic lesion formation, tooth development, neural plasticity, cancer, and Alzheimer disease (for a review, see Ref.
Kalinska M.
Meyer-Hoffert U.
Kantyka T.
Potempa J.
Kallikreins: the melting pot of activity and function.
Biochimie. 2016; 122: 270-282
). Moreover, kallikreins function in wound healing and proteinase-activated receptor signaling, they process antimicrobial peptides in the epithelial areas, and they affect the stability of some growth factors (
Gao L.
Chao L.
Chao J.
A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: activation of proteinase-activated receptor 1 and epidermal growth factor receptor.
Exp. Cell Res. 2010; 316: 376-389
Yamasaki K.
Schauber J.
Coda A.
Lin H.
Dorschner R.A.
Schechter N.M.
Bonnart C.
Descargues P.
Hovnanian A.
Gallo R.L.
Kallikrein-mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin.
FASEB J. 2006; 20: 2068-2080
KLKs are secreted as inactive proenzymes (pro-KLKs). With the exception of pro-KLK4, all zymogen forms of pro-KLK are then activated in the extracellular milieu by trypsin-like cleavage of the pro-peptide after either an Arg or Lys residue. The activation cleavage is exerted by the kallikrein itself or by still unknown endogenous proteases and constitutes a key mechanism regulating KLK activity in tissues (
Yousef G.M.
Diamandis E.P.
Expanded human tissue kallikrein family: a novel panel of cancer biomarkers.
Tumour Biol. 2002; 23: 185-192
Kallikrein-specific inhibitors, including some members of the serine protease inhibitor of Kazal-type (SPINK) family, control the activity of KLKs released into tissues (
Mägert H.J.
Ständker L.
Kreutzmann P.
Zucht H.D.
Reinecke M.
Sommerhoff C.P.
Fritz H.
Forssmann W.G.
LEKTI, a novel 15-domain type of human serine proteinase inhibitor.
). To date, at least 10 genes encoding SPINKs were identified in the human genome, and of them, SPINK5, 6, and 9 were reported to control the activity of different KLKs in different tissues. SPINK5 is detected ubiquitously in epithelial tissues and is composed of 15 protease Kazal-type inhibitory domains that inhibit KLK5 and KLK7. It is well known that mutations in the SPINK5 gene result in elevated activity of epidermal kallikreins and the development of Netherton syndrome, a severe skin disease manifested through abnormal skin desquamation, keratinization, hair follicle defects, and loss of skin barrier function (
Walley A.J.
Chavanas S.
Moffatt M.F.
Esnouf R.M.
Ubhi B.
Lawrence R.
Wong K.
Abecasis G.R.
Jones E.Y.
Harper J.I.
Cookson W.O.
Gene polymorphism in Netherton and common atopic disease.
). The expression of other SPINK family inhibitors is more tissue-specific. SPINK9 is exclusively found in the palms and soles of the feet (
Brattsand M.
Stefansson K.
Hubiche T.
Nilsson S.K.
Egelrud T.
SPINK9: a selective, skin-specific Kazal-type serine protease inhibitor.
J. Invest. Dermatol. 2009; 129: 1656-1665
Wu Z.
Fischer J.
Latendorf T.
Hansmann B.
Bartels J.
He Y.
Gläser R.
Schröder J.M.
Isolation of SPINK6 in human skin: selective inhibitor of kallikrein-related peptidases.
), and it seems to exclusively inhibit KLK5. In contrast, strongly conserved SPINK6 has a broad spectrum of action that controls kallikrein activity (KLK4, KLK5, KLK12, KLK13, and KLK14) not only in the skin (
Sperrhacke M.
Dimitrieva O.
Koblyakova Y.
Ahrens K.
Graumann N.
Baurecht H.
Reiss K.
Proksch E.
Characterization of Spink6 in mouse skin: the conserved inhibitor of kallikrein-related peptidases is reduced by barrier injury.
) but also in other tissues. This includes the salivary glands, where SPINK6 inhibits KLK5, 6, and 7, and the main salivary kallikrein, KLK13, which comprises 50% of the overall kallikrein activity in the glands (
Declercq W.
Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6.
Peptides. 2011; 32: 1187-1192
). Because the reported activity of KLK13 includes degradation of extracellular matrix proteins (
Kapadia C.
Ghosh M.C.
Grass L.
Human kallikrein 13 involvement in extracellular matrix degradation.
Biochem. Biophys. Res. Commun. 2004; 323: 1084-1090
), it is of no surprise that SPINK6 is cross-linked to fibronectin. This increases the local concentration of the inhibitor and protects the extracellular matrix from KLK-mediated damage (
Cross-linking of SPINK6 by transglutaminases protects from epidermal proteases.
An imbalance between SPINK proteins and proteases may cause severe diseases such as pancreatitis, celiac disease, Netherton syndrome, skin barrier defects, and cancer (
Witt H.
Luck W.
Hennies H.C.
Classen M.
Kage A.
Lass U.
Landt O.
Becker M.
Mutations in the gene encoding the serine protease inhibitor, Kazal type 1 are associated with chronic pancreatitis.
Gasiorowska A.
Talar-Wojnarowska R.
Czupryniak L.
Smolarz B.
Romanowicz-Makowska H.
Kulig A.
Malecka-Panas E.
The prevalence of cationic trypsinogen (PRSS1) and serine protease inhibitor, Kazal type 1 (SPINK1) gene mutations in Polish patients with alcoholic and idiopathic chronic pancreatitis.
Dig. Dis. Sci. 2011; 56: 894-901
Hoefnagel J.J.
Dijkman R.
Basso K.
Jansen P.M.
Willemze R.
Tensen C.P.
Vermeer M.H.
Distinct types of primary cutaneous large B-cell lymphoma identified by gene expression profiling.
Blood. 2005; 105: 3671-3678
Stenman U.H.
Role of the tumor-associated trypsin inhibitor SPINK1 in cancer development.
Asian J. Androl. 2011; 13: 628-629
Tüysüz B.
Ojalvo D.
Mat C.
Zambruno G.
Covaciu C.
Castiglia D.
D'Alessio M.
A new SPINK5 donor splice site mutation in siblings with Netherton syndrome.
Acta Dermato-Venereologica. 2010; 90: 95-96
Rockett J.C.
Patrizio P.
Schmid J.E.
Hecht N.B.
Dix D.J.
Gene expression patterns associated with infertility in humans and rodent models.
Mutat. Res. 2004; 549: 225-240
). Similarly, illegitimate, excessive activation of pro-KLKs by bacterial proteases accompanied by degradation/inactivation of SPINKs may result in damage of an infected connective tissue. This is a likely scenario to occur during progression of periodontitis.
Evidence accumulated to date has strongly implicated the contribution of proteolytic enzymes of subgingival plaque bacteria to the pathogenicity of periodontal disease, the most widespread chronic inflammatory condition in the human host. Importantly, in recent years, periodontal disease has been identified as a significant factor in the development of other systemic diseases, including rheumatoid arthritis, cardiovascular disease, aspiration pneumonia, preterm births, and low birth body weight in infants (
Olsen I.
Strategies for the inhibition of gingipains for the potential treatment of periodontitis and associated systemic diseases.
J. Oral Microbiol. 2014; 6: 24800
The key pathogen implicated in the development of chronic periodontitis is an anaerobic bacterium, Porphyromonas gingivalis. P. gingivalis secretes three related cysteine proteases, referred to as gingipains, that constitute its main virulence factors. Two gingipains are specific for Arg-Xaa peptide bonds (HRgpA and RgpB), whereas Kgp cleaves after a Lys residue (
Imamura T.
The role of gingipains in the pathogenesis of periodontal disease.
J. Periodontol. 2003; 74: 111-118
). Collectively, gingipains digest a broad spectrum of host proteins to provide the pathogen with nutrients for growth (
Sroka A.
Travis J.
Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis: structure, function and assembly of multidomain protein complexes.
Curr. Protein Pept. Sci. 2003; 4: 397-407
). More importantly, gingipains are involved in the disruption of host defense inflammatory reactions and hinder P. gingivalis clearance by the immune system (
Uehara A.
Naito M.
Nakayama K.
Takada H.
Dual regulation of interleukin-8 production in human oral epithelial cells upon stimulation with gingipains from Porphyromonas gingivalis.
J. Med. Microbiol. 2008; 57: 500-507
Nguyen K.A.
Dichotomy of gingipains action as virulence factors: from cleaving substrates with the precision of a surgeon's knife to a meat chopper-like brutal degradation of proteins.
Periodontology 2000. 2010; 54: 15-44
). This is accomplished by hijacking proinflammatory signaling pathways via cleavage and activation of the proteinase-activated receptor 2 (PAR-2) on human neutrophils (
Ford P.J.
Gamonal J.
Seymour G.J.
Immunological differences and similarities between chronic periodontitis and aggressive periodontitis.
Periodontology 2000. 2010; 53: 111-123
). Gingipains also affect many host proteolytic systems, affecting the homeostasis of the organism via complex interactions with host proteins, as exemplified by the activation of the kallikrein/kinin pathway and the resultant increase in vascular permeability (
Pike R.N.
Moore J.N.
Barton M.H.
Effect of free and vesicle-bound cysteine proteinases of Porphyromonas gingivalis on plasma clot formation: implications for bleeding tendency at periodontitis sites.
Infect. Immun. 1995; 63: 4877-4882
Dependence of vascular permeability enhancement on cysteine proteinases in vesicles of Porphyromonas gingivalis.
Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikrein/kinin pathway.
J. Clin. Invest. 1994; 94: 361-367
). However, activation of tissue prokallikreins, especially locally abundant KLK13, with simultaneous degradation of this protease endogenous inhibitor have not been investigated. This is of significant interest because recent discoveries implicate that infection with oral pathogens may promote tumor progression via facilitation of the epithelial-to-mesenchymal transition and an increase in the malignancy of the tumor (
Ha N.H.
Woo B.H.
Kim da J.
Ha E.S.
Choi J.I.
Kim S.J.
Park B.S.
Lee J.H.
Park H.R.
Prolonged and repetitive exposure to Porphyromonas gingivalis increases aggressiveness of oral cancer cells by promoting acquisition of cancer stem cell properties.
Tumour Biol. 2015; 36: 9947-9960
). Therefore, analysis of the interaction network of pathogen proteases and tumor-related proteinases of the KLK family may provide valuable insights into the mechanistic background of oral infection-cancer correlation.
Because SPINK6 is the main inhibitor of salivary kallikreins, especially KLK13, the inactivation of this molecule was our main focus in the context of P. gingivalis proteinase-mediated disruption of the KLK-inhibitor balance. Here we present, to our knowledge, the first report of the interplay of the tissue kallikrein system with P. gingivalis proteases, further adding to our understanding of the mechanisms of tissue disruption by this widespread oral pathogen.
SPINK6 Is Degraded by P. gingivalis Cultures
P. gingivalis produces many proteolytic enzymes, and, in addition to gingipains, it secretes several other proteases that may interact with SPINK6. Therefore, to investigate the ability of gingipains and other proteases to degrade SPINK6, the inhibitor was preincubated with wild-type P. gingivalis W83 in the presence or absence of gingipain-specific inhibitors or with the triple gingipain knockout mutant W83ΔkgpΔrgpAΔrgpB, completely devoid of gingipain activity (Fig. 1). The wild-type W83 strain completely degraded SPINK6 over the course of the experiment, as indicated by the absence of SPINK6 immunoreactivity on the Western blot of the samples. Preincubation of the W83 strain with Kyt inhibitors that are gingipain-specific, reversible, and do not affect the activity of other proteases, at least at the concentration used (
