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Bacteria in periodontal pockets develop complex sessile communities that attach to the tooth surface. These highly dynamic microfloral environments challenge both clinicians and researchers alike. The exploration of structural organisation and bacterial interactions within these biofilms is critically important for a thorough understanding of periodontal disease. In recent years, Filifactor alocis, a fastidious, Gram-positive, obligately anaerobic rod was repeatedly identified in periodontal lesions using DNA-based methods. It has been suggested to be a marker for periodontal deterioration. The present study investigated the epidemiology of F. alocis in periodontal pockets and analysed the spatial arrangement and architectural role of the organism in in vivo grown subgingival biofilms.
A species-specific oligonucleotide probe, FIAL, was designed and evaluated. A total of 490 subgingival plaque samples were submitted to PCR and subsequent dot blot hybridization to compare the prevalence of F. alocis in patients suffering from generalized aggressive periodontitis (GAP), chronic periodontitis (CP), and control subjects resistant to periodontitis. Moreover, a specially designed carrier system was used to collect in vivo grown subgingival biofilms from GAP patients. Subsequent topographic analysis was performed using fluorescence in situ hybridization.
While the majority of patients suffering from GAP or CP harboured F. alocis, it was rarely detected in the control group. In the examined carrier-borne biofilms the organism predominantly colonized apical parts of the pocket in close proximity to the soft tissues and was involved in numerous structures that constitute characteristic architectural features of subgingival periodontal biofilms.
F. alocis is likely to make a relevant contribution to the pathogenetic structure of biofilms accounting for periodontal inflammation and can be considered an excellent marker organism for periodontal disease.
Periodontitis is a chronic inflammatory bacterial infection leading to destruction of periodontal ligaments and supporting bone of the tooth. Its aetiology has been a field of intensive research in the past decades. As periodontal pockets accommodate a multitude of bacterial phylotypes, it is difficult to differentiate between mere commensals and true pathogens. During the 1970's, 80's and early 90's, research focused mainly on a number of culturable bacteria like Porphyromonas gingivalis, Prevotella intermedia, Aggregatibacter (Actinobacillus)actinomycetemcomitans, Tannerella forsythia and Treponema denticola that proved to be associated with the disease [1]. Studies have determined their relative prevalences, interactions and virulence factors [2-7]. By the end of the 1980's, the development of novel, culture-independent techniques allowed the identification of as-yet-unculturable and fastidious organisms in patients suffering from periodontitis and added new insight into bacterial communities in periodontal pockets [8-10]. In recent years, research has detected increasing numbers of bacterial species and phylotypes in subgingival plaque and other habitats of the human oral cavity [11-18]. There is little reason to believe that easily culturable bacteria contribute more to the development of periodontitis than fastidious organisms. Doubt has been raised whether the widely accepted periodontal pathogens P. gingivalis, P. intermedia and T. forsythia are appropriate diagnostic markers to differentiate between health and disease [19,20].
Along with these discoveries it became clear that the mere isolation and characterization of bacteria from diseased sites is not a sufficient approach to understand the complex pathogenesis of periodontitis. The organisms do not live in a planktonic form, but rather as a sessile community attached to the tooth surface in a matrix of extracellular polymers [21]. The structure and function of these bacterial biofilms are influenced both by bacterial interactions and host factors. Exploring the biofilm architecture and identifying its bacterial architects are pressing goals in current periodontal research.
Filifactor alocis (ATCC 35896T) was first isolated in 1985 from the human gingival crevice as Fusobacterium alocis [22] and later reclassified as Filifactor alocis [23]. It is a fastidious, Gram-positive, obligately anaerobic rod that possesses trypsin-like enzymatic activity [24], as do P. gingivalis and T. denticola [25,26]. In recent years, it has been discovered in patients suffering from chronic periodontitis (CP) [14,18,27,28], generalized aggressive periodontitis (GAP) [29] and endodontic infections [30]. Recently, F. alocis was detected in elevated numbers in CP patients with periodontal deterioration compared to patients with a stable periodontal condition and was therefore proposed as a potential marker for active disease [19].
The present study chose a DNA-based epidemiological approach utilizing dot blot hybridization to investigate the prevalence of F. alocis in subjects with GAP, CP, and in a subject group resistant to periodontitis. Furthermore, fluorescence in situ hybridization (FISH) was employed to analyse the spatial arrangement and the architectural role of F. alocis in periodontal pockets. For that purpose, a specially designed carrier system was used to collect in vivo grown biofilms from GAP patients [31].
Oligonucleotide probes
To detect F. alocis, a species-specific probe, FIAL (5'-TCTTTGTCCACTATCGTTTTGA-3') was designed after comparative sequence analysis of close phylogenetic neighbours to F. alocis. To ensure specificity, the probe sequence was compared to the sequences deposited in the Ribosomal Database Project II [32] and to all 16S rRNA entries at the EMBL and GenBank databases (as of August 2009) employing the Husar program package (DKFZ, Heidelberg, Germany). The probe was checked for its practical use in hybridization experiments with the program OLIGO (version 4.0). EUB 338, a probe complementary to a highly conserved region of the 16S rRNA gene in bacteria, was used in dot blot hybridization experiments to verify successful PCR amplification and in FISH experiments to detect and visualize large parts of the bacterial biofilm population [33]. For comparative purposes, probes POGI, PRIN, ACAC, TDEN, FUNU and B(T)AFO were employed in dot blot experiments to detect P. gingivalis, P. intermedia, A. actinomycetemcomitans, T. denticola, Fusobacterium nucleatum and T. forsythia, respectively. These probes have been published previously and deposited in ProbeBase [34].
Clinical samples for dot blot hybridization
A total of 490 subgingival plaque samples from 121 patients were examined and evaluated. Samples from GAP and CP patients were obtained from those reporting to the departments of periodontology of the Charité - Universitätsmedizin Berlin, the Dresden University of Technology, the University of Oslo and the University of Basel. These patients were diagnosed according to the criteria of the 1999 International Workshop for the Classification of Periodontal Diseases and Conditions [35] (see Table 1). Control samples were taken from elderly patients of a private periodontal practice in Berlin. These subjects, aged 65 years and older, had at least 20 natural teeth and displayed only mild periodontal disease. They had not received periodontal treatment previously, exhibited no sites with attachment loss of more than 2 mm or probing pocket depth (PPD) of more than 5 mm and will be referred to as periodontitis resistant (PR) patients in the following. Subjects suffering from chronic systemic disease were excluded from the study as well as pregnant or breast feeding women and patients who had received antiinflammatory or antimicrobial therapy within the past six months. Patient demographics are presented in Table 2. Ethical approval was given by the Ethical Committee at Charité - Universitätsmedizin Berlin. All patients signed informed consent forms. After removal of supragingival plaque the deepest periodontal pockets available were sampled. In GAP patients, additional samples were taken from shallow sites if present. None of the samples were taken from the same site in one patient. Three sterile paper points (ISO 35, Becht, Offenburg, Germany) were inserted into the pockets, removed after 10 seconds and placed immediately in 1 ml of reduced transport fluid (RTF) [36] containing 25% glucose.
Dot blot hybridization
DNA extraction from the 490 collected subgingival plaque samples, subsequent PCR amplification, preparation of dot blot membranes and dot blot hybridization experiments to analyse the prevalence of F. alocis were performed as published previously [37]. The broad range bacterial primers TPU1 5'-AGAGTTTGATCMTGGCTCAG-3' (corresponding to complementary positions 8-27 in the Escherichia coli 16S rRNA gene) and RTU3 5'-GWATTACCGCGGCKGCTG-3' (corresponding to positions 519-536 in E. coli 16S rRNA) were used to amplify part of the 16S rRNA gene out of the bulk DNA. Agarose gel electrophoresis confirmed successful amplification. Hybridizations with both EUB 338 and FIAL were carried out at 54°C, while stringency washes were performed at 58°C for EUB 338 and at 60°C for FIAL with a washing buffer containing 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) - 0.1% SDS for EUB 338 and 5× SSC - 0.2% SDS for FIAL. In all experiments, PCR-amplified products obtained from fixed cells of F. alocis, its closest cultured phylogenetic relative Filifactor villosus (ATCC 33388T), and a panel of 43 periodontal pathogens (see Figure 1 legend) and related bacteria were included as positive and negative controls, respectively. After hybridization, X-ray films were exposed for 2 to 30 hours. After stripping, all membranes were re-used for further experiments.
