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Adhesion and successful colonization of bacteria onto solid surfaces play a key role in biofilm formation. The initial adhesion and the colonization of bacteria may differ between the various types of surfaces found in oral cavity. Therefore, it is conceivable that diverse biofilms are developed on those various surfaces. The aim of the study was to investigate the molecular modifications occurring during in vitro biofilm development of Streptococcus mutans UA159 on several different dental surfaces.
Growth analysis of the immobilized bacterial populations generated on the different surfaces shows that the bacteria constructed a more confluent and thick biofilms on a hydroxyapatite surface compared to the other tested surfaces. Using DNA-microarray technology we identified the differentially expressed genes of S. mutans, reflecting the physiological state of biofilms formed on the different biomaterials tested. Eight selected genes were further analyzed by real time RT-PCR. To further determine the impact of the tested material surfaces on the physiology of the bacteria, we tested the secretion of AI-2 signal by S. mutans embedded on those biofilms. Comparative transcriptome analyses indicated on changes in the S. mutans genome in biofilms formed onto different types of surfaces and enabled us to identify genes most differentially expressed on those surfaces. In addition, the levels of autoinducer-2 in biofilms from the various tested surfaces were different.
Our results demonstrate that gene expression of S. mutans differs in biofilms formed on tested surfaces, which manifest the physiological state of bacteria influenced by the type of surface material they accumulate onto. Moreover, the stressful circumstances of adjustment to the surface may persist in the bacteria enhancing intercellular signaling and surface dependent biofilm formation.
Microbial adhesion onto surfaces and the subsequent formation of biofilms are critical concerns for many biomedical and dental applications. The initial adhesion and the successful colonization of bacteria onto solid surfaces play a key role in biofilm formation and the pathogenesis of infections related to biomaterials [1-4]. Many bacteria prefer to exist predominantly attached to surfaces in contact with liquids [5]. The advantages gained by the bacteria immobilized on surfaces are thought to include increased protection from the host's immune system, higher protection against antimicrobial agents, higher concentration of nutrients close to a surface, and easier inter cellular genetic and signal exchange [6].
The oral cavity is a unique environment, as different types of surfaces (hard, soft, natural and artificial) share the same ecological niche. In order to survive within this 'open growth system' and to resist shear forces, bacteria need to adhere either to soft or hard tissues [7,8]. Adhesion of oral bacteria to acquired enamel pellicle (AEP) leads to the development of the dental plaque biofilm. AEP is a-cellular film which results from selective adsorption of bacterial and host constituents such as salivary components. Among the artificial surfaces in the mouth one can find various types of restorative materials, which differ in chemical and physical properties. Although these surfaces occur in the same ecological niche, the attached biofilms are probably substantially different from one another, and each of these biofilms represents a unique micro-environment [9].
Potentially, each type of surface, such as implant, restorative material, or enamel, can be associated with the formation of different type of a biofilm. Clearly, controlling the initial adhesion into a biofilm depends mainly on the surface properties. While several dental materials promote selective adherence during early dental biofilm formation [10,11], other modified biomaterials may provide resistance to bacterial adhesion and biofilm formation [12,13]. Therefore, it is expected that diverse biofilms are developed on various surfaces. Previous studies have demonstrated that streptococci, including mutans streptococci, are the predominant colonizing microorganisms of oral surfaces. S. mutans is considered to be a most important etiological agent of diseases associated with dental caries. On teeth, it is one of the species which form biofilm causing dissolution of enamel by acid end-products resulting from carbohydrate metabolism [14-16].
In nature, acclimation of bacteria to any type of biofilm environment is probably associated with a change in gene expression [17-19]. However, in contrast to other areas, less is known about the gene expression of bacteria immobilized on different dental surfaces. It is compelling that adaptation of oral bacteria to the different types of dental surfaces may also be associated with different patterns of gene expression, especially those genes associated with biofilm regulation, formation and bacterial physiology. The aim of this study was to identify transcriptional modifications that accompany the formation of in vitro biofilms by S. mutans on a variety of dental surfaces.
The tested surfaces
Dental restorative material - composite Filtek Z250 (60% zirconia/silica, average particle size 0.01-3.5 microns; BIS-GMA, UDMA and BIS-EMA resins (3 M Dental Products, St Paul, MN, USA)).
Ti disks tested in this study were Ti alloy (TiAl(6)V(4)) disks (6 mm diameter) with machined type of surface modifications manufactured by Alpha-Bio implant company (Petach Tikva, Israel).