Kadowaki T.
Baba A.
Abe N.
Takii R.
Hashimoto M.
Tsukuba T.
Okazaki S.
Suda Y.
Asao T.
Yamamoto K.
Suppression of pathogenicity of Porphyromonas gingivalis by newly developed gingipain inhibitors.
Mol. Pharmacol. 2004; 66: 1599-1606
), protected the majority of SPINK6 from degradation (Fig. 1). Some cleavage of SPINK6 was observed, apparently due to residual activity of the gingipains in the presence of Kyt inhibitors, which were used at low micromolar concentration (5 μm). Exclusive gingipain involvement in degradation was confirmed by the finding that SPINK6 incubation with the triple gingipain mutant W83ΔkgpΔrgpAΔrgpB did not affect the stability of the inhibitor. Moreover, when SPINK6 was incubated with another cysteine proteinase of P. gingivalis, the streptopain-like TPR, no degradation was observed, even in the presence of a 100 nm concentration of the protease (data not shown). Taken together, these results indicate that wild-type bacteria efficiently degrade SPINK6 and that three gingipains (Kgp, RgpB, and HRgpA) are the enzymes responsible for this activity.
FIGURE 1SPINK6 is effectively degraded by P. gingivalis bacterial cultures. SPINK6 (200 ng) was incubated with 106 cells of P. gingivalis strains for 1 h at 37 °C. The samples were processed for Western blotting using an anti-SPINK6 antibody. Samples of untreated SPINK6, SPINK6 treated with wild-type (W38) bacteria in the presence or absence of inhibitors, and SPINK6 treated with the gingipain triple mutant bacteria (W83ΔkgpΔrgpAΔrgpB) are shown.
Gingipains Degrade SPINK6 in a Time- and Concentration-dependent Manner
P. gingivalis gingipains degraded SPINK6 in a concentration-dependent (Fig. 2, A–F) and time-dependent (Fig. 2, G–L) dependent manner. The Lys-specific gingipain Kgp (20 nm) degraded SPINK6 nearly completely, as visualized by silver-staining SDS-PAGE gels. By contrast, SPINK6 appeared intact on non-reducing gels following incubation with Arg-specific gingipains, even at relatively high enzyme concentrations (10 nm RgpB and 50 nm HRgpA). However, SDS-PAGE under reducing conditions revealed the accumulation of the lower molecular weight proteolysis product during incubation with 2 nm RgpB and 10 nm HRgpA.
FIGURE 2Gingipains degrade SPINK6 in a concentration- and time-dependent manner. A–F, SPINK6 (200 ng) was incubated with the indicated concentration of respective gingipain for 1 h at 37 °C. The reactions were stopped, and the samples were resolved using SDS-PAGE. The gels were silver-stained. All SDS-PAGE separations were performed under reducing and non-reducing conditions. G–L, SPINK6 was incubated with 50 nm Kgp, 2 nm RgpB, or 20 nm HRgpA for the indicated times. SDS-PAGE followed by silver staining was used to analyze the degradation.
Similarly, the time course analysis showed progressive degradation by Kgp (50 nm) with no significant differences between reducing and non-reducing conditions. After 45 min, only traces of the native inhibitor could be detected by SDS-PAGE. Incubation with the Arg-specific gingipains led to the accumulation of the nicked inhibitor, represented by two polypeptide chains resolved only by reducing SDS-PAGE. For both RgpB (2 nm) and HRgpA (20 nm), a 30-min incubation was long enough for almost complete cleavage of the inhibitor. Cumulatively, these results are congruent with the gingipain specificities and the SPINK6 structure, which contains six Lys residues scattered throughout the polypeptide chain and only a single Arg residue at the P1 position in the reactive site loop of the inhibitor stabilized by three disulfide bridges.
Gingipain-treated SPINK6 Is Inactive as a KLK13 Inhibitor
The reactive site loop of SPINK6 is stabilized by three disulfide bridges that hold in close proximity the P1 Arg and P1′ Glu residues. Because Kazal inhibitors nicked at the P1-P1′ bond may retain inhibitory activity (
Rawlings N.D.
Tolle D.P.
Barrett A.J.
Evolutionary families of peptidase inhibitors.
), we tested the ability of gingipain-treated SPINK6 to inhibit KLK13 (Fig. 3). The results indicated that the apparently stable, Rgps-nicked SPINK6 displayed no inhibitory activity when incubated in a 2:1 molar ratio with KLK13, in contrast to the intact inhibitor, which diminished KLK13 activity by ∼90%.
FIGURE 3Gingipain degradation leads to loss of the inhibitory properties of SPINK6. SPINK6 was incubated for 1 h at 37 °C with each of the indicated gingipains (20 nm) or with a mixture of all three gingipains (4 nm each). The reactions were stopped by addition of 5 μm Kyt-1 and/or Kyt-36, and KLK13 was added to the samples. KLK13 activity was measured using the fluorogenic substrate Boc-VPR-AMC. Results were normalized to the uninhibited KLK13 as 100% and compared with SPINK6 incubated alone. Statistical significance: **, p < 0.05. RFU, relative fluorescence unit.
Limited Proteolysis of SPINK6 by Gingipains
To further characterize SPINK6 interactions with gingipains, the cleavage sites within the inhibitor were identified. As suggested by SDS-PAGE analysis, reverse-phase HPLC chromatography of Kgp-treated SPINK6 (Fig. 4A) confirmed the complete fragmentation of the inhibitor. Six discrete fragments of the SPINK6 polypeptide chain generated by Kgp were identified, resulting from the four Lys-Xaa peptide bond cleavages as identified by MS/MS analysis (Fig. 4G). As expected, none of the degradation products showed any inhibitory activity toward the KLK13 protease (Fig. 4D).
FIGURE 4Gingipains target the reactive site loop of SPINK6. SPINK6 (10 μg) was incubated with 100 nm Kgp, RgpB, or HRgpA for 1 h at 37 °C. The reactions were stopped by acidification with TFA (0.1% final concentration), and the samples were resolved by HPLC (A–C). Eluted peaks were pooled, tested for their inhibitory activity against KLK13 (D–F), and analyzed by MS (G–I). Ctrl, control. AU, absorbance units; Frac., fraction.
The pattern of degradation products for the Arg-specific HRgpA and RgpB differed significantly from that of Kgp. Five main peaks were identified (Fig. 4, B and C), and none of them contained the unmodified inhibitor. All of the products were consistent with a single cleavage after Arg42. Importantly, the SPINK6 structure is stabilized by three disulfide bridges that keep the polypeptide fragments together after cleavage with gingipains. However, 2 mm l-cysteine required for gingipains activity reduced the disulfide bridges in the SPINK6 molecule already destabilized by a proteolytic cleavage. In the case of multiple cleavages of the inhibitor polypeptide chain by Kgp, there were no intact bridges left to keep the fragments together. Conversely, a single cleavage at Arg42 by Rgps generated predominantly the double-chain, full-length molecule, as demonstrated by the +18-Da mass shift compared with the native unmodified inhibitor. However, in an unstable two-chain structure of SPINK6, disulfide bridges holding the molecule together must be partially reduced, yielding N- (SPINK61–42) and C-terminal (SPINK643–80) fragments. It can be speculated that the rearrangement of the disulfide bridges in these fragments during sample preparation for HPLC analysis led to two different molecular conformations of identical mass (MS/MS data) but differed in the retention times on HPLC (Fig. 4, H and I). Nonetheless, the fragmentation of the inhibitor chain seems to be required, as the intact inhibitor was unaffected by 2 mm l-cysteine used in the assay and eluted as a single peak, retaining the full inhibitory activity.
To test for residual activity among the cleavage products, the inhibitory potential of all of the Kgp-, RgpB-, and HRgpA-derived products was evaluated (Fig. 4, D–F). Peak areas were used to calculate the amount of peptide in each peak. Reactions were carried out under more rigorous conditions than previous assays, using a 10:1 molar ratio of the given inhibitor fragment to KLK13 (compared with the 2:1 molar ratio in the experiments above). The N-terminal SPINK61–42 and the C-terminal SPINK643–80 fragments were completely inactive as inhibitors, whereas the full-length, +18-Da, single hydrolysis-modified peptide displayed 50% inhibitory activity of the control SPINK6 when incubated with KLK13 under the same reaction conditions. This result indicates that this product is a relatively weak KLK13 inhibitor, as a 10-fold molar excess did not fully inhibit the target enzyme compared with native SPINK6, which inhibited 90% of the protease activity at a 2:1 molar ratio in the experiments above.
SPINK6 Acts as a Potentially Important Inhibitor in Human Saliva
Human saliva displays a significant, trypsin-like proteolytic activity. As SPINK6 remains cross-linked to the ECM and provides localized inhibitory protection, the fraction of saliva activity susceptible to the SPINK6 control was estimated. Hence, a saliva mixture from five healthy individuals was titrated with recombinant SPINK6 using the Boc-Val-Pro-Arg-AMC fluorogenic substrate. Saliva (5 μl) was diluted to a final volume of 200 μl (40-fold dilution), and proteolytic activity was measured in the presence of 0–100 nm SPINK6 (Fig. 5B). In addition, the susceptibility of the saliva of each individual was tested separately (Fig. 5A). The 50 nm SPINK6 was able to inhibit ∼60% of trypsin-like activity in each of the individual saliva samples. Moreover, the Arg-specific proteinase activity in the saliva mixture decreased in a SPINK6 concentration-dependent manner until 75% reduction. Nonetheless, ∼25% of the trypsin-like activity in the tested sample remained unaffected by SPINK6. The overall SPINK6-sensitive activity was determined as ∼1 μm in the undiluted saliva by SPINK6 active site titration (data not shown). This result indicates that inactivation of SPINK6 could render a major fraction of protease activity uncontrolled, with the potential to disrupt signaling pathways and damage tissues.
FIGURE 5The majority of trypsin-like proteinase activity in healthy saliva is sensitive to SPINK6 inhibition. A, saliva of each healthy individual (5 μl, n = 5) was diluted 40× in reaction buffer, and 50 nm SPINK6 was added to each sample. Activity was measured using the fluorescent trypsin substrate Boc-Val-Pro-Arg-AMC. The results were normalized, and activity in the SPINK6-untreated sample was set as the 100% control (ctrl) for each individual. Significantly different means were identified using Mann-Whitney test. B, the saliva mixture from healthy individuals (5 μl) was diluted 40× in reaction buffer and incubated with increasing concentrations of recombinant SPINK6. Residual trypsin-like protease activity was measured using the Boc-Val-Pro-Arg-AMC substrate and was plotted as percent of control sample activity (no inhibitor added). Data are presented as mean ± S.D.
Kgp Activates pro-KLK13
The majority of the kallikrein family proteases contain an Arg or Lys residue at the profragment cleavage site (
Yoon H.
Laxmikanthan G.
Lee J.
Blaber S.I.
Rodriguez A.
Kogot J.M.
Scarisbrick I.A.
Blaber M.
Activation profiles and regulatory cascades of the human kallikrein-related peptidases.