Figure 1. Dot blot hybridizations of identical membranes with EUB 338 (a) and the species-specific probe FIAL (b). PCR-amplified products from F. alocis (field A1) and its closest cultured relative F. villosus (A2) served as positive and negative controls, respectively. Additionally, products from the following bacteria were applied as negative controls: Centipeda periodontii (DSM 2778) (A3), Selenomonas noxia (DSM 19578) (A4), Selenomonas ruminantium (DSM 2150) (A5), Selenomonas lacticifex (DSM 20757) (A6), Selenomonas sputigena (DSM 20758) (A7), Eggerthella lenta (ATCC 25559) (A8), Peptostreptococcus anaerobius (ATCC 27337) (A9), and Actinomyces viscosus (ATCC 15987) (B1), Streptococcus intermedius (ATCC 27335) (B2), Streptococcus mutans (ATCC 35668) (B3), Neisseria lactamica (ATCC 23970) (B4), Flavobacterium odoratum (ATCC 4651) (B5), Fusobacterium necrophorum (NCTC 25286) (B6), Fusobacterium periodonticum (CCUG 14345) (B7), Fusobacterium simiae (CCUG 16798) (B8), F. nucleatum (ATCC 25586) (B9), Klebsiella pneumoniae (ATCC 23357) (C1), Veillonella dispar (ATCC 17748) (C2), Veillonella parvula (ATCC 10790) (C3), Kingella kingae (ATCC 23330) (C4), Eikenella corrodens (CCUG 2138) (C5), Bacteroides fragilis (ATCC 25285) (C6), Bacteroides gracilis (ATCC 33236) (C7), Campylobacter concisus (ATCC 33236) (C8), Campylobacter rectus (ATCC 33238) (C9), Capnocytophaga gingivalis (ATCC 33624) (D1), Capnocytophaga sputigena (ATCC 33612) (D2), Capnocytophaga ochracea (ATCC 27872) (D3), Prevotella buccalis (ATCC 33690) (D4), Prevotella oralis (MCCM 00684) (D5), Prevotella nigrescens (NCTC 9336) (D6), Porphyromonas asaccharolytica (ATCC 25260) (D7), P. intermedia (ATCC 25611) (D8), P. gingivalis (ATCC 33277) (D9), Haemophilus paraphrophilus (ATCC 29241) (E1), Haemophilus aphrophilus (NCTC 55906) (E2), Haemophilus influenzae (clinical isolate) (E3), Haemophilus influenzae (ATCC 33391) (E4), Pasteurella haemolytica (ATCC 33396) (E5), Leptotrichia buccalis (MCCM 00448) (E6), A. actinomycetemcomitans (MCCM 02638) (E7), A. actinomycetemcomitans (ATCC 33384) (E8) and A. actinomycetemcomitans (ATCC 43718) (E9). In columns 10-17 and in lanes F to J of columns 1-9 PCR products from patient samples of the different diseased groups and the periodontitis resistant (PR) group were applied. (a): Signals in all fields prove successful PCR-amplification. (b): Absence of signals in all bacterial controls along with strong signal in field A1 proves specificity of the experiments. Prevalences of F. alocis in all diseased collectives exceed the prevalence in the PR group.
Statistical analysis
Statistical evaluation of the dot blot hybridization results was performed using the exact chi-square test. The prevalence of F. alocis in different patient groups was compared. Moreover, the presence of F. alocis in relation to the PPD was analysed. P values below 0.05 were considered statistically significant.
Clinical samples for FISH
A carrier system designed to collect biofilms grown in vivo in periodontal pockets was used for sampling [31]. Ethics approval for subgingival sample collection was given by the Ethical Committee at Charité - Universitätsmedizin Berlin. Expanded polytetrafluoroethylene (ePTFE) membranes were placed in periodontal pockets of GAP patients for 7 to 14 days and colonized by the subgingival bacterial flora. Strips of ePTFE measuring 3 mm in width were wrapped around and attached to rigid plastic tips (Plast-O-Probe; Maillefer, Ballaigues, Switzerland), which permitted the insertion of these strips down to the bottom of the pocket, therefore allowing the complete extension of the membrane over the entire probing depth. One side of the double bent strip faced the soft tissue and the other side, slightly longer, faced the root surface. This longer cervical end was fixed to the tooth with cyanoacrylic glue (Tesa, Beiersdorf, Hamburg, Germany) to stabilize the position of the carrier. After removal, carriers were fixed for at least 3 h with 3.7% (v/v) formaldehyde in phosphate-buffered saline (pH 7.4) and embedded in cold polymerizing resin (Technovit 8100, Kulzer, Wehrheim, Germany) as reported previously [38]. Sectioning into slices of 2-3 μm was performed as previously published [39]. A total of 28 carriers from 11 GAP patients seeking treatment at the Charité - Universitätsmedizin Berlin were examined. These patients met the same inclusion criteria as the GAP patients selected for dot blot hybridization and likewise signed informed consent forms. See Table 2 for patient demographics. Additionally, a gingival biopsy of a GAP patient obtained during periodontal surgery was processed in the same manner and included in the FISH experiments.
FISH experiments were performed as described previously [40] apart from using Vectashield containing DAPI (4,6-Diamidino-2-Phenylindoldihydrochlorid) (Vector Laboratories, Orton Southgate, UK) as mounting medium. The probes were synthesized commercially (biomers.net, Ulm, Germany). EUB 338 was 5' end-labelled with fluorochrome Cy5 (indodicarbocyanine) while FIAL was 5' end-labelled with fluorochrome Cy3 (indocarbocyanine). Differential labelling allowed simultaneous hybridization with both probes.
Optimization of probe FIAL for FISH
The stringency of FIAL was adjusted by incubating fixed cells of F. alocis and its closest cultured relative, F. villosus with different hybridization mixes. The formamide concentrations covered a range from 0% (v/v) to 75% (v/v), rising in steps of 5% (v/v). At each level of formamide, a series of images of each bacterial species was taken with a fixed exposure time. The software daime [41] was used to measure the light intensities emitted by both species for each concentration of formamide. While the signal intensity of F. villosus did not reach 50 Relative fluorescence Units (RU) at any level of formamide due to unspecific binding of the probe, the intensity of F. alocis remained constantly above 150 RU using formamide concentrations of up to 20% (v/v) (see Additional file 1). In addition, fixed cells of 16 different bacterial species, most of them periodontal pathogens, were incubated with FIAL at 20% (v/v) formamide as negative controls, namely F. nucleatum (ATCC 25586), Eikenella corrodens (CCUG 2138), Kingella kingae (ATCC 23330), Veillonella parvula (ATCC 10790), Veillonella dispar (ATCC 17748), P. gingivalis (ATCC 33277), A. actinomycetemcomitans (ATCC 33384), Pasteurella haemolytica (ATCC 33396), T. forsythia (ATCC 43037), Haemophilus aphrophilus (NCTC 55906) P. intermedia (ATCC 25611), Campylobacter rectus (ATCC 33238), Capnocytophaga sputigena (ATCC 33612), Capnocytophaga gingivalis (ATCC 33624), Eggerthella lenta (ATCC 25559), and Peptostreptococcus anaerobius (ATCC 27337). As none of the controls were detected by FIAL, all further experiments were performed with 20% (v/v) of formamide, including F. alocis as positive and F. villosus as negative control.
Additional file 1. Optimization of probe FIAL for FISH using the program daime. FISH was performed incubating fixed cells of F. alocis and F. villosus with different hybridization mixes. Signal intensities (Relative fluorescent Units, RU) emitted by F. alocis and F. villosus at different formamide concentrations were calculated from images taken with a fixed exposure time. Due to unspecific binding of FIAL, the light emission of F. villosus cells remained below 50 RU at every level of formamide. The signal emitted by F. alocis cells was considered sufficient using formamide concentrations of up to 20% (v/v).
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Epifluorescence microscopy
After hybridization, carrier and biopsy sections were analysed using an epifluorescence microscope (AxioPlan II, Zeiss, Jena, Germany) equipped with a 100 W high pressure mercury lamp (HBO 103W/2, Osram, Munich, Germany) and 10×, 40× and 100× objectives. DAPI, Cy3 and Cy5 signals were analysed by narrow band filter sets HQ F31-000, HQ F41-007 and HQ F41-008, respectively (AHF Analysentechnik, Tübingen, Germany). Image acquisition was performed with an AxioCam MRm (Zeiss) making use of the AxioVision 4.4 software.
Dot blot hybridization
When carried out with the probe EUB 338 (specific for most bacteria), dot blot hybridization experiments indicated the presence of bacteria in all 490 patient samples as well as in the positive (F. alocis) and negative controls (see Figure 1 legend) and thus confirmed successful PCR amplification (Figure 1a). The Filifactor alocis-specific probe FIAL clearly detected F. alocis, while neither the closest phylogenetic neighbour F. villosus nor any of the organisms in the panel of oral bacteria (see Figure 1 legend) yielded a signal, thus indicating specific hybridization conditions (Figure 1b).
Taking all the collected samples into consideration, F. alocis could be identified in 77.8% of the 330 samples from 72 GAP patients, 76.7% of the 78 samples from 30 CP patients and 15.8% of the 82 samples from 19 PR patients (Table 2; Figure 2a). The prevalence of the organism was highest in the Oslo CP collective (87.5%), followed by the Basel GAP collective (80.0%), and the Dresden GAP collective (77.8%) (data not shown). As the number of samples per patient varied between the different collectives, statistical evaluation focused on the deepest pocket of each patient. Prevalence rates were 68.1% for the GAP group, 66.7% for the CP group and 5.3% for the PR group. While detection frequencies did not differ significantly between GAP and CP patients, both diseased groups harboured F. alocis significantly more often than the PR group (p < 0.001) (Figure 2b).
Figure 2. Prevalence of F. alocis. (a): Prevalence of F. alocis in all of the samples collected from GAP patients, CP patients and PR subjects as determined by dot blot hybridization using oligonucleotide probes. (b): Prevalence of F. alocis (F. a.), P. gingivalis (P. g.), P. intermedia (P. i.), A. actinomycetemcomitans (A. a.), T. denticola (T. d.), T. forsythia (T. f.), and F. nucleatum (F. n.) in the deepest pocket of each patient. Asterisks (*) indicate statistically significant differences (p < 0.05) between the GAP and PR groups. Crosses (†) indicate statistically significant differences (p < 0.05) between the CP and PR groups. (c): Percentage of samples positive for F. alocis at probing pocket depths 4-6 mm and 7-9 mm. Statistical analysis was limited to one pocket per patient and depth group. Asterisks (*) indicate statistically significant differences (p < 0.05) between the GAP and PR groups. Crosses (†) indicate statistically significant differences (p < 0.05) between the CP and PR groups.