Hydroxyapatite (HA) tablets were prepared by the following procedure: 340 mg of HA beads (Bio-Rad Laboratories, Hercules, CA, USA) of particle size diameter 80 μm, surface area 40 m2/g, were pressed at a pressure of 8 tons for 20 sec by a single-punch machine (Erweka, Frankfurt, Germany). The punch diameter was 1.2 cm. Before every preparation of tablets the punch (all the surface and inside) was cleaned with ethanol (70%) and stearic acid (5%).
Following the sterilization the Ti, HA, and the composite materials were placed into the 20-mm diameter and 15-mm deep polystyrene multidishes (NUNCLON-143982, Roskilde, Denmark); consequently, the polystyrene multidishes were used as a non-dental reference surface.
Bacterial strains and culture conditions
S. mutans UA159, a serotype c strain, was obtained from Robert Burne (University of Florida, Gainesville). The planktonic S. mutans UA159 was grown in Brain Heart Infusion Broth (BHI, Difco Labs, Detroit, USA) at 37°C in 95% air/5% CO2 (v/v) in polystyrene tubes for 24 h. For biofilm generation, S. mutans culture was seeded in 20-mm diameter, 15-mm deep sterile polystyrene multidishes (NUNCLON-143982, Roskilde, Denmark), and cultivated with fresh BHI medium at 37°C in 95% air/5% CO2 (v/v) for 18 h. For generation of the biofilm on different surfaces, we placed the Ti, HA, or the composite into the polystyrene multidishes. Each experiment was performed in three independent biological repetitions in duplicates.
Analysis of biofilm construction
The 18 h grown biofilms developed on the different surfaces were analyzed for depth and bacterial vitality using a confocal laser scanning microscope (CLSM). The biofilm was stained with LIVE/DEAD BacLight fluorescent dye (Molecular Probes, OR) (1:100) for 10 min. Fluorescence emission of the PBS washed samples was measured using a CLSM (Zeiss LSM 510, Carl Zeiss Microscopy, Jena, Germany). In each experiment, exciting laser intensity, background level, contrast and electronic zoom size were maintained at the same level. At least three random fields were analyzed in each experiment. A series of optical cross-sectional images was acquired at 6.9- μm depth intervals from the surface through the vertical axis of the specimen, using a computer-controlled motor drive. 3-D confocal images were reconstituted and processed for display using Adobe Photoshop ver. 7.0 software (Shemesh et al., 2007).
RNA extraction
Extraction of total RNA from S. mutans cells was performed as described previously [20]. In brief, biofilm-grown cells were suspended in TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) and dislodged by scraping into a 2-ml microcentrifuge tube containing 0.4 ml 1-mm-diameter glass beads (Sigma-Aldrich). The cells were disrupted with the aid of a Fast Prep Cell Disrupter (Bio 101, Savant Instruments, Inc., NY, USA), centrifuged and the RNA containing supernatant was supplemented with 1-Bromo-3-Chloropropane (BCP) (Molecular Research Center, Cincinnati, OH, USA). The upper aqueous phase was precipitated with isopropanol. After centrifugation, the resulting RNA pellet was washed with ethanol and resuspended in diethyl pyrocarbonate (DEPC)-treated water.
Because of the sensitivity of the PCR, residual contaminatingDNA was eliminated by incubation of the sample with RNase-free DNase (Promega, Madison, WI, USA). The DNase was then inactivated by incubation at 65°C for 10 min, and the RNA was precipitated with ethanol and suspended in diethyl pyrocarbonate (DEPC)-treated water. The RNA concentration was determined spectrophotometricallyusing the Nanodrop Instrument (ND-1000, Nanodrop Technologies, Wilmington, DE, USA). The integrity of the RNA was examined by agarose-gel electrophoresis (data not shown).