) and, therefore, may be susceptible to activation by gingipains. Because KLK13 contains a Lys residue at the activation site, the ability of Kgp, a Lys-specific gingipain, to generate the mature form of the protease was tested (Fig. 6). Increasing concentrations of Kgp were incubated with pro-KLK13, and KLK13 activity was measured using the Boc-Val-Pro-Arg-AMC substrate (Fig. 6A), which is not hydrolyzed by Kgp. To ensure that no HRgpA/RgpB contamination affected the results, the assay was performed in the presence of the Rgps inhibitor Kyt-1. As shown in Fig. 7A, Kgp activated proKLK13 in a concentration-dependent manner, with a maximal effect at 5 nm Kgp. Further increases in Kgp concentration (50 and 100 nm) resulted in a gradual loss of activity that could be the result of degradation of the mature KLK13 by high concentrations of gingipains. Nonetheless, the activity was fairly stable, leaving ∼80% of maximal KLK13 activity even at 100 nm Kgp. Similarly, the active site labeling by FP-biotin indicated the proper binding of the biotinylated inhibitor, as presented in Fig. 6B. Increasing concentrations of Kgp resulted in the appearance of the band, corresponding to the active KLK13. Already at 0.1 nm Kgp concentration, binding of the FP-biotin was visible, and with further increases up to 30 nm Kgp, the band was saturated. Finally, the activation of KLK13 by Kgp was confirmed by the removal of the N-terminal His tag attached to the KLK13 profragment (Fig. 6C). Similarly, the Western blotting analysis using anti-His tag antibody revealed a decrease in the signal with increasing Kgp concentration, resulting in near-complete disappearance of the respective band at 50 nm Kgp concentration. This indicates that increasing concentrations of Kgp indeed facilitate the removal of the N-terminal profragment in the nanomolar range of concentrations and, coupled with the activity and FP-biotin labeling results, confirm the ability of Kgp to activate KLK13 and to release a functional protease. This indicates that low nanomolar levels of Kgp activate proKLK13, disrupting the protease-inhibitor balance already affected by SPINK6 proteolytic inactivation by gingipains.
FIGURE 6Lys-specific gingipain K activates pro-KLK13. Pro-KLK13 was incubated with the indicated concentrations of Kgp in the presence of the Arg-specific gingipain inhibitor Kyt-1 for 1 h at 37 °C. A, after incubation, samples were diluted into Boc-Val-Pro-Arg-AMC solution, and the rate of substrate hydrolysis was measured. Results were plotted as percent of maximal KLK13 activity versus Kgp concentration. B, Western blotting analysis of pro-KLK13 incubated with increasing concentrations of Kgp, followed by active site labeling by FP-biotin, results in appearance of a band corresponding to active KLK13. C, Western blotting analysis with anti-His tag antibodies shows the removal of the N-terminal His tag upon incubation with Kgp in a dose-dependent manner.
FIGURE 7Gingipains affect SPINK6 integrity and function in the presence of saliva. SPINK6 (10 μg) was incubated with a 25% saliva mixture from five healthy donors in the presence of the indicated gingipain (50 nm). After 1 h, the reaction was stopped by addition of gingipain-specific inhibitors and analyzed. A, the ability of the reaction products to inhibit KLK13 was evaluated in 25% saliva in the presence of gingipain inhibitors. A 2-fold molar excess of the SPINK6 reaction products was added to KLK13 solution, and activity was measured using fluorescent monitoring of the Boc-VPR-AMC substrate. B, Western blotting analysis with anti-His tag antibodies shows that HRgpA and RgpB fully degraded SPINK6. Kgp was less active, leaving a small fraction of the inhibitor intact. Ctrl, control. Statistical significance: *, p < 0.01; **, p < 0.05.
Gingipains Degrade SPINK6 in the Presence of Saliva
Finally, the in vivo environment contains not only saliva but numerous other proteins, possibly acting as competing substrates for gingipains. Therefore, the degradation of SPINK6 was analyzed in the presence of 25% saliva (a mixture from five healthy volunteers). SPINK6 (10 μg) was incubated for 1 h with 50 nm gingipains in the presence of the 25% saliva mixture diluted in reaction buffer. Samples were analyzed by Western blotting with anti-His tag antibodies. Additionally, the residual inhibitory activity of the degradation mixture was verified using the KLK13 inhibition assay (Fig. 7A). Western blotting results (Fig. 7B) confirmed the superior activity of the Arg-specific HRgpA and RgpB, which were able to completely degrade 10 μg of SPINK6 in the presence of 25% saliva. Similarly, the presence of HRgpA or RgpB blocked inhibition by SPINK6 because >60% of KLK13 activity in saliva was restored. Kgp again was proven to be the least effective, with residual levels of SPINK6 detected by Western blotting and only 15% of KLK13 activity restored. These data indicate that Arg-specific gingipains may specifically target SPINK6 in the complex protein mixture, whereas Kgp degradation could be regarded as a rather nonspecific attack on the exposed Lys residues, limited by the presence of other saliva-derived proteins.
Our results indeed confirm the ability of P. gingivalis to affect the kallikrein-inhibitor balance, adding to the well known pathological interactions of this bacterium with the protease control system of the host. Biochemical and mutant strain analyses using healthy saliva mixtures in vitro showed that gingipains were the sole proteases responsible for this activity. Gingipain concentrations in the nanomolar range were effective, and a combination of gingipains was found to be most efficient. Moreover, the Lys-specific Kgp not only degraded the inhibitor but was also capable of activating the proform of KLK13, as opposed to Arg-specific HRgpA and RgpB.
This important observation indicates that gingipains indeed possess the potential to activate the main kallikrein proteinase, KLK13, present in human saliva (
Shaw J.L.
Distribution of 15 human kallikreins in tissues and biological fluids.
Clin. Chem. 2007; 53: 1423-1432
). Additionally, our results confirm the ability of gingipains (both Lys- and Arg-specific) to activate other members of KLK family of proteases, an observation pursued in our future work.
K. Plaza, M. Kalinska, O. Bochenska, U. Meyer-Hoffert, Z. Wu, J. Fischer, K. Falkowski, L. Sasiadek, E. Bielecka, B. Potempa, A, Kozik, J. Potempa, and T. Kantyka, unpublished results.
Moreover, the complex protein mixture present in saliva did not block the inactivation of SPINK6 by Arg-specific gingipains, indicating a targeted activity of these pathogen-derived proteinases.
Additionally, our results indicate a substantial level of salivary trypsin-like activity, of which 75% is susceptible to SPINK6-mediated inhibition. This allows the estimation of the total KLK activity in saliva approximating 1 μm, an amount left uncontrolled after the SPINK6 inactivation. Taken together, these data indicate that the kallikrein-inhibitor balance is one of the targets of P. gingivalis proteases in producing inflammation.
Gingipains are well established virulence factors of P. gingivalis and are often described as an archetype of protease involvement in bacterial virulence. These aggressive enzymes, with strict P1 specificity toward Arg (HRgpA and RgpB) or Lys (Kgp) residues, target a number of essential components of human immunity, coagulation cascade, and regulatory pathways (
The biphasic virulence activities of gingipains: activation and inactivation of host proteins.
). Their impact on host protease-inhibitor systems has been reported previously, as gingipains inactivate the α1-protease inhibitor (
Meyer J.
Guessous F.
Huynh C.
Godeau G.
Hornebeck W.
Giroud J.P.
Roch-Arveiller M.
Active and α-1 proteinase inhibitor complexed leukocyte elastase levels in crevicular fluid from patients with periodontal diseases.
), secretory leukocyte peptidase inhibitor (
Into T.
Inomata M.
Kanno Y.
Matsuyama T.
Machigashira M.
Izumi Y.
Nakashima M.
Matsushita K.
Arginine-specific gingipains from Porphyromonas gingivalis deprive protective functions of secretory leucocyte protease inhibitor in periodontal tissue.
Clin. Exp. Immunol. 2006; 145: 545-554
), and, as reported previously by our group, elafin (
Wiedow O.
Dubin G.
Elafin is specifically inactivated by RgpB from Porphyromonas gingivalis by distinct proteolytic cleavage.
Biol. Chem. 2009; 390: 1313-1320
). The common feature of the aforementioned inhibitors is that all three, working in concert, control the proteolytic activity released from neutrophils and, thus, prevent damage of connective tissue during inflammation. This report provides evidence that gingipains can efficiently degrade SPINK6, a Kazal-type inhibitor of the tissue kallikrein family of proteinases. To our knowledge, this is the first example of the gingipain-mediated disruption of a system that controls the activity of these tissue-specific enzymes.
All three gingipains are capable of inactivation of SPINK6. As the sequence of SPINK6 (Fig. 8) contains six Lys residues, it is not surprising that Lys-specific gingipain K was able to fragment the protein into small peptides unable to inhibit the target enzyme. More importantly, however, cleavage by Arg-specific HRgpA and RgpB also led to the loss of inhibitor activity. SPINK6 contains only a single Arg residue, located in the P1 position of the reactive site loop. Moreover, the disulfide bridges in the polypeptide stabilize the inhibitor against dissociating into separate fragments (Fig. 8). Several reports indicate that these inhibitors can maintain their function after cleavage of the P1-P1′ peptide bond, possibly with lowered efficiency (
Laskowski M.
Qasim M.A.
What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes?.
Biochim. Biophys. Acta. 2000; 1477: 324-337
). Indeed, as mass spectrometry analysis confirmed that the main product of HRgpA or RgpB degradation of SPINK6 was the nicked inhibitor with the reactive site loop held together by disulfide bridges, and this polypeptide retained partial inhibitory activity. Two other peptides were identified, fragments SPINK61–42 and SPINK643–80, both with the oxidized disulfide bridges, and were inactive as proteinase inhibitors. Each fragment eluted from an HPLC column with two distinct retention times and with the oxidized ("bound") disulfide bridge state. Therefore, as the MS/MS sequence analysis confirmed the identity of these peptides, we hypothesize that the distinct elution times for each of the peptide fragments correspond to the different disulfide bridge configurations within the given fragment.
FIGURE 8SPINK6 is cleaved by gingipains. Kgp cleavage was detected at four positions: on the C-terminal side of Lys(24–1), Lys37, Lys58, and Lys71 (numbering of the full-length SPINK6 according to the Uniprot database). The hydrolysis after Lys(24–1) is located at the enterokinase cleavage site in the N-terminal extension of the protein (gray). All other Kgp recognition sites are within the sequence of the native SPINK6 protein. HRgpA and RgpB hydrolyze the single peptide bond on the C-terminal side of Arg42. An additional unassigned hydrolysis site at Gln53 is marked (asterisk) and may be an artifact of the MS analysis. The disulfide bond scaffold binds N-terminal and C-terminal regions of the molecule, hence dissociation of the SPINK61–42 and SPINK643–72 requires reorganization of the disulfide bridges.