The signal intensity of the FIAL-positive patient samples varied between the three groups, suggesting a higher number of Filifactor in GAP and CP pockets than in PR pockets tested positive for the organism. Nonetheless, as hybridizations were carried out on PCR-amplified bacterial DNA, no further analysis of signal intensities was performed.
Detection frequencies of P. gingivalis, P. intermedia, A. actinomycetemcomitans, T. denticola, T. forsythia, and F. nucleatum in the three patient groups are displayed in Figure 2b.
To investigate the prevalence of F. alocis in relation to the PPD, the donor sites were divided into four groups (I: 1-3 mm, II: 4-6 mm, III: 7-9 mm, IV: > 9 mm). As there is a certain degree of interdependency between pockets belonging to the same patient, statistical analysis was limited to one pocket per patient and probing depth group. Although a slightly higher percentage of group III pockets than group II pockets was positive for Filifactor in both the GAP and the CP patients, these differences were not statistically significant. Similarly, analysis revealed no statistically significant differences in the prevalence of the organism in GAP patients compared to CP patients in both pockets of 4-6 mm and pockets of 7-9 mm. In contrast, the prevalence of F. alocis in pockets of 4-6 mm differed significantly between both PR and GAP patients (p < 0.001) and PR and CP patients (p < 0.001) (Figure 2c). Insufficient numbers or complete absence of pockets of 1-3 mm in GAP and CP patients, pockets of 7-9 mm in PR patients and pockets deeper than 9 mm in CP and PR patients did not permit further statistical analysis.
F. alocis was reliably detected by both the species-specific probe FIAL and the eubacterial probe EUB 338. The negative control F. villosus was not targeted by FIAL but only by EUB 338, thus confirming specific hybridization conditions (Figure 3). In all of the periodontal ePTFE carriers from GAP patients as well as in the gingival biopsy gained during periodontal surgery, the bacterial biofilms could be visualized by FISH with EUB 338 and displayed characteristic features like densely-packed mushroom-like protuberances and signal-free channels [42]. F. alocis could be detected in 9 out of 11 carrier patients (in 17 out of 28 carriers) as well as in the examined gingival biopsy.
Figure 3. Specificity of FISH experiments. Hybridization of fixed cells of F. alocis (a and c) and F. villosus (b and d) was performed with probes EUB 338-Cy5 (magenta, a and b) and FIAL-Cy3 (bright orange, c and d). (a and c): Identical microscopic fields show detection of F. alocis by both EUB 338 (a) and FIAL (c) whereas detection of F. villosus by EUB 338 only (b) and not FIAL (d) proves specificity of the FISH experiment.
In the carrier-grown biofilms, the organism could be visualized in those areas that had grown in the depth of the pocket, but rarely in areas corresponding to the cervical part of the pocket and rarely on the very tip of the carrier. In most cases, Filifactor colonized the side of the carrier facing the soft tissue (Figure 4c) and could only be found in few numbers or not at all on the carrier side facing the root (Figure 4b). Many parts of the biofilm showed F. alocis as a short rod of 1-2 μm length, whereas at some sites the organism appeared longer, extending to 7-8 μm (Figure 5a). While in some areas Filifactor cells seemed to be scattered within the biofilm without any recognizable pattern, numerous sites clearly showed a higher degree of organisation. Repeatedly, F. alocis could be found in densely packed groups (Figure 4c), arranged in concentrical structures (Figure 5d) or grouped in "test-tube brush" formations [43] around signal free channels (Figure 5c). Figure 5b shows the radial orientation of F. alocis towards the surface of a mushroom-like protuberance of the biofilm.
Figure 4. Carrier grown biofilm visualized by FISH. Hybridization was performed with the probes EUB 338-Cy5 (magenta) and FIAL-Cy3 (bright orange) along with DAPI staining (blue) on a carrier after 7 days of attachment to the mesial aspect of tooth 16 in a GAP patient. (a): Collage of several microscopic fields in low magnification. The overlay of Cy3, Cy5 and DAPI filter sets shows the bacterial biofilm that grew in the depth of the pocket. EUB 338 visualizes large parts of the bacterial community, while FIAL detects only F. alocis. DAPI stains both host cell nuclei and bacteria. The carrier tip (1) and the carrier side facing the tooth (2) show little or no presence of F. alocis. The bright orange signal on the carrier side facing the pocket epithelium (3) reveals a strong presence of Filifactor in the part of the biofilm indicated by the arrow. Arrowheads on the tooth side (2) point to artifacts caused by upfolding of the embedded carriers. (b and c): Higher magnifications of the inserts. (b) shows the biofilm on the tooth side of the carrier without F. alocis among the bacteria. (c) shows F. alocis in densely packed groups among the organisms on the epithelium side and host cell nuclei (blue).
Figure 5. Formations of F. alocis in carrier-borne biofilms. FISH on different carriers with GAP biofilms using the probes EUB 338-Cy5 (magenta) and FIAL-Cy3 (bright orange) along with DAPI staining (blue). EUB 338 detects the whole bacterial population while FIAL visualizes F. alocis specifically. DAPI stains both bacteria and host cell nuclei. High magnifications show F. alocis in different areas of the biofilms. (a): Overlay of Cy3, Cy5 and DAPI filter sets. In some regions of the biofilm Filifactor rods can reach a considerable length. (b and c): Overlay of Cy3 and DAPI filter sets. (b) shows the radial orientation of F. alocis and other organisms on the surface of a mushroom-like protuberance of the biofilm. (c) shows F. alocis forming test-tube-brush-like structures around a signal-free channel. (d): Overlay of Cy3 and Cy5 filter sets. F. alocis and fusiform bacteria form concentrical structures.
Similar formations that indicate ultrastructural organisation of the biofilm could be observed in the gingival biopsy. In several areas, F. alocis formed branch-like structures within the affected tissue (Figure 6a) or palisades around large rodshaped bacteria (Figure 6b). Again, Filifactor was observed among the organisms in concentric bacterial aggregations (Figure 6c).
Figure 6. Formations of F. alocis in periodontal tissue. FISH on a biopsy gained during periodontal surgery using the probes EUB 338-Cy5 (magenta) and FIAL-Cy3 (bright orange) along with DAPI staining (blue). EUB 338 visualizes the entire bacterial community, while FIAL detects only F. alocis. DAPI stains both host cell nuclei and bacteria. High magnifications depict F. alocis in different parts of the biopsy. (a): F. alocis forms tree-like structures among coccoid and fusiform bacteria and autofluorescent erythrocytes. (b) shows F. alocis forming palisades with fusiform bacteria around large rodshaped eubacterial organisms. (c) shows F. alocis being part of concentrical bacterial aggregations resembling those detected in GAP carriers.
To our knowledge, the present study is the first to analyse the prevalence of F. alocis in samples from both GAP and CP patients, and subjects with apparent periodontitis resistance. The detection of the organism in 77.8% of the GAP patients and in 76.7% of those suffering from CP is convincing evidence that suggests an involvement of F. alocis in periodontal disease. Equally striking is the low prevalence of Filifactor in the PR group. All of these patients had reached the age of 65 years and were in good periodontal condition without the help of extensive therapeutic efforts. Even if a multitude of factors including oral hygiene and immune response contributed to their periodontal status, one would assume that frequent detection of an organism in the GAP and CP groups along with scarce detection in PR patients, as is the case for F. alocis, indicates pathogenic rather than commensal behaviour.
One can argue that deep periodontal pockets harbour increased numbers of bacteria and that any organism inevitably should be isolated more constantly from CP patients (mean pocket depth: 7.13 mm, 1.4 mm SD) and especially GAP patients (7.81 mm, 2.48 mm SD) than from PR patients (3.63 mm, 0.79 mm SD). However, dividing the entirety of the sampled sites into four groups according to the measured PPD (I: 1-3 mm, II: 4-6 mm, III: 7-9 mm, IV: > 9 mm), statistical analysis of pockets between 4 and 6 mm still reveals a significantly higher prevalence of F. alocis in both the GAP and the CP group compared to the PR group. In addition, the organism was not detected significantly more frequently in deeper pockets (7-9 mm) than in rather shallow pockets (4-6 mm) in both GAP and CP patients. Although a connection between PPD and bacterial load cannot be denied, these findings indicate that the influence of pocket depth does not invalidate the aforementioned results.
If one compares the prevalence rate of F. alocis to those of the widely accepted periodontal pathogens P. gingivalis, P. intermedia, A. actinomycetemcomitans, T. denticola, F. nucleatum, and T. forsythia (see Figure 2b), investigated in these very samples using identical methods, Filifactor is the third most prevalent for GAP and second most prevalent for CP patients and is thus at eye level with organisms that are considered key players in periodontal disease. At the same time, F. alocis shows the lowest prevalence in the PR group of all analysed organisms. Together with F. nucleatum, F. alocis is the only organism to show a significantly higher detection frequency in both GAP and CP patients compared to the PR group.
Using PCR-based identification methods may introduce bias, since structurally different organisms could exhibit different copy numbers of ribosomal genes and will generally respond differently to DNA isolation and the chosen set of broad range bacterial primers [44]. However, the relevance of F. alocis is supported by several other epidemiological studies conducted in the past years using DNA-based techniques. F. alocis was detected in GAP patients as well as in CP patients with prevalence rates varying between 45% [29] and 90% [28], depending on the methods employed. Some authors propose F. alocis as a marker organism for periodontal disease [28] and even for the shift from periodontal health to disease [19].
Our data strongly support the findings of these studies and motivated the attempt to visualize F. alocis within the periodontal biofilm of GAP patients using FISH. The organism could be detected in high numbers in the majority of the examined carriers. The percentage of positive patients approximately matches the dot blot results. Strikingly, several areas of the biofilm show F. alocis in densely packed groups (Figure 4c) or as a part of concentric bacterial agglomerations (Figure 5d) - formations that suggest a certain degree of organisation to the observer. Moreover, the organism could be visualized in structures that are considered characteristic architectural features of periodontal biofilms. F. alocis is among the bacteria in mushroom-like protuberances on the surface of the biofilm (Figure 5b) and it contributes, grouped around what might be diffusion or convection channels, to the formation of structures reminding of test-tube brushes (Figure 5c). The close colocalization of F. alocis with other periodontal pathogens suggests that Filifactor might be involved in coaggregation events that take place during the establishment and maturation of the biofilms and that are thought to play a crucial role in biofilm formation [45]. Moreover, the tight colocalization might indicate necessary symbiotic relationships that could help to explain the fastidiousness of Filifactor.
Just like group I treponemes [31], F. alocis predominantly colonizes the apical and middle third of the carriers and could only casually be detected in the cervical third. Most interestingly, the organism preferably settles on the side of the carrier facing the soft tissues and is thus in immediate contact to the host's immune defence. All these observations point to a causal involvement of F. alocis in the formation and maintenance of the analysed biofilms.
However, one might question whether these carrier-borne biofilms accurately model the unperturbed biofilms in periodontitis patients. Wecke et al. [31] compared the bacterial load after 3 and 6 days and showed that the biofilm mass covering the carriers increases with time. The presence of F. alocis on only one side of the membranes is further evidence that these samples are not simply fragments of biofilm torn out of the pocket during the removal of the carriers, but in fact newly grown biofilms that form while the carriers are in situ. Although FISH reveals structural elements specific to periodontal biofilms, one cannot deny that the introduction of the carrier into the periodontal pocket creates an artificial environment. The barrier between root surface and pocket epithelium might hamper access of the immune system to the bacteria on the tooth side, while only the biofilm growing on the soft tissue side actually faces the host. Moreover, these biofilms do not form on natural substrate but instead on ePTFE membranes. However, it seems likely that the substrate is of minor importance to the biofilm development. Wecke et al. [31] did not observe differences between biofilms grown on different carrier materials, and it is likely that the acquired pellicle, which covers both the root and the membrane, renders colonization conditions on a broad range of materials alike. This claim is supported by microscopic examination of the biopsy submitted to FISH. F. alocis could be visualized in high numbers and detected in arrangements similar to those seen in carrier-borne biofilms. Thus, a contribution of Filifactor to the structural organisation of 'naturally' grown biofilms seems highly probable.
The applied carrier system proves to be a valuable tool for the exploration of periodontal biofilms as it allows to investigate topographic relations within the pocket without invasive treatment. Subsequent FISH permits to analyse the distribution and colocalization of potential pathogens within the biofilm and can thus contribute to a better understanding of the complex host-microbe interactions that lead to periodontal destruction.
The prevalence of Filifactor alocis in both GAP and CP patients was found to be elevated as compared to PR control. F. alocis thus seems to be a powerful diagnostic marker organism for periodontal disease. FISH revealed the involvement of F. alocis in numerous structural arrangements that point to its potential role as one of the architects of structural organisation within periodontal biofilms. Filifactor alocis should be considered an important periodontal pathogen and warrants further research.
Authors' contributions
SS assisted in designing the study, designed and optimized the oligonucleotide probe FIAL, participated in patient sample preparation, carried out dot blot and fluorescence in situ hybridizations, evaluated the data and drafted the manuscript. BR collected patient samples for dot blot hybridization, performed statistical analysis and helped to draft the manuscript. ALG provided the initial idea and participated in designing the study. AP participated in patient sample preparation, dot blot hybridizations and FISH probe optimization. JH provided the gingival biopsy, participated in patient sample preparation and FISH experiments. MB assisted in probe design and dot blot hybridizations. AF developed the periodontal carriers and collected subgingival biofilms for FISH experiments. UBG was involved in designing the study and supervised the work. AM designed and supervised the study and the experiments, analysed the data and participated in writing. All authors read and approved the final manuscript.
We thank Eva Kulik, University of Basel, and Eivind Strøm, University of Oslo, for providing clinical samples, Cindy Hefenbrock and Marie Knüver for excellent technical assistance, Derek Ramsey for proof reading, and Dr. Wolf-Ulrich Klotz for his support.
This work was supported by the Sonnenfeld-Stiftung, Berlin, Germany, and by a Rahel-Hirsch grant from Charité - Universitätsmedizin to AM.
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Microbiol Immunol 2000, 44:643-652. PubMed Abstract
Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, Sahasrabudhe A, Dewhirst FE: Bacterial diversity in human subgingival plaque.
J Bacteriol 2001, 183:3770-3783. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Paster BJ, Falkler WA Jr, Enwonwu CO, Idigbe EO, Savage KO, Levanos VA, Tamer MA, Ericson RL, Lau CN, Dewhirst FE: Prevalent bacterial species and novel phylotypes in advanced noma lesions.
J Clin Microbiol 2002, 40:2187-2191. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE: Defining the normal bacterial flora of the oral cavity.
J Clin Microbiol 2005, 43:5721-5732. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL, Boches SK, Dewhirst FE, Griffen AL: Molecular analysis of bacterial species associated with childhood caries.
J Clin Microbiol 2002, 40:1001-1009. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kumar PS, Griffen AL, Moeschberger ML, Leys EJ: Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis.
J Clin Microbiol 2005, 43:3944-3955. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kumar PS, Leys EJ, Bryk JM, Martinez FJ, Moeschberger ML, Griffen AL: Changes in periodontal health status are associated with bacterial community shifts as assessed by quantitative 16S cloning and sequencing.
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Riep B, Edesi-Neuss L, Claessen F, Skarabis H, Ehmke B, Flemmig TF, Bernimoulin JP, Gobel UB, Moter A: Are putative periodontal pathogens reliable diagnostic markers?
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Donlan RM, Costerton JW: Biofilms: survival mechanisms of clinically relevant microorganisms.
Clin Microbiol Rev 2002, 15:167-193. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Cato EP, Moore LVH, Moore WEC: Fusobacterium alocis sp. nov. and Fusobacterium sulci sp. nov. from the human gingival sulcus.
Jalava J, Eerola E: Phylogenetic analysis of Fusobacterium alocis and Fusobacterium sulci based on 16S rRNA gene sequences: proposal of Filifactor alocis (Cato, Moore and Moore) comb. nov. and Eubacterium sulci (Cato, Moore and Moore) comb. nov.
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Maiden MF, Tanner A, Macuch PJ: Rapid characterization of periodontal bacterial isolates by using fluorogenic substrate tests.
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Kumar PS, Griffen AL, Barton JA, Paster BJ, Moeschberger ML, Leys EJ: New bacterial species associated with chronic periodontitis.
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Dahlen G, Leonhardt A: A new checkerboard panel for testing bacterial markers in periodontal disease.
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Hutter G, Schlagenhauf U, Valenza G, Horn M, Burgemeister S, Claus H, Vogel U: Molecular analysis of bacteria in periodontitis: evaluation of clone libraries, novel phylotypes and putative pathogens.
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Siqueira JF Jr, Rocas IN: Detection of Filifactor alocis in endodontic infections associated with different forms of periradicular diseases.
Oral Microbiol Immunol 2003, 18:263-265. PubMed Abstract | Publisher Full Text
Wecke J, Kersten T, Madela K, Moter A, Gobel UB, Friedmann A, Bernimoulin J: A novel technique for monitoring the development of bacterial biofilms in human periodontal pockets.
FEMS Microbiol Lett 2000, 191:95-101. PubMed Abstract | Publisher Full Text
Maidak BL, Cole JR, Lilburn TG, Parker CT Jr, Saxman PR, Farris RJ, Garrity GM, Olsen GJ, Schmidt TM, Tiedje JM: The RDP-II (Ribosomal Database Project).
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Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA: Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.
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Loy A, Horn M, Wagner M: probeBase: an online resource for rRNA-targeted oligonucleotide probes.
Nucleic Acids Res 2003, 31:514-516. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Armitage GC: Development of a classification system for periodontal diseases and conditions.
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Syed SA, Loesche WJ: Survival of human dental plaque flora in various transport media.
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Moter A, Hoenig C, Choi BK, Riep B, Gobel UB: Molecular epidemiology of oral treponemes associated with periodontal disease.
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Moter A, Leist G, Rudolph R, Schrank K, Choi BK, Wagner M, Gobel UB: Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions.
Microbiology 1998, 144(Pt 9):2459-2467. PubMed Abstract | Publisher Full Text
Schlafer S, Nordhoff M, Wyss C, Strub S, Hubner J, Gescher DM, Petrich A, Gobel UB, Moter A: Involvement of Guggenheimella bovis in digital dermatitis lesions of dairy cows.
Vet Microbiol 2008, 128:118-125. PubMed Abstract | Publisher Full Text
Sunde PT, Olsen I, Gobel UB, Theegarten D, Winter S, Debelian GJ, Tronstad L, Moter A: Fluorescence in situ hybridization (FISH) for direct visualization of bacteria in periapical lesions of asymptomatic root-filled teeth.