As cysteine proteinases, gingipains are dependent on reducing agents in the reaction buffer, and, thus, it is possible that reorganization of the disulfide bridges is the result of the initial reduction of the protein. Nevertheless, our data confirm full activity of SPINK6 under optimized experimental conditions with buffer containing 2 mm l-cysteine (data not shown). Moreover, all control samples in the experiments were incubated in reaction buffer and, as confirmed by HPLC and MS, maintained full activity with no changes in the structure or disulfide bridges. Therefore, we conclude that reorganization of the disulfide bridge pattern is most likely a result of internal instability in the cleaved inhibitor. This model is in agreement with the standard mechanism of inhibition where a target protease forms a pocket tightly binding the inhibitor and preventing its free dissociation, hence stabilizing the cleaved form in the binding cleft. By contrast, hydrolysis by gingipain R may allow release of the internal tension of the molecule by disulfide bridge reorganization. As mentioned above, this hypothesis is supported by the detected state of disulfide bridges in the isolated peptides that, despite 2 mm l-cysteine in the reaction buffer, were all oxidized.
The inactivation of SPINK6, a potent, tightly binding inhibitor of kallikrein family proteinases (
), is an important observation. SPINK6 is present in epithelial tissues, including skin and gingiva, in two forms: fully soluble and transglutaminated by its N-terminal glutamine to extracellular matrix proteins, like fibronectin (data not shown and Ref.
). This diversification of the inhibitor forms provides protection for the extracellular matrix, locally increasing the inhibitory capacity because of covalent linkage of the active inhibitor to the ECM proteins. As suggested for other tightly binding tissue inhibitors of proteases (e.g. elafin (
Guyot N.
Zani M.L.
Maurel M.C.
Dallet-Choisy S.
Moreau T.
Elafin and its precursor trappin-2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase.
Biochemistry. 2005; 44: 15610-15618
)), the protective system is constituted by the initial response molecule, a tightly binding, locally produced inhibitor, which later transfers the protease molecule onto the final, serum-derived, irreversible inhibitor (either serpin or α2-macroglobulin). Hence, the tightly binding inhibitor provides the necessary quick response and protects essential components of the tissue. The fact that gingipains not only inactivate SPINK6, as presented here, but are also capable of direct degradation of ECM proteins (
Banbula A.
Role of bacterial proteinases in matrix destruction and modulation of host responses.
) likens them to an enzymatic arsenal that is capable of both inactivation of host protease inhibitors and degradation of the ECM matrix itself.
Tissue kallikreins are a family of 15 extracellular serine proteases with trypsin- and chymotrypsin-like specificity. Their functions are not well defined, but recent studies indicate that kallikreins 5, 7, and 14 regulate epithelial desquamation (
Simon M.
Jonca N.
Guerrin M.
Haftek M.
Bernard D.
Caubet C.
Schmidt R.
Serre G.
Refined characterization of corneodesmosin proteolysis during terminal differentiation of human epidermis and its relationship to desquamation.
); KLK3, 7, and 13 may activate plasminogen; and KLK2, KLK3, KLK6, KLK7, and KLK14 may degrade ECM proteins (as reviewed in Refs.
Lundwall A.
Kallikrein-related peptidases.
Cell. Mol. Life Sci. 2008; 65: 2019-2038
Kontos C.K.
Scorilas A.
Kallikrein-related peptidases (KLKs): a gene family of novel cancer biomarkers.
Clin. Chem. Lab. Med. 2012; 50: 1877-1891
). Moreover, KLK5 and KLK7 are enzymes that process both precursor and mature forms of antimicrobial cathelicidine hCAP18 in the skin (
), and KLK5 and KLK14 regulate inflammation through activation of proteinase-activated receptors (
Roosterman D.
Kempkes C.
Bocheva G.
Steinhoff M.
Activation of proteinase-activated receptor-2 by human kallikrein-related peptidases.
J. Invest. Dermatol. 2008; 128: 18-25
). Furthermore, our results implicate KLK13, one of the main salivary kallikreins, as an important enzyme in the regulation of epithelial wound healing, most likely via processing of the growth factor precursors.4 Additionally, kallikreins have been implicated in numerous reports as important factors and biomarkers in the development and progression of tumors (as reviewed in Ref.
Borgoño C.A.
The emerging roles of human tissue kallikreins in cancer.
Interestingly, the development of periodontitis and bacterial infections of the oral cavity have been linked to an increased risk of oral cancer (
Tezal M.
Grossi S.G.
Genco R.J.
Is periodontitis associated with oral neoplasms?.
Sullivan M.A.
Hyland A.
Marshall J.R.
Stoler D.
Reid M.E.
Loree T.R.
Rigual N.R.
Merzianu M.
Hauck L.
Lillis C.
Wactawski-Wende J.
Scannapieco F.A.
Chronic periodontitis and the incidence of head and neck squamous cell carcinoma.
Cancer Epidemiol. Biomark. Prev. 2009; 18: 2406-2412
). More recent reports further strengthen the correlation between P. gingivalis infection and oral and stomach cancer development in vitro and in vivo (
Gao S.
Li S.
Ma Z.
Liang S.
Shan T.
Zhu X.
Zhang P.
Liu G.
Zhou F.
Yuan X.
Jia R.
Scott D.A.
Lamont R.J.
Wang H.
Presence of Porphyromonas gingivalis in esophagus and its association with the clinicopathological characteristics and survival in patients with esophageal cancer.
Infect. Agents Cancer. 2016; 11: 3
). This effect is usually attributed to the direct effect of bacterial toxins and virulence factors promoting the development of chronic inflammation. Our results propose a novel explanation of the periodontitis-cancer correlation. The inactivation of a kallikrein-specific inhibitor may be beneficial for bacteria, as it affects inflammation, tissue reconstruction, and antimicrobial-peptide processing; however, the long-term increase in kallikrein activity could eventually lead to the development of oral cancer via activation of growth factors, facilitation of chronic inflammation, and promotion of metastasis by ECM matrix remodeling.
Our report provides evidence for the involvement of P. gingivalis gingipains in destabilization of the regulation of KLK activity through inactivation of SPINK6, the epithelium-specific inhibitor of kallikreins. This result not only adds to the known tendency of P. gingivalis to disrupt the proteolytic balance in infected tissues but also provides a possible mechanistic link between the development of periodontitis and oral cancer progression. As the role of the salivary kallikreins in the development of oral cancer is not known, the reported data provide a basis for further investigation of P. gingivalis infection and kallikreins in carcinogenesis and tumor metastasis in the oral cavity.
The Pichia pastoris expression system was obtained from Invitrogen. Gingipains and proKLK13 were purified as described previously (
Purification and characterization of gingipains.
Curr. Protoc. Protein Sci. 2007; (>Chapter 21, Unit 21 20)
Chang A.
Sotiropoulou G.
Soosaipillai A.
Xing X.
Howarth D.H.
Human kallikrein 13: production and purification of recombinant protein and monoclonal and polyclonal antibodies, and development of a sensitive and specific immunofluorometric assay.
Clin. Chem. 2003; 49: 77-86
). As HRgpA and Kgp were purified from whole culture medium, cross-contamination was possible. Therefore, the Arg gingipain-specific (Kyt-1) and Lys gingipain-specific (Kyt-36) (
) small molecule, tightly binding inhibitors were used in all gingipain experiments to ensure inhibition of undesired cross-reactivity in the samples. All reagents were obtained from Sigma-Aldrich (Taufkirchen, Germany) unless otherwise indicated.
Expression and Purification of Recombinant SPINK6
A DNA sequence encoding SPINK6 (GenBank accession no. GQ504704.1) was amplified from keratinocyte cDNAs in three consecutive PCR reactions that introduced an EcoRI cleavage site and histidine tag- and enterokinase cleavage site-coding sequence at the 5′ end and a NotI recognition site at the 3′ end. The final amplicon was ligated into the pPIC9 vector following the α-factor secretion sequence and used to transform Escherichia coli strain Dh5α. Positive clones were sequenced to verify the sequence, and the plasmid was linearized with SalI (Thermo Scientific) and electrotransformed into P. pastoris strain GS115 to obtain the Mut+ phenotype. Following addition of 1 m sorbitol, cells were plated on MD plates (1.34% yeast nitrogen base, 0.00004% biotin, and 2% glucose) and grown for 48 h at 30 °C. The Mut+ phenotype was verified by the ability to grow in the presence of methanol. Positive recombinants were chosen for expression cultures. BMGY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 1% ammonium sulfate, 0.0004% biotin, 100 nm potassium phosphate buffer (pH 6.0), and 1% glycerol) was inoculated with a single colony and incubated at 30 °C for ∼16 h with shaking (250 rpm), and, after reaching A600 = 4.0, the cells were harvested by centrifugation (5 min, 3000 RCF) and resuspended in BMMY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 1% ammonium sulfate, 0.0004% biotin, 100 nm potassium phosphate buffer (pH 6.0), and 0.5% methanol) at A600 = 1.0 to induce expression. Cultures were incubated for 72 h at 30 °C with shaking at 250 rpm for protein production. Every 24 h, aliquots of 100% methanol were added to maintain 0.5% final concentration. Cells were then harvested by centrifugation (15 min, 3000 RCF), and the supernatant was collected, filtered, and mixed with 50 mm sodium phosphate buffer (pH 7.5), 2.5 m NaCl, and 25 mm imidazole at a ratio of 4:1 (supernatant:buffer, v:v). Finally, equilibrated nickel-Sepharose resin (GE Healthcare) was added and incubated with the supernatant for 16 h at 4 °C with gentle agitation. Histidine-tagged protein was eluted with increasing concentrations of imidazole. The fraction of interest was then acidified by adding TFA to a final concentration of 0.1%. The sample was loaded onto an equilibrated, semipreparative HPLC column (Jupiter C18, 5 μm, 10/100 mm, Phenomenex). Protein was eluted using a 25–35% gradient of phase B (0.08% TFA, 80% acetonitrile) in 17 column volumes using an ÄktaMicro chromatography unit (GE Healthcare). Fractions containing the inhibitor were collected and dried using a CentriVap concentrator (Labconco) to remove the solvent.
Degradation of SPINK6 by Bacterial Cultures