Microbiology 2003, 149:1095-1102. PubMed Abstract | Publisher Full Text
Daims H, Lucker S, Wagner M: daime, a novel image analysis program for microbial ecology and biofilm research.
Environ Microbiol 2006, 8:200-213. PubMed Abstract | Publisher Full Text
ten Cate JM: Biofilms, a new approach to the microbiology of dental plaque.
Odontology 2006, 94:1-9. PubMed Abstract | Publisher Full Text
Listgarten MA: Structure of the microbial flora associated with periodontal health and disease in man. A light and electron microscopic study.
J Periodontol 1976, 47:1-18. PubMed Abstract
Marchesi JR, Sato T, Weightman AJ, Martin TA, Fry JC, Hiom SJ, Dymock D, Wade WG: Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA.
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Rickard AH, Gilbert P, High NJ, Kolenbrander PE, Handley PS: Bacterial coaggregation: an integral process in the development of multi-species biofilms.
Trends Microbiol 2003, 11:94-100. PubMed Abstract | Publisher Full Text


text:
In Agatha Christie - Dead Mans Folly you're invited to a Murder Hunt. Join famed detective Hercule Poirot and guests as you discover that this charade could be the perfect disguise for a devious scheme. Find hidden clues, decode messages and solve puzzles to unravel a story of deception, secrets and a fatal end.
Cock's Revenge is a shooting game. The cock's have been bothered for a long time by cats. They were tired of this bothering so they decided to get revenge. Try to kill all cats so the cock's can live peacefully in this free online shooting game. Click the left mouse button and drag to activate the power.
Pics Print - The Digital Photo Printing Solution for Windows
Hen Coops Game is a memory game in which a funny cock will show you an egg under a hen and then it will shuffle the three hens from one side to the other. When he finishes, you must say under which hen the egg is. You will have a couple of seconds to select a hen, and the faster you provide the right answer, the higher your score will be.
Add2it PostIt Free is a cool tool which can help you to write notes about anything you want. Is easy to install and it can be manipulated by everyone. You can select the place where you want to keep the note, the language you want to use. It is very helpful for students, when they have to wrote projects, business mans, old mans etc.
Picture Resize Genius is a useful and feature-rich tool to work with images. Though its primary function is image resizing, it also lets you work with image files in several other ways as it includes an image renamer, converter, editor, among other features.
Webpage Builder For Digital Pics lets you easily generate HTML Pages with your digital pictures with just a few mouse clicks. This product is faster and easier than any product we have seen for generating web pages to display digital pictures. It is by far faster and easier than any generic html editors for creating basic web pages.
The PICS editor has been developed to simplify the process of generating a PICS declaration for a mobile that can be used to generate an applicability list based on a specific version of 51.010-2.
A user-friendly windows application that it is based on a database of tables extracted from the 3GPP 51.010-2 release documents.
Flash and Pics Control is a free add-on for Internet Explorer. This full-featured extension works exclusively on Internet Explorer and IE-based browsers. With this add-on integrated in your Internet Explorer you will be able to handle many aspects of your Web browsing such us enabling/disabling Flash animations, images, cookies, ActiveX controls, Java Scripts and Java Applets.
Import Pics for PowerPoint allows you to import all readable images in a folder with a single mouse click. All images are automatically sized and centered on a slide with one image per slide. Also the program enables you to move, resize and crop all images in a series.
Tux Paint 0.9.19 is a drawing program for children ages 3 to 12.
When you start the program, you will be presented with a white space, in which you can draw using the tools on your left. When you choose a tool, the different types of this tool that are available will appear on your right. You can pick the color that the chosen tool will use at the bottom of the screen.
Flash & Pics Control is an Internet Explorer add-on that allows you to enable/disable pictures and flash animation. The program adds new item to the Tools menu of Internet Explorer to quickly enable/disable pictures or flash. Having flash and pictures disabled lets you reduce the amount of incoming traffic and, thus, save your money.
You can share a screenshot with your iMobsters clan and get instant reactions from your group. Not your thing? How about sending pics of your new sneakers and getting those oohhhs and aahhs from your B-Boys group. Groups accommodate up to 2,000 members. Browse our listings and join some that you like the look of or create your own today.
Shape Collage is a free automatic photo collage maker that lets you create picture collages in a variety of shapes in less than a minute with just a few mouse clicks.
Magic Jigsaw features a good number of classic jigsaw games, that guarantees fun for all your family. It includes more than ten beautiful photographs and the possibility of transforming your own pictures into a new jigsaw game. The full version of Magic Jigsaw is absolutely free.
Picture Merge Genius is a photo collage creator for Windows. It is an application that allows you to combine several pictures into one image that you can share with your family and friends or use to preserve memories in a special way. The application is simple enough and offers two ways of creating photo montages.
STOIK image editing program - easy image viewer and pics editor, video editor. Quick to learn and easy to use, STOIK Imagic makes managing your photos and videos fun. Bundling a blazing fast image viewer with convenient image organizer and comprehensive still and video editor, STOIK Imagic has everything to keep, share and perfect your memories.
Ashampoo Photo Converter is an image conversion tool for Windows. The application allows you to convert from and to all the popular image file types, including BMP, GIF, JPG, TIF, PNG, PDF, supporting a total of 18 formats. As a plus, the app includes a photo editor to enhance and apply effects to your pics.

subset: commoncrawl


text:
Renewing Our Commitment to Catholic Elementary and Secondary Schools in the Third Millennium
Young people are a valued treasure and the future leaders of our Church. It is the responsibility of the entire Catholic community--bishops, priests, deacons, religious, and laity--to continue to strive towards the goal of making our Catholic elementary and secondary schools available, accessible, and affordable to all Catholic parents and their children, including those who are poor and middle class. All Catholics must join together in efforts to ensure that Catholic schools have administrators and teachers who are prepared to provide an exceptional educational experience for young people--one that is both truly Catholic and of the highest academic quality.
In 1990, the Catholic bishops of the United States issued the statement In Support of Catholic Elementary and Secondary Schools. In it we affirmed our strong conviction that Catholic elementary and secondary schools are of great value to our Church and our nation; and that, in our role as chief teachers, we are each responsible for the total educational ministry of the local Church. We affirmed that "the entire ecclesial community ... is called to value ever more deeply the importance of this task and mission, and to continue to give it full and enthusiastic support." These Catholic schools afford the fullest and best opportunity to realize the fourfold purpose of Christian education, namely to provide an atmosphere in which the Gospel message is proclaimed, community in Christ is experienced, service to our sisters and brothers is the norm, and thanksgiving and worship of our God is cultivated (p. 2).
In that statement we pointed to the great value and the many successes of Catholic schools and the numerous challenges that they face. We unequivocally committed ourselves and the whole Catholic community to the following set of goals:
* Catholic schools will continue to provide a Gospel-based education of the highest quality.
* Catholic schools will be available, accessible, and affordable.
* The bishops will launch initiatives in both the private and public sectors to secure financial assistance for parents, the primary educators of their children, so that they can better exercise their right to choose the best schools for their children.
* Catholic schools will be staffed by highly qualified administrators and teachers who would receive just wages and benefits, as we expressed in our pastoral letter Economic Justice for All.
Much has changed in our Church and our nation in the ensuing years. Catholic schools continue to be valued and successful; but they still encounter numerous challenges. The bishops have addressed many of the goals that we set in 1990, but much is still left to be done. Therefore, we believe that the time has come to revisit and reaffirm our commitment to Catholic elementary and secondary schools as invaluable instruments in proclaiming the Good News from one generation to the next. This catechesis is a privileged way of "initiating the hearers into the fullness of Christian life" and is "intimately bound up with the whole of the Church's life" (Catechism of the Catholic Church, nos. 5, 7). We believe that now is the appropriate time to renew our challenge to the entire Catholic community to join in this critical endeavor. We are convinced that Catholic schools continue to be "the most effective means available to the Church for the education of children and young people" who are the future of the Church (To Teach as Jesus Did, no. 118).
Young people of the third millennium must be a source of energy and leadership in our Church and our nation. Therefore, we must provide young people with an academically rigorous and doctrinally sound program of education and faith formation designed to strengthen their union with Christ and his Church. Catholic schools collaborate with parents and guardians in raising and forming their children as families struggle with the changing and challenging cultural and moral contexts in which they find themselves. …


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How can I delete all email in my Inbox?
If you want to delete all email in your Inbox, telnet to traveler.ilstu.edu to easily delete all your email at once.
Before You Begin
The most common reason to delete all email is that you want to "start over" with a fresh Inbox, free of old email and spam. If you have hundreds or thousands of emails in your Inbox, RedbirdMail does not allow you to easily delete all email in your Inbox.
The easiest way to delete all your email is to use PINE on traveler.ilstu.edu. Using a telnet client, you can connect to Traveler, a UNIX server, and perform the mass delete. Because Traveler is UNIX-based, all commands are issued with the keyboard only (mouse interaction is not available).
Telnet to traveler.ilstu.edu
First, you need to telnet to traveler.ilstu.edu. This requires a telnet client. Mac OS X has a built-in telnet client. You must install telnet on Windows Vista/7 before you can use it.
Windows Vista/7
Before you can use Telnet in Windows Vista/7, you must install it. Refer to 1290: Installing Telnet on Windows Vista/7/8.
To run telnet on Windows Vista/7, do the following:
- Click the Start button.
- Type
telnet traveler.ilstu.eduin the search box where it says, Start Search, and press Enter.
Mac OS X
To run telnet on Mac OS X, do the following:
- Open your hard drive.
- Double-click Applications > Utilities > Terminal.
- In the upper left-hand corner click Terminal > Preferences...
- Select xterm from the drop-down menu next to Declare terminal as: under the Advanced section of the Settings tab.
- Quit the Terminal program and re-open it.
- Type
telnet traveler.ilstu.eduand press Enter.
Log In and Open Your Inbox
Once you are connected to traveler.ilstu.edu, you will be prompted to log in. Follow the directions below.
- Type your ULID and press Enter. (Do not use the Backspace key on Windows; use the Delete key instead.)
- Type your password and press Enter. The flashing cursor will not move as you type your password; this is normal.
- PINE, a UNIX mail client, will open, and you will be prompted to log in again.
- Your ULID will already be present where it says, ENTER LOGIN NAME. Press Enter.
- Type your password and press Enter. Like before, the flashing cursor will not move as you type your password.
- The FOLDER LIST line is already selected. Press Enter.
- The Mail on mail.ilstu.edu:143/IMAP line is already selected. Press Enter.
- The Inbox is already selected. Press Enter.
- Your Inbox will open and display your email. You may use the up and down arrow keys on your keyboard to scroll up and down. Press Enter when a message is selected to view it.
Delete All Email from Your Inbox
Once you've connected to traveler.ilstu.edu and opened your Inbox, you can delete all your email. Follow the instructions below.
- Commands are displayed at the bottom of the telnet window.
- Press O to see Other Commands.
- Press O again to see more commands.
- Press ; (semi-colon) to Select emails in your Inbox.
- Press A to Select All emails in your Inbox.
- Press A again to Apply an operation to the selected email messages.
- Press D to mark all email for deletion. All of your emails should now be flagged with an X and a D on your telnet screen.
- When you are ready to permanently delete all messages you've marked for deletion (i.e. all the email in your Inbox), press X to Expunge them.
- It may take a few minutes to expunge all your deleted emails, depending on how many you have to delete. The telnet window may be unresponsive while the server performs the operation.
- After a few moments, you will see a message that indicates the number of emails deleted.
After deleting all emails from your Inbox, you may close the telnet window.


text:
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Abdul Haseeb Basit is AHB Photography. A freelance photographer in London and covering the UK, specialising in headshots, commercial photography and stills photography for films and music.
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text:
Why that chin's a pointer to evilComment on this story
London - From Captain Hook to Dick Dastardly and Cruella De Vil, their faces portray evil.
Now scientists have worked out what scares us most about a cartoon baddie – their pointy chin.
Warwick University researchers found a downward pointing triangle can make us feel threatened in the same way as an angry face. They believe it is no coincidence the ultimate baddies often have a sinister sharp chin and eyebrows sloping towards a downward point in the middle.
Thirty volunteers were asked to rate hundreds of pictures of faces appearing on a screen as good or bad, kind or cruel, pleasant or unpleasant.
Each picture was flanked by a large picture of a triangle on each side, which, without the participants' knowledge, worked as a "distractor".
When a face was surrounded by downward pointing triangles, people were slower to identify it as positive and quicker to identify it as negative.
By contrast, left and right pointing triangles had no effect at all on the results, and upwards pointing triangles had a slight positive effect.
It is known from previous studies that people pick negative faces – such as sad or angry expressions – out of a crowd more quickly than positive ones.
Dr Derrick Watson, of the psychology department, said simple geometric shapes have an emotional effect on us, as they "carry the features present within negative or positive faces". He added: "Our study shows downward pointing triangles in particular convey negative emotions and we can pick up on them quickly and perceive them as a threat."
This is the first time this link has been made, although previous research has shown angular lines are seen as negative.
Co-author Dr Elisabeth Blagrove said: "If we look at cartoon characters the classic baddie will often be drawn with the evil eyebrows that come to a downward point in the middle.
"This could go some way to explain why we associate the downward pointing triangle with negative faces."
The study is published in the journal Emotion, published by the American Psychological Association. - Daily Mail
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text:
Golden Eagles quarterback Austin Davis
- Filed Under
Only one college football game will be played today, and that's the Hawaii Bowl that pits the Southern Miss Golden Eagles against the Nevada Wolf Pack.
Southern Miss (11-2) is the Conference USA champion; Nevada (7-5) is a member of the Western Athletic Conference, but will join the Mountain West Conference next season.
Both teams have been in Hawaii most of the week, practicing and enjoying the sights from Pearl Harbor to Waikiki Beach. ...


text:
Newport hooligan in court after Crawley Town fight
Posted: October 27, 2010|
FOOTBALL FIGHT: Newport County fan Michael Nixey
A HOOLIGAN who started crowd trouble at a Crawley Town game has been banned from all football matches for the next three years.
Newport County fan Michael Nixey started a fight with Reds fans before invading the pitch at the league clash on October 16.
The 41-year-old appeared before Crawley Magistrates' Court last Thursday, when he was fined and given the banning order.
Nixey, of Newport, pleaded guilty to a charge of disorderly conduct provoking unlawful violence and trespassing on the playing area at a football match.
The court heard he was part of a group of County fans who had managed to get into a home supporters' area during the game at the Broadfield Stadium, which the away team won 3-2.
When Newport scored, the group celebrated and a fight broke out.
Nixey was spotted on CCTV by police "exchanging punches with other men".
The Newport fans were moved to another area of the ground, with their own fans, but trouble flared again and Nixey ran on to the pitch.
Richard Lynn, prosecuting, said: "The defendant climbed over a barrier and went on the pitch where he was arrested and detained.
"In interview, speaking about the fight, he admitted words had been exchanged and accepted that he had thrown a few punches, but said he couldn't remember if he had connected with anyone.
"He said he had been hit in return and left swollen above his right eyebrow.
"His team had scored and he was celebrating."
Tanya Price, defending, said Nixey, who works in Wales as a gardener, was "extremely embarrassed" at his "stupidity" on the day.
Miss Price said: "Emotions were running high and clearly inappropriate behaviour ensued.
"He has a three-year-old and another child on the way. This is not the sort of behaviour he is accustomed to. This is extremely out of character."
Nixey's three-year banning order means he is banned from all football grounds in England and Wales.
He was also ordered to pay £200 costs and warned he may be forced to surrender his passport before international games to stop him leaving the country.


text:
Sleep Disorders in Women (cont.)
Web Links
National Sleep Foundation, Women and Sleep
National Heart, Lung, and Blood Institute, National Center on Sleep Disorders Research, Sleep, Sex Differences, and Women's Health
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Brain Basics: Understanding Sleep
American Academy of Sleep Medicine, Find a Sleep Center
Medically reviewed by Peter O'Connor, MD; American Board of Otolaryngology with subspecialty in Sleep Medicine
Medically Reviewed by a Doctor on 7/29/2014
Medical Author:
Gila Hertz, PhD, ABSM
Mary E Cataletto, MD
Medical Editor:
Anthony M Murro, MD
Medical Editor:
Mary L Windle, PharmD
Medical Editor:
Carmel Armon, MD, MHS, MSc
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Sleep Dysfunction in Women »
Women are twice as likely as men to have difficulties falling asleep or maintaining sleep, although before puberty no significant differences are apparent.


text:
Why I have no common sense
This story is an example of why I lack any common sense whatsoever.
Yesterday at work a guy came in and said he was delivering phone books. Okay, no problem, bring them in. Oh wait—they are heavy. Okay, no problem, I'll help you. Oh wait—there's a lot of them. Okay, I'll open up receiving.
Now at this point I'll admit I thought something was unusual. But I didn't question anything and proceeded to help these two guys unload 650 telephone books into our receiving area. With a staff of about 12, we really don't need those many books. It was only after they had left and I went back to the front desk that I realized the order was probably meant to go to the college nearby and we got it by mistake.
After a quick call to the phone company we sorted out the problem, and the books will be picked up today. I feel sorry for those two guys though, because after I help them load up the phone books, they're going to have to unload them a third time at the college.
This is just another example of how very intelligent people often lack basic common sense (I think it ties in the poor social skills thing too). If I had any common sense I would have stopped them and then asked someone why we were getting 650 books. But did I do that? Nooo. What an idiot!
It does make for a good story though.
The long night has ended.
Two good events have occurred in the past week to counterbalance this annoying cough that's plagued me. Firstly, my new computer arrived on the 21st! Secondly, I am now officially done with high school.
With university approaching, I wanted a new computer. My former one, a Toshiba Satellite A70, was … performance-challenged, to say it nicely. It was a nice computer in its own right, but clunky from the start and it did not improve. While it did what you asked, the battery life was poor, and it ran too hot. It would not make a good computer for commuting between university and home. Time to upgrade!
With Seth's assistance I went through the customization process on Dell's website and had soon ordered my own Inspiron 6400: Intel Core 2 Duo, 2 GB RAM, 160 GB hard drive. Oh, and Windows Vista. It was a tense two and a half weeks waiting for it to arrive. No one was home when the courier came, though, so I had to go pick it up at the depot. Luckily I've got a great boss who let me drop by the depot while I was working to pick up my computer before the depot closed.
So on Thursday night, my computer sat in a box on my bed. I opened it, and there it was. Shiny. It was up and running in a few minutes, although getting all of my programs installed took considerably longer. Unfortunately, I didn't pay very much attention to the Stargate series finale.