P. gingivalis wild-type W83 and W83ΔkgpΔrgpAΔrgpB strains were cultured overnight in Schaedler medium supplemented with hemin (1 mg/ml), vitamin K (0.5 mg/ml), and cysteine (0.05 mg/ml) under anaerobic conditions (90% N2, 5% CO2, 5% H2) at 37 °C. Overnight cultures were diluted into fresh medium to A600 = 0.1 and cultured as above until reaching A600 = 1.0. Subsequently, 200 ng of SPINK6 was added to 1 × 106 bacteria in culture medium and incubated under anaerobic conditions for 1 h at 37 °C. The reaction was terminated by addition of SDS-PAGE sample buffer containing DTT and boiling for 5 min. The products of the reaction were separated by SDS-PAGE, and proteins were electrotransferred onto a PVDF membrane (Immobilon-P, Millipore). The membrane was blocked with 3% skim milk in TTBS buffer (50 mm Tris-HCl (pH 7.5), 500 mm NaCl, and 0.05% Tween 20) for 1 h at 37 °C. The blocked membrane was incubated overnight at 4 °C with primary antibody (1:1000, goat anti-human SPINK6, purified as described before (
)) and then washed four times with TTBS and incubated with a secondary antibody (HRP-conjugated anti-goat IgG (1:30,000, catalog no. A8919, Sigma) for 1 h at room temperature. Pierce ECL (Thermo Fisher Scientific) was used as the chemiluminescence substrate, and images were developed using Medical X-Ray-Film Blue (Agfa).
Analysis of SPINK6 Degradation
Before each experiment, gingipains (HRgpA, RgpB, and Kgp) were activated for 15 min at 37 °C in TNCT buffer (0.2 m Tris-HCl, 0.15 m NaCl, 5 mm CaCl2, and 0.05% Tween 20 (pH 7.5)) supplemented with 10 mm l-cysteine. For analysis of concentration dependence, SPINK6 (200 ng) was incubated with 0, 2, 5, 10, 20, 50, or 100 nm of each gingipain for 1 h at 37 °C in TNCT buffer containing 2 mm l-cysteine. The reaction was terminated by addition of Kyt-1 or Kyt-36 (PeptaNova GmbH, Sandhausen, Germany), which specifically inhibit R- or K-type gingipains, respectively (5 μm final concentration). Samples were separated by SDS-PAGE and silver-stained. Based on the results obtained, concentrations of gingipains were chosen to perform the time course analysis of SPINK6 degradation. For the degradation time course, SPINK6 (200 ng) was incubated with activated Kgp (50 nm), RgpB (2 nm), or HRgpA (20 nm) for 0, 5, 15, 30, 45, 60, or 90 min at 37 °C in TNCT buffer supplemented with 2 mm l-cysteine. After the indicated time intervals, the reaction was stopped by addition of Kyt-1 or Kyt-36 to samples with R- or K-type gingipains, respectively. Samples were resolved using SDS-PAGE, and protein bands were visualized by silver staining.
Samples for separation by SDS-PAGE were prepared under reducing and non-reducing conditions. In both cases, sample buffer (4% SDS, 60% glycerol, 0.3 m Tris (pH 8.0), and 0.01% bromphenol blue) was added to reaction mixtures at a 1:3 volume ratio. For reducing conditions, sample buffer was supplemented with 100 mm DTT, and samples were boiled for 5 min at 100 °C. Electrophoresis was run in a Schagger/von Jagow Tricine system with a two-layer separating gel: 16% (T:C 8.7:1) and 10% (T:C 16.5:1). Protein size was estimated using the PageRuler Plus prestained protein ladder (Fermentas) after silver staining.
Inhibition Assays
SPINK6 (40 nm) was incubated with each gingipain (20 nm) or with an equimolar mixture of three gingipains, each at 4 nm final concentration for 1 h at 37 °C in a total volume of 50 μl of TNCT containing 2 nm l-cysteine. The reactions were terminated by the addition of 5 μl of Kyt-1 or Kyt-36 to samples with R or K gingipains, respectively, or both into samples of SPINK6 incubated with the mixture of gingipains and the KLK13 control (4.5 μm final concentration of each Kyt). After incubation for 15 min at 37 °C, 45 μl of KLK13 in TNET buffer (100 mm Tris, 150 mm NaCl, 5 mm EDTA, and 0.05% Tween 20 (pH 7.5)) was added, and samples were incubated for another 15 min at 37 °C. The final concentration of enzymes, inhibitors, and substrate (in 100 μl of reaction mixture) was 20 nm SPINK6, 10 nm gingipains (or 2 nm each gingipain in a mixture), 20 nm KLK13, and 2.4 μm Kyt inhibitor. Finally, 100 μl of a fluorogenic substrate, N-t-Boc-Val-Pro-Arg-AMC (40 μm, Sigma) in TNET buffer was added, and hydrolysis of the substrate was recorded for 60 min as the increase of fluorescence intensity (an excitation wavelength of 355 nm and an emission wavelength of 460 nm) using a microplate fluorescence reader (SpectraMax Gemini EM, Molecular Devices). Of note, the kinetic parameters of substrate hydrolysis by KLK13 were determined previously: Km = 10.5 × 10−5 ± 1.7 × 10−5 m and kcat/Km = 4.66 × 103 s−1·M−1.
HPLC Separation and Analysis of Degradation Products
SPINK6 (10 μg) was incubated with 100 nm Kgp, RgpB, or HRgpA in TNCT buffer containing 2 mm l-cysteine for 1 h at 37 °C. The reaction was stopped by acidification of the samples with TFA (0.1% final concentration (v/v)). Samples (100 μl) were loaded onto a column equilibrated with 0.1% TFA (AERIS C18 peptide, 3.6 μm, 4.6/150 mm, Phenomenex). Elution was performed using a 10–80% gradient of phase B (0.08% TFA, 80% acetonitrile) in 19 column volumes. Collected fractions were dried using a CentriVap concentrator (Labconco). Each fraction was resuspended in TNET buffer to a final concentration of 1 μm based on the peak integration data. The sample (20 nm final concentration) was then mixed with KLK13 (2 nm final concentration) and incubated in TNET buffer for 15 min at 37 °C (100-μl total volume). Subsequently, 40 μm substrate was added (100 μl), and fluorescence intensity was measured as described above. The final mixture contained 10 nm SPINK6 or its degradation products, 1 nm KLK13 (10:1 molar ratio), and 20 μm substrate in a final volume of 200 μl.
MS Analysis of the Degradation Products
Peptide fractions were dried by evaporation, redissolved in 30% methanol supplemented with 0.1% (v/v) formic acid, and analyzed using an HTC Ultra ETD II mass spectrometer (Bruker) equipped with an electrospray ionization source and an ion trap analyzer. The samples were directly injected using a syringe pump (KD Scientific) at a flow rate of 180 μl/h and analyzed in positive ion mode with a capillary voltage of 3.5 kV, a nebulizer pressure of 10 psi, a drying gas flow of 5 liters/min, and an ion source temperature of 300 °C. Spectra were acquired in MS and MS/MS modes in the range of 100–3000 m/z with three different fragmentation types: collision-induced dissociation, collision-induced dissociation/electron transfer dissociation, or electron transfer dissociation/proton transfer reaction. MS data were processed manually using the DataAnalysisTM 4.0 and BiotoolsTM 3.2 software.
Activation of pro-KLK13 with Kgp
Kgp was activated in TNCT buffer supplemented with 10 mm l-cysteine for 15 min at 37 °C. A sample of 200 nm pro-KLK13 (expressed in P. pastoris) was incubated with 0, 0.5, 1, 5, 10, 50, or 100 nm Kgp in TNCT containing 2 mm l-cysteine and 5 μm Kyt-1 for 1 h at 37 °C. The reactions were terminated by addition of Kyt-36 (5 μm) to inhibit Kgp. After 15-min incubation, 40 μm N-t-Boc-Val-Pro-Arg-AMC substrate was added, and fluorescence intensity was recorded as described above. The final mixture contained 100 nm pro-KLK13/KLK13, 1 nm Kgp, 1 μm Kyt-1, 1 μm Kyt-36, and 20 μm substrate in TNET buffer (200-μl total volume). Simultaneously, to verify the formation of the functional active site of KLK13 and to monitor removal of the N-terminal His tag, a separate reaction was performed. Pro-KLK13 (2 μg, 3.5 μm) was incubated with 0, 0.1, 5, 10, 20, 40, or 50 nm of activated Kgp in TNCT buffer containing 2 mm l-cysteine for 1 h at 37 °C. The reactions were terminated as described above. These samples were divided, and the first set of samples (0.5 μg of proKLK13) was incubated with 1 μm FP-biotin (Santa Cruz Biotechnology Inc.) for 30 min at 37 °C. The second set containing 1 μg of proKLK13 was used for an anti-His tag Western blot. Both sets of samples were separated by SDS-PAGE and electrotransferred onto a PVDF membrane (Immobilon-P, Millipore). Nonspecific binding sites were blocked with 5% BSA in TTBS. The FP-biotin-labeled samples were incubated with HRP-conjugated streptavidin (Sigma-Aldrich) diluted 1:25,000 in TTBS. Similarly, the second set of samples was developed using a monoclonal, HRP-conjugated anti-His tag antibody (catalog no. A7058, Sigma-Aldrich) diluted 1:20,000 in TTBS. After incubation, the antibodies were washed, and bands were visualized as described above.
Titration of Trypsin-like Activity in the Saliva
Saliva collected from five healthy individuals was pooled. The mixture was centrifuged for 30 min at 12,000 × g, and 5 μl of supernatant was diluted 10-fold in 45 μl of TNET buffer on a microtiter plate. To wells containing diluted saliva, 50 μl of solution containing 0, 2, 8, 20, 40, 80, 200, or 400 nm SPINK6 was added. After a 15-min preincubation, 100 μl of 40 μm N-t-Boc-Val-Pro-Arg-AMC substrate was added, and residual trypsin-like activity was measured. In addition, separate saliva samples from healthy donors were incubated with 50 nm SPINK6 and analyzed, as above, to provide information about individual variations in SPINK6-sensitive protease content.
Degradation of SPINK6 in the Presence of Saliva
Saliva was clarified by centrifugation as described above and diluted 2-fold with sterile PBS. Gingipains were preactivated in TNCT buffer supplemented with 10 mm reduced l-cysteine (Sigma-Aldrich) for 15 min at 37 °C. To eliminate potential cross-contamination of Rgp with Kgp and vice versa, Kyt inhibitors (2 μm final concentration) were added to eliminate any contaminating activity: Kyt-1 to the Kgp sample and Kyt-36 to the HRgpA and RgpB samples. SPINK6 (10 μg) was incubated with each gingipain (50 nm final concentration) in the presence of 25% saliva (a mixture of five healthy donor samples) for 1 h in 37 °C in TNCT buffer containing 2 mm cysteine. The reaction was stopped by adding Kyt-1 or Kyt-36 (2 μm final concentration) into samples with HRgpA/RgpB or Kgp, respectively, and incubating at 37 °C for 5 min. The integrity of SPINK6 was determined by both Western blotting analysis and by its ability to inhibit the enzymatic activity of KLK13. Samples separated by SDS-PAGE were transferred onto PVDF (Bio-Rad). Nonspecific binding sites were blocked with 5% skim milk in TTBS, followed by incubation with a monoclonal HRP-conjugated anti-His tag antibody (catalog no. A7058, Sigma-Aldrich) diluted 1:20,000 in TTBS. Alternatively, a 2-fold molar excess of the reaction products (as estimated by the original SPINK6 amount in the sample) was added to a 10 nm KLK13 solution in 0.1 m Tris-HCl (pH 7.5) supplemented with 2 μm Kyt-1 and Kyt-36. Kallikrein activity was measured by monitoring the fluorescence intensity as described above.
K. P. and M. K. performed the majority of the experimental work. K. P. performed the inhibition and degradation experiments, and M. K. was responsible for human saliva preparation and analysis. K. P. and M. K. participated in the preparation of the manuscript. O. B. and A. K. performed the mass spectrometry experiments and data analysis. U. M. H., Z. W., and J. F. were responsible for isolation of the SPINK6 gene and establishment of the expression system of the inhibitor. K. F., L. S., and E. B. performed expression and purification of recombinant (pro)KLK13. B. P. was responsible for purification of the bacterial gingipains. J. P. and T. K. were equally responsible for the experimental design, data analysis, work coordination, and preparation of the manuscript.
Received in revised form: May 26, 2016
Received: March 3, 2016