On Friday I transferred my personal files over from the old computer, and then everything was set up how I liked it. So far I am uber-impressed by the hardware. My battery life is around 4 hours. The fan is silent, but the computer doesn't burn my legs—it's a little hot, but all things considered, this is more than acceptable. The software, however, is annoying. Vista's interface is great, but its functions are less than robust. Just yesterday I had to boot into safe mode because in Vista Home Premium you cannot enable the Local Users & Groups snap-in for the management console. It doesn't let you.
Little does Microsoft know, this only motivates me to get kozier with Kubuntu.
And now I'm done school. Today was my data management exam, which was very easy. It was my last exam, and my last time in Westgate as a student. Next year I'll return as an annoying alumnus who bothers his former teachers.
To all my friends south of the border
Please, when you come to Canada, change your money. It isn't that hard, and it will save me some headaches.
Thunder Bay is close to the border, so we get frequent American visitors to the art gallery. For some reason, they believe it's fine to just hand us American money. Canada's just the 51st state anyway, right? I know that when I go to the States, I don't flash my Canadian cash all around the place. I trade my money in for your pallid green bills.
Our cash register is not a hi-tech computer with a flat panel display and a high speed Internet connection. It's a box with lights and a few buttons. The exchange rate is currently set to about 62 American cents for every Canadian dollar, and our boss has to change it manually. I honestly don't even know how to do the conversion on the machine (there's a button, but I'm never sure when to push it during the transaction…).
So please, take it from someone who has to deal with your cash. I'll be happier if you change it to Canadian money. Our bills are shiny and colourful—you'll like them as souvenirs. And if I'm happier, it means I'll be nicer to you. And if I'm nicer to you, you'll be happier. Welcome to the circle of life.
We have slogan!
With my last full day of high school tomorrow (well, today), it seems only fitting that I announce a slogan for this summer.
It is: "Unite the plots."
No, it probably doesn't mean much to you. But I'm going to put it up on a banner on my wall and stare at it every day, for it shall be my overriding goal as I venture into the scary task of writing book 2. Three plots, lots of characters—and they shall be united around a centre of creamy moral ambiguity goodness.
I can't wait for this week to end!
That is all.
America of the Future
2010: Dateline NBC: To Catch a Terrorist airs.
2015: Citizens may purchase and carry personal nuclear weapons as per the second amendment, as it is everyone's right to protect his or herself from terrorism.
2017: Bleeding heart liberals demand tougher nuke-control laws after numerous drive-by nukings.
2020: The CW airs America's Next Top Terrorist.
2025: Government introduces "Adopt a Terrorist" program. Families may "adopt" a well-known terrorist and attempt to eliminate him or her using any means possible. To help, government also legalizes sale and personal ownership of cruise missiles.
2030: Donald Trump launches TerroristMarket.com. Users may "invest" in a terrorist organization (like buying stocks in a company) and receive dividends if the organization is successful.
2032: Government legalizes terrorism in order to tax it. Experts call move brilliant, citing terrorism as a "reinforcing team values" and "encouraging conformity to a single, unifying set of social morals".
Behold, I have finished War and Peace
You heard me. I'm now among one of the elite who have read War and Peace from beginning to end. Not only that, but I am among the elite who read it for pleasure instead of being forced to read it for some other purpose. If only I could read Russian. This actually happened last week, but I never got around to blogging about it.
Yes, War and Peace was excruciatingly boring at times—particularly when Tolstoy described the battles. I don't care about battles, but at least make it interesting, man! Don't describe the formations. Give me something to stimulate my imagination, honestly! The interaction among most of the main characters was great though.
After reading this, it strikes me that Tolstoy is essentially the Dickens of Russia. He is an excellent storyteller, but a terrible writer. Sometimes he gets lost in the social commentary and forgets that he does have to advance the plot once and a while; that's what made it boring at times. After all, the last part of the epilogue is just an indulgent treatise on his views of history and how events happen.
It is worth it, though. It is boring, and it takes way too long to read, but if you sit down and can finish it, then it's actually pretty good. Maybe you should try it the next time you've got nothing to do on a rainy day.
Class of 2007
School is not over yet; exams have yet to come, but tonight I went through the complicated tribulation of the graduation ceremony and emerged (although somewhat tired) unscathed. The ceremony was long, and at times dull, but it was an interesting experience nonetheless. I cried when our teachers sang, because not only did they sing well, but I thought about all they've done for us during our schooling. It's part of the reason I'm going into teaching, I think—not to sing, but just to affect others' lives in such a profound manner. I've been lucky to have a great number of dedicated teachers who do more than impart facts. They've supported me and shaped me as a person. Similarly, valedictorian Cassie Graham's speech was moving and flawlessly delivered. I mean, Dr. Seuss?! That alone deserves mucho kudos—there are very few people wiser than that man, and the quotation that Cassie chose was spot-on. But it was much more than that. It was personal. There was something in there that let everyone stop and nod and say, "That was me."
It was surreal, in a sense, as I walked away from the stage, proceeding out the auditorium doors and into the much-needed fresh air. I've graduated high school. It's one of these seminal moments that define our culture, the kind of special ceremony portrayed with honour and reverence in TV shows and movies. It is our coming of age. I'm also so focused on the future that I forget the present. I'm a seventeen-year-old who has just graduated high school.
I'm scared. I've been coddled for the last 17 years. Now I'm on the cusp of adulthood; now I have to make my own choices and actually guide my own life. While I am intellectually ready to move on to higher learning, I'm going to miss the social setting of high school. I am not a terribly social person, but I still have my little group of close friends, and they mean a lot to me—and I'm not sure if I express that enough.
I'm going to Lakehead University next year, so I won't even be leaving the city. My goal is to become a high school teacher. Rather simple, eh? But it's what I want to do (and write novels!). Many of my friends are staying here to go to LU or Confederation College, so I will still see them. Two of my closest friends, Cortney and Vivike, are leaving for southern Ontario to pursue their goals, and I'm going to miss them so much—and this is an awareness that has only quickened recently as the end of the year approaches.
For you see, I am afraid of change. A basic aspect of my personal philosophy has and always will be that change is good. Change is a fundamental aspect to humanity without which we would not survive. We should embrace change. Yet I'm afraid of it all the same. I'm scared of what's out there, this big unknown in front of me. What am I going to do?
This isn't unique, I know. And right now, that's the only thing keeping me on track: the fact that I know I'm not alone. It's just an expression of what everyone goes through at multiple points of their life, when they reach this crucial turning point that will define a new portion of their story. It's not even poignant or profound, but give me a break—after all, I just sat through a three hour ceremony. My diction isn't exactly well-rested.
I'm not even sure what I wanted to say anymore. I just wanted to collect my thoughts about this whole graduation experience. After all—now we're people. And I just wanted to say how immeasurably proud I am of my peers, and especially of my friends, for all of our accomplishments, and for our bright prospects.
I'm going to miss high school, and I'm going to love university. I'm sad to go and happy to move on…. I don't know. I'm going to stop now, because I'm not making any sense, I don't think. There's just too many emotions clashing in my heart right now to talk about any particular feeling. I've done my best, too, to avoid as many of these cliches that seem to dogmatically follow graduation (although I'm sure there's a few in here somewhere).
Here's a random thought! We're six billion apes crammed onto a moist rock hurtling through the vast expanse of space. No steering wheel, no brakes. We think we're special because we've got fire and a stick.
And yeah, I think that digital watches are a neat idea. I'm Ben Babcock, and I just graduated high school. Now someone gimme a stick. It's my turn to make fire.


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A recent study has shown that milk may play a role in the preventing the onset of type 2 diabetes. According to a study that was published earlier on the American Journal of Clinical Nutrition, a there was a link between type 2 diabetes prevention and the drinking of milk.
The study indicated that the danger of developing type 2 diabetes among teenagers who have the habit of drinking milk was lower by around 43 percent. This habit may also be carried by the teenagers once they become adults, which would play a factor in type 2 diabetes prevention.
This comes as the Centers for Disease Control and Prevention indicated that the danger of developing diabetes has increased among children who do not consume sufficient amounts of low fat milk. According to the CDC report although milk drinkers among children have reached almost 73 percent, only 20 percent of these children consume low fat milk.
However the same report also pointed out that there were some inconsistencies in the consumption of low-fat milk due to income and ethnicity. Children of the higher income range consume more low fat milk compared to children from the lower income group.. While Hispanic and non-Hispanic black children consume less low fat milk compared to non-Hispanic white children
The study had around 37,000 women as participants while another study included around 440,000 individuals revealed that the consumption of low fat milk as a source of protein instead of meat will reduce the danger of developing type 2 diabetes by around 17 percent.
Milk drinkers will develop a habit that will allow them to prevent the onset of type 2 diabetes better compared to individuals who do not drink milk. Due to this, it has become advisable to encourage milk drinking among children in order to help them fight against the onset of type 2 diabetes later on in their life.


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Enable IPC CEO David Walker recently released an on-demand Webcast designed as a 5 minute introduction of the Company's management, subsidiary operation SolRayo and current projects and opportunities to interested individuals. Additionally, the Webcast provides a brief description of the Company's technologies:
The Webcast is available now at: http://www.enableipc.com/presentations_intro.html and can be viewed at your convenience.
Enable IPC's Ultracapacitors Used in High Profile Renewable Energy Project
Enable IPC has recently completed ultracapacitor electrode shipments to IMDEA Energia in Madrid, Spain for a renewable energy demonstration project. The electrodes will be incorporated into a power conditioning unit by IMDEA and Green Power, a Spain-based renewable energy manufacturer, for a demonstration as part of the SA2VE project -- a Spanish government-sponsored program focused on new energy solutions, particularly relating to "green" power. If the project is successful, the Company will have a multi-million dollar opportunity in renewable energy.
The Company's ultracapacitor technology combines nanoparticles with common carbon sheets for a low cost, easy-to-implement process that improves the performance of ultracapacitors as clean energy storage devices. The enhanced ultracapacitors are simpler, cheaper and longer lasting than conventional devices, including some batteries, but perform just as well for many applications including renewable energy.
$600 Million Ultracapacitor Market
Enable IPC / SolRayo's ultracapacitor technology is aimed at a market estimated to grow to over $600 million by the year 2012. While the company has been mostly focusing on the use of ultracapacitors in renewable energy, there are also huge opportunities for this technology in consumer, other industrial and transportation applications as well.
Enable IPC provides efficient, streamlined strategies for turning technologies into products and bringing them to market. Enable IPC's growing portfolio currently includes the exclusive rights to two break-through energy technologies: a nanoparticle-based ultracapacitor and a nanowire-based microbattery.
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MJ AFT '06 -'09 (Denali National Park, AK); LAURA ALEXANDER '08, '09 (Corvallis, OR); STACIE ANDERSON '09 (Prescott, AZ); ANNE BEAULAURIER '02 -'09 (Denali National Park, AK); KC BOEHLY '09 (Salt Lake City, UT); MATT CAHILL '09 (Amherst, NH); JILL CARTER '09 (Gloucester, MA); LINDSEY CARTER '09 (Panton, VT); LINDSAY CHRONISTER '08, '09 (Seattle, WA); WALLACE & JERRYNE COLE (Denali National Park, AK); DIANE CURRAN '09 (Bangor, ME); FRANK DeSALVO '09 (Conway, NH); WILL DUNCAN '08, '09; (Walpole, NH); FIONA GALBRAITH '07 -'09 (Nelson, BC); SHARYN GERHARDT '95 -'09 (Everett, WA); CHRIS GOCHENOUR '06, '09 (Overland Park, KS); JOSEPH GOSS '09 (Thomson, GA); RUSSELL GRIGGS '07 -'09 (Logan, UT); DANIEL HAGAMAN & ESTHER ALSUM '07 -'09 (Hotchkiss, CO); SIMON, JENNA, & DANIKA HAMM (Denali National Park, AK); AUSTIN HICKS '08, '09 (Seattle, WA); KEN HOCKERT '04, '05, '07-'09 (San Francisco, CA); ELLEN HORBETT '04 -'09 (Denali National Park, AK); ALEX HUGHES '09 (College Station, TX); FRANKLIN JEFFERS '09 (Redmond, OR); JOHN KAHLE '05 -'09 (Denali National Park, AK); TIERNAN MARTIN '08, '09 (Cuttingsville, VT); MATTHEW & MEGAN MARTZ '05 -'09 (Angola, IN); AMY MATTHEWS '09 (Indianapolis, IN); DREW McCARTHY '07 -'09 (Anchorage, AK); BRIAN McCORMICK '95 -'09 (Seattle, WA); IAN & TAYLER McEWEN '07 - '09 (Anchorage, AK); BRIAN McKINNEY '08, '09 (Bellingham, WA); KATHRYN McKINNEY '05, '07 -'09 (San Francisco, CA); MARTHA McPHEETERS '06-'09 (Denali National Park, AK); PHYLLIS MILLER '09 (Decatur, GA); DERIC MIZOKAMI '09 (Fontana, CA); SCOTT PETERS '08, '09 (Denali National Park, AK); ANDREW RECUPERO '07 -'09 (Ferrisburg, VT); DANIEL RODRIGUEZ '09 (Sylmar, CA); KATHY SCHAKE '05 -'09 (Portland, OR); JULIANN SCHAMEL '05, '07, '09 (Fairbanks, AK); ANYA SCHWARTZ '08, '09 (Burlington, VT); LAUREN SIMAS '08, '09 (Berkeley, CA); JULIA SMITH '04 -'09 (Bradenton, FL); TYLER SOFIE '09 (Bellingham, WA); HERTHEL STEPHENS '06, '09 (Girdwood, AK); JAN TOMSEN '03, '04, '06 -'09 (Denali National Park, AK); VIKI VANCUROVA '07 -'09 (Liptovsky Hradok, Slovakia)
MARIA BERGER (Fairbanks, AK); JAKE PEDERSON (Tacoma, WA); AMANDA SOLONIKA (Portland, OR); FRITZ WITTWER (Denali National Park, AK)
CARLEY DUNN (Anchorage, AK)
Vol. XXXIX
November 2014
Edited by Jan Tomsen
P.O. Box 67
Denali National Park, AK 99755
(907) 683-2290
The collective knowledge, talent, and warm hospitality of our staff are what make our guest experience so memorable.
General staff positions are available for the 2015 season, as well as professional-seasonal positions.
Registered Nurses and EMTs are encouraged to apply for any of our positions.
If you know of someone who would be a good fit in our community, encourage him or her to view the employment pages of our website.
In 1964, the Wilderness Act was signed into law. At the time it protected over nine million acres of federal land according to rigorous standards that represent the highest level of federal land protection in the United States. Fifty years later, 109.5 million acres have Wilderness designation, 52% of those in Alaska, including Denali's original, two million-acre core.
For five days in July 1963, the Executive Council of The Wilderness Society held their annual meeting at Camp Denali. In attendance was a truly impressive list of people well-known for their pioneering work in land and wildlife conservation: Olaus and Margaret Murie, Adolf and Louise Murie, Howard Zahniser, and Sigurd Olson, among others. In the Tundra Telegram from that year Ginny describes what a good show the park put on for the group:
The weather cooperated and so did the caribou migration. A highlight of the meeting was the Friday trip to Eielson Visitor's Center, from which point hikers scattered in all directions, following caribou bands or simply exploring the canyons, ridges, and the Thorofare River bar.


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Choosing the right path: enhancement of biologically relevant sets of genes or proteins using pathway structure
- Equal contributors
1 Environmental Systems Biology Group, Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, RTP, NC 27709, USA
2 Department of Biology, Sacred Heart University, Fairfield, CT 06825, USA
Genome Biology 2009, 10:R44 doi:10.1186/gb-2009-10-4-r44Published: 24 April 2009
A method is proposed that finds enriched pathways relevant to a studied condition using the measured molecular data and also the structural information of the pathway viewed as a network of nodes and edges. Tests are performed using simulated data and genomic data sets and the method is compared to two existing approaches. The analysis provided demonstrates the method proposed is very competitive with the current approaches and also provides biologically relevant results.


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Greece is the Word
Forget Spain for now, events in Greece over the next few days will be setting the tone for Europe for better or for worse.
Greek voters go back to the polls on Sunday and clearly face a tough choice, caught between a rock and a hard place. Surveys have suggested growing support for pro-bailout parties but with no opinion polls allowed in the last 2 weeks of electioneering, we're going in to the countdown blind.
Parts of the Greek property market have all but seized-up as the election has approached but if we're lucky, the result could pull us back from the brink if it is seen as an endorsement of efforts to stay in the euro. Equally however it could edge us closer to what many see as an inevitable break-up.
Either way, we shouldn't perhaps get our hopes up that the end of the problem is nigh. Uncertainty is not likely to fade any time soon and greater electoral tests may lie ahead in Germany and Italy.
A flight of capital may be seen if anti-bailout parties prevail on Sunday but whoever wins some let-up in austerity seems likely as the growing stresses in Greek business and society demand a response. Nonetheless, a Greek euro-exit may not be a foregone conclusion, even though the potential for one or more members to leave at some point is greater than it seemed a few months back.
Contingency planning is clearly called for but when businesses consider this, they are often finding more questions than answers. Perhaps the key property issue is what will happen to leases if the currency changes. No one seems to know but many have an opinion!
The safest assumption may be that those with most market power will prevail – and for now that means tenants given that Greece is a market with falling rents, rising availability, limited demand and tenant-friendly leases which can be broken at 3 months notice with just one month's rent penalty, once passed the first anniversary.
At the same time, if the currency depreciates, so too will asset values for those looking from abroad. Hence even though few leases have a clause to deal with currency change, it is not surprising that some corporates and investors are asking for one. More occupiers are also looking to renegotiate existing leases and in our opinion this will continue as the market and tenant needs change.
It has to be remembered however that Greek lease law offers a high level of security of tenure and those landlords with the luxury to choose will be wary of locking in too low a rent for the long term. There also tends to be less flexibility to renegotiate in the shopping centre sector than elsewhere. Hence the best advice for landlords and tenants is to consider each property and lease separately, be ready to act and renegotiate but not commit too far in to the future.
What is more, while all market players need to hunker down to wait out the crisis period, changing conditions will create an opportunity for some. However it may be a little while before some, investors in particular, have the confidence to act and in the near term, uncertainty will only put more investor emphasis on the safest, top prime global markets.
David Hutchings, European Research Group, London


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- Filed Under
Long ago, my Uncle Cary bought this new and totally cool '67 Thunderbird. He was cool himself. The car was silver with a black top, and in the back had the so-called suicide doors hinged on the rear.
It obviously was a Batmobile, so we used it to play Batman whenever he - my uncle, not Batman - drove it to my grandmother's house. We'd run around sing-screaming the television show's signature anthem: Dadadadadadadada - Batman!
I saw an identical T-Bird the other day, during something Mississippi Gulf Coast promoters call Cruisin' the Coast. They bill it as America's Largest Block Party - and ...