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Batya Ungar-Sargon Bio, Net worth, Wiki, Age, Height, Husband
Batya Ungar-Sargon is an American author and journalist. She is deputy opinion editor of Newsweek and former opinion editor fof The Forward, America's leading Jewish news organization. She has written for The Daily Beast, The New York Times, Washington Post, the Los Angeles Times, Foreign Policy, Newsweek, and the New York Review of Books.
Batya Ungar-Sargon Career
Batya Ungar-Sargon Biography
Batya Ungar-Sargon Family
Batya Ungar-Sargon Net Worth
Batya Ungar-Sargon Social media
Facts About Batya Ungar-Sargon
Batya Ungar-Sargon was born in Gaza, Israel probably in 1981 and is a member of the Jewish faith. Due to the little information given, we cannot determine her nationality.
According to her Twitter account, she lives in Brooklyn and had a grandfather "Willy Ungar" who was killed when she was a teenager. Also, according to her tweet, her grandmother is Indian. Batya attended a Jewish school and college for her education. She also has a Ph.D. from the University of California, Berkeley.
Tim Yakteen Biography, Net worth, Wiki, Age, Height, Wife
Sargon has written for The New York Times, Washington Post, Foreign Policy, and other publications while staying true to her identity and ethics. She has also made occasional appearances on MSNBC, NBC and other news outlets. Her dissertation is entitled "Compulsive Lust: The Power and Form of the Roman 1719-1740".
She brings her perspective as an Opinion Editor that has nothing to do with the publications. Shifting her focus from wine to social and political issues, she received more public attention than ever. Their work is often political, racial, social and ideological in nature.
Real name Batya Ungar-Sargon
place of birth Gaza, Israel
nationality Israeli or American
job Deputy Opinion Editor
religion Judaism
sun sign Not known
shoe size 7 (USA)
University University of California
marital status singles
Is she lesbian? no
Batya Ungar-Sargon's Husband Not known
About her private life, there is no information on whether she was married or not. As such, it's quite difficult to dig up her past as no reports are available from reliable sources that include a name that we can connect to her. She seemed more concerned with her work than her marriage.
As a result of her hard work, Batya Ungar-Sargon has acquired considerable wealth. However, her true net worth and earnings are yet to be discovered on the internet. Her estimated net worth is around $1 million. She is keeping her second source of income secret for the time being.
Batya isn't that active on social media, so we couldn't find her on either Instagram or Facebook. Despite this, she likes to stay on top of world events and is therefore active on Twitter, where she is known as @burgarsargon with more than 87.2K followers.
She was previously Opinion Editor for Forward, America's leading Jewish news organization
Her work mainly deals with political, social and racial issues.
Marsai Martin Biography, Net worth, Wiki, Age, Height, Boyfriend
Melissa Navia Bio, Net worth, Wiki, Age, Height, Boyfriend
1 thought on "Batya Ungar-Sargon Bio, Net worth, Wiki, Age, Height, Husband"

redpajama_set_name: RedPajamaCommonCrawl


Raised in Australia's multicultural, tropical city of Darwin, Kath moved to Adelaide to study contemporary jewellery. She graduated from the South Australian School of Art in 2000.
Kath continued to develop her practice by working from a number of studios, including the renowned Gray Street Workshop, JamFactory's Metal Design Studio and Soda and Rhyme. Kath now lives in the beautiful Adelaide Hills with a studio nearby at The Barn.
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Thyme Tea Recipe May Beat Green For Antioxidants & Health
Superfoodly July 5, 2017
In 2012, Pope Benedict bestowed her the title Doctor of the Church. Now that title has to do with her contribution to doctrine and theology, but back when she was alive nearly one thousand years ago, Saint Hildegard of Bingen was also involved in medicine and botany.
That's why she's often credited as being Germany's founder of scientific natural history.
In her writings about medicinal plants, she spoke very highly of this evergreen herb. Based on her descriptions, it's possible she might have valued thyme tea for fibromyalgia, MS, arthritis, lupus and/or other degenerative diseases with similar symptoms, because she said things like this:
"…if someone is worn out with palsy or with a stitch or by that disease that so wears out his or her members as if they were corroded or eaten, take sage, twice as much tithymal, three times as much thyme as tithymal, and cook these in water."
Now that particular recipe was for turning the tea into a topical ointment, by combining it with goat tallow. She also said it "kills lice on a person, if the person rubs himself or herself with it." You can read about this in Hildegard's Healing Plants.
Benefits of thyme tea
Today many purport this warm beverage helps with cough and nasal congestion. Drinking the tea is said to act as an expectorant and loosen mucus in the lungs from bronchitis and upper respiratory tract infections. It is true that the plant's main constituent – thymol – has demonstrated antiviral, antibacterial, and antifungal activity in lab experiments. However there is too little clinical research to know whether or not these same benefits occur in the human body when consumed as a food or drink. It has never been tested in humans with colds or the flu. (1) (2) (3)
What can be said with certainty is that tea made with thyme offers a satisfying savory flavor, unmatched by the common varieties of herbal teas.
Some say it's good for weight loss – which is also unproven – but being that it's a zero calorie beverage, you really can't go wrong with it while dieting! And being that there's no sugar, it's perfect for type 1 and type 2 diabetes.
Impressive antioxidant potential
Which tea has the most antioxidants? Most people assume it must be green tea. Some get more specific and say it's the Japanese matcha form which is best for anti-aging.
What is matcha? It's the youngest leaves of the Camellia sinensis tea plant, grown partly in shade which produces that bright vivid green.
How much antioxidants does it have? An ORAC value of 168,500 is the highest we've ever come across for it. Keep in mind that's a very expensive ceremonial grade which is stone ground by hand. It's quite different than the powder you will find for sale at Whole Foods!
The ORAC value of cheap dried thyme is almost the same as the highest quality, most expensive matcha in the world.
With a value of 157,380 for dried thyme, it's basically on par with the best matcha in the world. When you compare it to the quality of matcha you're more likely to encounter at a health food store or tea shop, then thyme is probably even higher!
Now keep in mind these two ORAC values are not for brewed beverages.
They're based off of 100 grams of dry matcha powder and the same amount for the dry spice. Since you only use about 2 grams per brewed cup, it means a standard serving of matcha tea will be around 1/50th of that ORAC value… that's around 3,000 (168,500 dividend by 50). If you were to make thyme tea with the same 2 gram amount, then its ORAC could be comparable. But only if you do it the right way…
How to make thyme tea
There are two ways of making it.
The most common is to steep or boil the fresh and raw plant material in hot water and then filter it prior to drinking.
The second way involves no filtering, because you make it using a finely ground powder which you drink.
Both green tea and matcha tea come from the exact same plant, Camellia sinensis. So why does the latter have almost double the antioxidant content per cup?
Part of the reason is because you are ingesting the ground leaves, not just steeping them. Only so much of the phytonutrients will come out from them while they soak in hot water.
Now to be clear, that's not the only reason. Matcha also uses leaves which have more chlorophyll. But if you were to brew them whole like you do regular green, the ORAC value would not be as impressive.
To maximize the benefits of thyme tea for health and nutrient content, you should make it the same way as matcha – using the ground powder and drinking it unfiltered.
Using dried vs. fresh thyme?
Here are the ORAC values for 100 grams of each:
Fresh = 27,426
Dried = 157,380
The dried spice has almost 500% more antioxidants!
Why? Unlike fresh blueberries which after just 3 days, will have their vitamin C and anthocyanin content plummet by 67% and 80%, respectively, the beneficial compounds in the Thymus vulgaris plant remain largely intact, even after processing. Its rosmarinic acid – a major antioxidant – holds up well in the dried spice. (4)
It's not that the dried has more than the fresh, but rather that its water content has been removed, and hence, the well-preserved phytonutrients are very concentrated in the spice.
That's the first advantage of using powder versus fresh thyme for your tea. The other benefit is that you can drink it like you do matcha. Accomplishing the same with the fresh herb is practically impossible, as you would not be able to chop or blend it into such tiny pieces.
The tough part? Finding it for sale in this finely ground form.
At Whole Foods in Redondo Beach, CA they only sold this tiny container – merely 3/10th of an ounce – for $2.99. So expensive, your homemade drink may be comparable to the cost of a Starbucks latte!
All of their normal-sized bottles of thyme were for the bigger leaf pieces, which are not suitable for tea.
On Amazon, a bag of powder that's 48x larger (1 lb) can be purchased from Starwest Botanicals for an excellent value. That's the smarter way to do it.
Once you have your finely ground powder, you are ready to make your drink…
Thyme Tea Recipe
How to make it doesn't get any easier than this! In just 3 minutes, your throat will be soothed by a hot cup. 0 calories and 0 grams of fat.
½ to 1 teaspoon of finely ground thyme powder
1 cup filtered water
Measure powder into tea cup. Use more for a stronger flavor
Pour hot water into cup.
Stir and enjoy!
What does it taste like? The fact that it's savory – and not trying to be sweet like most herbal teas – works quite well for thyme. It's like drinking a good savory soup. Salty like celery, yet there's no sodium added.
There may not be medical data of it helping for weight loss, but what can be said is that after drinking a warm cup of this, it feels like you just ate something savory and satisfying. Might that help curb your appetite?
What is it good for?
You may have heard about people using thyme tea for cough and asthma. There are a few pieces of lab research which suggest there might be mechanisms to explain why that is:
In a study with mice, the thymol component was found to reduce allergic airway inflammation and help block the NF-KB pathway. (5)
In a study with rats, thyme extract was found to help with the side effect of hyper-sensitivity of the endothelin cells in their trachea. The scientists compared that symptom to that which is also present in asthma and COPD. (6)
In Switzerland, a trial was conducted with 62 participants to test an herbal cough syrup made with ivy and thyme. Based on symptom scoring and safety, they claimed the formula "seems to alleviate cough in consequence of common cold, bronchitis or respiratory tract diseases with formation of mucus." (7)
Asthma and allergies can be triggered by chitins, which are particles produced by fungi, insects, and other organisms. The same pro-inflammatory cytokines activated by these chitins were found to be counteracted by thymol in the cultured cells tested, as shown below. (8)
Above you see charts for the inflammatory signaling pathways miR-155 and miR-146a. They are increased with the chitins and two other known triggers (the positive controls). However when thymol was added to the cells, there appears to be less expression of these "bad" pathways associated with allergies and asthma.
While there is this preliminary evidence about the potential for lung benefits, there is too little research to claim any of these effects are proven. For arthritis joint pain, multiple sclerosis, lupus, fibromyalgia, Candida infections and other things you may read about on home remedy blogs, there is little to no related research about them.
Therefore, this tea should not be used as a treatment for any disease or illness. Instead, just enjoy it as the healthy and tasty beverage it naturally is!
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25 Lauric Acid Foods & Oils: High Sources To Supplement With
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ORAC Values of 72 Essential Oils
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Taylor sweeps the board at Palmer Park
CLASS ACT - Brian Taylor won every event he entered at Palmer Park on Monday
Racing against the same faces every week can sometimes make one rather apathetic, but Brian Taylor (VC Londres) - of National Derny Champion fame - was in rampant form at the Reading Track League at Palmer Park this week.
Taylor was back in action after a successful racing trip to Belgium at the weekend.
A feature of his racing style is aggression and he showed that in abundance to finish top spot in every race in which he competed - which was all of them!
Youngster Lewis Atkins (PCA Ciclos Unos) was for once, outclassed by the older, more experienced Taylor.
And even when it came down to a full-on head-to-head sprint, the older man still displayed the class to take the win, albeit it only just!
As usual the youths kicked off the action with a Hare and Hounds.
Chris Bradbury from Palmer Park Velo showed how much he has improved recently with wins in all his events and he comfortably won the Hare and Hounds from under-16 lady Bridie Hindle.
Sam Serruya and Stephen Bradbury (both Palmer Park Velo) each won their age categories in the under-14s and under-12s respectively.
With the marked increase in the number of ladies present, separate events were run, which produced some tactical, gutsy and aggressive riding.
In the keirin, again Hindle (Dataphonics RT) showed the senior girls a clean pair of heels as she caught the wheel of the Derny motorcycle from the off and kept the pace high enough on the final lap to prevent anyone passing her.
She looked very comfortable winning this event and she also went on to win the Devil Take the Hindmost in a closely-fought sprint with Reading regular Clara Beard (Team Quest).
Although training to be a veterinary surgeon, Beard still finds time to put in some quality training and showed how strong she is with an excellent ride in the 44-lap endurance event, staying in, while many others packed, to finish as the sole lady.
Another rising star, Keir Stone Brown of Chippenham Wheelers), also showed his mettle with a fine ride in this event.
He mixed it with the seniors, doing a fair share of the workload to pull back local strongman Rob English, riding in Cambridge University colours, who was away at the front for the majority of the 44 laps.
That was until Taylor, Atkins and Alex Peterson (Reading CC) made an acceleration that saw everyone else off, rapidly catching English, who was able to hang on, to form the winning break with just eight laps to go.
When the bell was rung, the watching and tactics started.
Taylor took the wheel of Atkins while English watched them and this would have been a good time for Peterson to attack as the others seemed engaged with each other.
But he hesitated a moment too long, allowing Taylor and Atkins to keep his wheel.
Down the finishing straight, Taylor found a new level and took three metres out of Peterson in no time.
Atkins could do nothing as the three crossed the line in the space of two bike lengths with Taylor victorious, Peterson having hung on and Atkins ruing the error of his tactics.
Overall the racing was made far more competitive by a few riders who are not regular Reading racers, particularly Taylor.
It is a credit to the man that he can come back from a weekend where he has been overseeing the racing of some of his youth and junior ladies in criteriums in Belgium to take on the best Reading Track League has to offer and come away with the spoils in fine style.


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text:
package org.apache.camel.test.blueprint.converter2;

import org.apache.camel.Converter;
import org.apache.camel.TypeConverters;

public class MyConverters implements TypeConverters {

    @Converter
    public Country toCountry(String iso) {
        Country answer = new Country();
        answer.setIso("en");
        answer.setName("England");
        return answer;
    }

    @Converter
    public String toIso(Country country) {
        return country.getIso();
    }
}

redpajama_set_name: RedPajamaGithub


The new segment, which is part of the Phase-III of the DMRC network would cover areas from Majlis Park to Shiv Vihar.
New Delhi: The Delhi Metro is all set to open the 9.7-km Lajpat Nagar-Mayur Vihar Pocket 1 section of Pink Line on December 31 as a New Year gift for its riders. The corridor has three underground stations; Lajpat Nagar, Vinobapuri, Ashram and Hazrat Nizamuddin, and two elevated stations, Mayur Vihar Ph-I and Mayur Vihar Pocket-1.
The Lajpat Nagar-Mayur Vihar Pocket 1 section of Pink Line was first inspected by the Commissioner of Metro Railway Safety (CMRS), who approved its passenger operations, stated a senior DMRC official. The Pink line is 59 kms long.
"This extension of the Delhi Metro's Pink Line will be formally flagged off by Union Minister of State (Independent Charge) Hardeep Singh Puri and Delhi Deputy Chief Minister Manish Sisodia on December 31 at 11 AM from Metro Bhawan. Passenger services will commence on this section on the same day from 4 PM onwards," an official told PTI.
The DMRC commenced operations on December 24, 2002. It was inaugurated by former prime minister Atal Bihari Vajpayee. Presently, it has 231 stations spanning a network of 317 km. The new segment, which is part of the Phase-III of the DMRC network, would cover areas from Majlis Park to Shiv Vihar.


After making his debut in Wii's Super Smash Bros. Brawl, legendary game designer Hideo Kojima has explained that it is unlikely that Solid Snake will make a return in the upcoming Wii U and 3DS titles.
Although, that isn't to say that he isn't less keen for the character's inclusion. Speaking within a Twitch interview with Geoff Keighley, the Metal Gear creator expressed: "I'm not working on that game, but I don't think it's likely. If Mr. Sakurai is watching this, hopefully we see Snake".
Konami's gruff-voiced icon had been joined in Brawl by other newcomers, including Pit from Kid Icarus, Pikmin's Olimar, Wario, and Sonic the Hedgehog. That eagerness for new characters has seen the forthcoming Wii U and 3DS titles secure Little Mac, Rosalina & Luma, Villager, Mega Man, and the Wii Fit Trainer.


As with code evaluation, Amherst provides agency negotiations with regard to repairs. Many times when a contractor is faced with obtaining permits for a repair they are required to perform additional work as a result of code compliance that is not always required by the Uniform Building Code. Each State and/or public agency follows different guidelines requiring different interpretations. Amherst can provide a real-time third party evaluation of the current code and negotiate a reasonable interpretation and/or requirement whereas a contractor may not have an incentive to do so.


text:
Home | Ronald Kessler
Tags: zawahiri | weapons | mass | destruction | fbi | kessler | wmd
FBI: 100 Percent Chance of WMD Attack
By Ronald Kessler Monday, 14 February 2011 06:47 PM Current | Bio | Archive
The probability that the U.S. will be hit with a weapons of mass destruction attack at some point is 100 percent, Dr. Vahid Majidi, the FBI's assistant director in charge of the FBI's Weapons of Mass Destruction Directorate, tells Newsmax.
Such an attack could be launched by foreign terrorists, lone wolves who are terrorists, or even by criminal elements, Majidi says. It would most likely employ chemical, biological, or radiological weapons rather than a nuclear device.
As it is, Majidi says, American intelligence picks up hundreds of reports each year of foreign terrorists obtaining WMD. When American forces invaded Afghanistan, they found that al-Qaida was working on what Majidi calls a "nascent" weapons of mass destruction effort involving chemical and biological weapons.
In every other case so far, the reports of foreign terrorists obtaining WMD have turned out to be unfounded. However, Majidi's directorate within the FBI investigates more than a dozen cases in the U.S. each year where there was intent to use WMD.
For example, in 2008, the FBI arrested Roger Bergendorff, who was found to have ricin and anarchist literature. Ricin kills cells by inhibiting protein synthesis. Within several days, the liver, spleen, and kidneys of a person who inhales or ingests ricin stop working, resulting in death.
"The notion of probability of a WMD attack being low or high is a moot point because we know the probability is 100 percent," Majidi says. "We've seen this in the past, and we will see it in the future. There is going to be an attack using chemical, biological or radiological material."
Even a WMD attack that does not kill a great number of people would have a crushing psychological impact.
"A singular lone wolf individual can do things in the dark of the night with access to a laboratory with low quantities of material and could hurt a few people but create a devastating effect on the American psyche," Majidi says.
As described by Majidi, who was previously the chemistry division leader at Los Alamos National Laboratory, the WMD Directorate was established in 2006 to coordinate all elements of the FBI that deal with WMD cases.
Regarding a subject that is full of hype and misinformation, it is rare for an official who is an expert in the field and has full access to current classified information to talk about it for publication.
Majidi says the kind of threat that keeps him awake at night is one from a lone wolf. That's because the FBI, along with the CIA and foreign partners, has developed a number of ways to detect plots by al-Qaida and other foreign terrorists. Besides intercepting their communications and infiltrating their organizations, the FBI gets reports when people purchase materials that could be used in a WMD attack. These techniques are known as trip wires.
For my book "The Terrorist Watch: Inside the Desperate Race to Stop the Next Attack," Arthur M. "Art" Cummings II, who headed FBI counterterrorism and counterintelligence investigations, gave an example of the FBI's use of trip wires.
When the FBI got a report of a man buying chemicals that could be used for explosives, it investigated. In this case, it would have been easy to dismiss the purchases as innocent, since the man was buying the supplies from a swimming pool company, and his business shipped pool supplies.
"That explanation wasn't good enough," Cummings says. "It's not OK to say, It looks like pool supplies, we're done. You don't finish there. Who at the pool company, specifically, did he buy them from? What specifically was the transaction, and what happened from there? Is it a friend; is it an associate; is it somebody who wants to do us harm? There was a day we would have said, It's a commercial transaction, don't worry about it. Each and every lead is followed all the way down to the most minute detail."
Majidi says three agents from his directorate have been assigned to FBI offices overseas — known as legal attaché offices or legats — in countries like Georgia to work with foreign intelligence authorities on possible attacks.
Currently, Majidi is working to develop ways to detect development of new organisms that could be used in a biological attack. By definition, there would be no way to detect a new organism or to develop an antidote before it is unleashed.
"We are not sitting on our hands waiting to predict what will happen based on what happened yesterday," Majidi says. As an example, he says, "You can design an organism de novo that never existed before. While there is no known articulated threat, this is something that we feel is a technology or science that potentially can be misused, either accidentally or on purpose."
The FBI is working with the synthetic biology community to develop ways to zero in on any hint that someone could be developing such an organism that could become a threat.
"We're not there to stop the science but to integrate our activities within their portfolio so that when the threat does develop or may develop over a long arc of time, we are ahead of those issues," Majidi explains.
Majidi says the most remote threat is an attack with a nuclear device. A terrorist bent on detonating a nuclear weapon would have to successfully negotiate a series of steps, Majidi says. He would have to find an expert with the right knowledge. He would have to find the right material. He would have to bring the device into the country, and he would have to evade detection programs.
"While the net probability is incredibly low, a 10 kiloton device would be of enormous consequence," Majidi says. "So even with those enormously low probabilities, we still have to have a very effective and integrated approach trying to fight the possibility."
Experts are constantly being quoted with estimates of the amount of enriched uranium that could be unaccounted for from Soviet Union stockpiles and could be used to make nuclear weapons. Majidi says no one knows the actual amount.
"I know there is a hobby of guessing, and different folks give you a different number," he says. "All I can tell you is that from the interdictions that we have had in the past decade, the quantities have been sufficient of highly enriched uranium that I clearly worry about this material on a global scale. How much is there? Any amount is too much."
A terrorist who stole a nuclear weapon from a country that has one would have an easier time than if he tried to make one. "One of the things you have to understand is that nuclear markets are very ambiguous markets," Majidi says. "There are as many bad guys trying to sell material as there are good guys trying to make sure that that doesn't happen."
While terrorists talk about using WMD, the preferred method for attack so far has been explosives. Majidi cites two examples: the Christmas Day bomber, Umar Farouk Abdulmutallab, a Nigerian citizen who boarded a Northwest Airlines flight to Detroit on Dec. 25, 2009, and tried to detonate explosives sewn into his underwear; and the Times Square bomber, Faisal Shahzad, a Pakistani immigrant who attempted to set off a car bomb in Times Square.
"While all of these guys are still interested in potentially using chemical, biological, or radiological weapons wherever it is possible, the pragmatic approach that they have taken is to use what has worked for them best, which is various forms of explosives and improvised explosives," Majidi says.
"The latest round is concealing explosives coming through the commercial shipping environment," Majidi notes. "That brings to the fore the fact that explosives are something that we're not going to get away from any time soon. It's the modality that is most often preferred by a pragmatic adversary."
Given the sensitivity and complexity of the subject, Majidi says he tries to present all the issues in context: "One of my jobs is to make sure I put all of these things in an appropriate light, because if you were in my job you would see that everyone always tries to elevate things to a tremendous level."
Of one thing Majidi is sure: "There's a probability of 100 percent that a WMD event will happen."
Ronald Kessler is chief Washington correspondent of Newsmax.com. View his previous reports and get his dispatches sent to you free via e-mail. Go here now.
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RonaldKessler
The probability that the U.S. will be hit with a weapons of mass destruction attack at some point is 100 percent, Dr. Vahid Majidi, the FBI s assistant director in charge of the FBI s Weapons of Mass Destruction Directorate, tells Newsmax. Such an attack could be launched...
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Monday, 14 February 2011 06:47 PM
The Gipper's Corner
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Democrat-Media Alliance Has Tied Itself to a Sinking Ship
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Argentic edition highlighted with pastel, on argentic paper.
Titled "Easy rider", dated, signed, numbered and artist's stamp on the back.
Peter Zupnik is a photographer born in 1961 in Slovakia.
He began his studies in applied photography at the secondary school of arts and crafts in Košice from 1976 to 1980. He then joined the FAMU University in Prague to pursue a course in artistic photography until 1986.
After finishing his studies, he became an independent photographer to live out of his passion and left Prague to live in Paris in 1995.
His work has been exhibited throughout the world and his photographs have been part of several private and public collections such as the one at the Maison Européenne de la Photographie and at the Pompidou Center.


With categories ranging from Periodic Table to Celebrity News, trivia app QuizUp has garnered many devotees, especially among members of the quiz bowl team.
Deciding to attend the tournament without its best, most experienced players, the quiz bowl team returned home with a new sense of experience and confidence. On Saturday, Oct. 12, the quiz bowl team participated in a tournament at Seven Lakes High School in Katy, the team's first competition of the year. The teams placed 10th and 12th.


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Tag Archives: Halstead
February 17, 2020 Ospreyshire Origins	Acoustic, Amazing, Ballad, Cher Lloyd, Dear Innovare...The Souls of Ignored Pioneers Shall Be Renowned, Ed Sheeran, England, Essex, Europe, Experimental, Folk, Halstead, Halstead's Trophy, Ipswitch, Keyboards, Lyrics, Matt Cardle, Music Plagiarism, One Direction, Ospreyshire Origins, Photograph, Plagiarism, Pop, Pop Stars, Story Behind the Song, Ukulele, United Kingdom, X Factor 2 Comments
Ospreyshire Origins: Halstead's Trophy
I no longer wanted to be down
After you took my crown and cashed it in for millions of pounds
So shamelessly
I had to strengthen my heart
To withstand all your darts that multiplied from the start
Of your thievery
Originator be my guide
May justice be on my side
Halstead will be filled with pride with the trophy that's rightfully mine
You think you can do what you will
I made you admit your guilt
How does it make you feel that you're forced to know my name?
Take a picture now (X6)
To immortalize your shame
Do you want to know what can really suck about plagiarism cases? It can force me to actually defend mainstream pop stars on certain occasions when I would never do so otherwise.
This is one of those times.
Meet Matt Cardle. He's an acoustic pop singer/songwriter hailing from Halstead, Essex, England. Yes, the name of the song refers to his hometown. Cardle has been quite popular in the UK even though he's unknown in America. He got his big break after winning on X Factor, so he certainly has several ears throughout Good Ol' Blighty. He had a song on his first album called "Amazing" which was a minor hit in the UK back in 2011. Wouldn't it be crazy if another Brit were to steal his song? Whoever could it be?
THIS GUY!
That's right. Ed Sheeran stole someone else's song! That is just annoying since his fanbase defended him and called Matt Cardle some nobody. Sure, Matt Cardle doesn't have as much of a worldwide popularity as the Ipswitch-based pop star, but I wouldn't call him obscure especially when it comes to English music. Matt Cardle has sold over 2 million records, has been on major labels even to this day, and like I said earlier: he's an X Factor winner and I know that show is popular in the United Kingdom. Here's some more context. Do you want to know who Matt Cardle beat out in the finals of X Factor in the same season he won it? Cher Lloyd and this one boy band that no one's heard of called ONE DIRECTION! Yeah, think about that for a minute.
In case you're wondering, the song that ripped off "Amazing" is "Photograph". Sure, it wasn't as big of a hit in America compared to "Sing" or "Thinking Out Loud" which were on the same album, but I know I heard that song playing on the radio whenever I was shopping or eating at some restaurants. To be just, the verses themselves are independent, but those choruses…WOW, Ed didn't even try besides having different lyrics. Here's a video of both song's choruses and tell me he didn't listen to this Essex crooner's song!
Matt Cardle's co-writers/producers actually sued Ed Sheeran and the beat him down in court! Sheeran was forced to give writing credits and royalties to everyone involved who made "Amazing". Good on them for doing that. I never thought I would have to defend someone who won a freaking music reality show, but that was the day.
Much like both songs, I decided to do a light acoustic ballad, but with my ukulele as a main instrument and I got to use my "pop star" voice which is a rarity as Ospreyshire for obvious reasons. I hope you also appreciate the stealth puns with both songs in Halstead's Trophy.
You're welcome, Essex!
The album cover of Matt Cardle is from Wikipedia and is property of Syco music.
The photo of Ed Sheeran is from The Independent.


Dimensions: 40" x 38" x 18"
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