Microarrays design, cDNA labeling and hybridization
Figure S1 shows schematically the construction of DNA-microarray experiments for gene expression studies of biofilm on representative surfaces. The arrays consisted of 1948 70-mer oligonucleotides representing 1960 open reading frames (ORF) of S. mutans UA159 and additional control sequences. The probe labeling, hybridization and array data normalization were carried out as previously described [21]. In brief, cDNA was generated with random primers from total RNA and labeled indirectly with cy3 or cy5 dye. Hybridizations were performed against the samples from the polystyrene and composite surfaces in a reference design manner (Additional file 1, Figure S1). Slides were scanned using a Genepix 4000B scanner (Axon Ltd). Fluorescence intensities were quantitatively analyzed using GenePix Pro 4.1 software (Axon). The result files (gpr) produced by GenePix were analyzed utilizing the LIMMA [22] software package, available from the CRAN site http://www.r-project.org webcite. Spots flagged as not found or absent in GenePix were removed by filtering. Another filter was applied for saturated spots. After filtering, the data within the same slide were normalized using global loess normalization with the default smoothing span of 0.3 [23]. To identify differentially expressed genes, a parametric empirical Bayesian approach implemented in LIMMA was used [24]. According to this approach, data from all the genes in a replicate set of experiments are combined into estimates of parameters of a priori distribution. These parameter estimates are then combined at the gene level with means and standard deviations to form a statistic B that is a Bayes log posterior odds [24]. B can then be used to determine whether differential expression has occurred. A moderated t test was performed in parallel, with the use of a false discovery rate [25] correction for multiple testing. TIGR arrays included four replicates for each gene. Instead of taking the average of replicate spots, we used the duplicate correlation function [26] available in LIMMA to acquire an approximation of gene-by-gene variance. This method greatly improves the precision with which the gene-wise variances are estimated and thereby maximizes inference methods designed to identify differentially expressed genes. A P value < 0.05 confidence level was used to pinpoint significantly differentiated genes. Genes had to have an A-value (A = log2 [Cy3 × Cy5]/2), the average expression level for the gene across all arrays and channels) of more than 8.5, thus omitting genes with an average intensity in both channels of less than 256.
Additional file 1. Figure S1. Schematic diagram showing construction of DNA-microarray experiments for gene expression studies of biofilms on various surfaces.
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Reverse transcription and real-time quantitative PCR
The quantitative SYBR green PCR assays employing an ABI-Prism 7000 Light Cycler System (Applied Biosystems, Foster City, CA, USA) was performed as described previously [14]. The corresponding oligonucleotide primers were designed using the algorithms provided by Primer Express (Applied Biosystems) for uniformity in size (≈ 90 bp) and melting temperature. For each set of primers, a standard amplification curve was plotted (critical threshold cycle against log of concentration) and only those with slope ≈ -3 were considered reliable primers. The expression levels of all the tested genes for real-time RT-PCR were normalized using the 16S rRNA gene of S. mutans (Acc. No. X58303) as an internal standard (Additional file 2, Table S1). Each assay was performed with at least two independent RNA samples in duplicate.
Additional file 2. Table S1. Nucleotide sequences of primers for genes whose expression was compared. Table S2. The differentially expressed (P < 0.05) genes of S. mutans biofilms on HA vs. polystyrene surfaces. Table S3. The differentially expressed (P < 0.05) genes of S. mutans biofilms on composite vs. polystyrene surfaces. Table S4. The differentially expressed (P < 0.05) genes of S. mutans biofilm on Ti vs. polystyrene surfaces.
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Autoinducer-2 (AI-2) assay
It has been suggested [27,28] that AI-2 signaling may play an important role in the biofilm formation of S. mutans. It is conceivable that, the challenge of stressful condition during the transition to a new surface may alter the quorum sensing (QS) process in the bacteria. Consequently, we tested the secretion of AI-2 signal molecule by S. mutans immobilized in biofilms formed on the different surfaces to determine the impact of the tested material surfaces on the physiology of the attached bacteria. The AI-2 luminescence reporter assay was performed [29] to detect AI-2 secretion levels, in cell-free conditioned medium of S. mutans biofilms formed on the four tested surfaces. At the end of the biofilm incubation period, a supernatant fluid was collected and filtered through a 0.22 μm-pore size filter (Millipore). The cell-free conditioned medium was either used immediately or stored at -20°C. To determine the amount of AI-2, an overnight culture of Vibrio harveyi MM77, a mutant strain which does not produce either AI-1 nor AI-2, was diluted 1:5,000 in a mixture of 90% (v/v) fresh AB medium and 10% (v/v) conditioned medium to a total volume of 200 μl per well. The negative control contained bacteria in fresh AB medium alone and the positive control contained bacteria, fresh AB medium and 10% v/v spent medium containing AI-2 of V. harveyi BB152 (AI-1-, AI-2+). Readings were performed in triplicate in white 96-well plates with an optic bottom (NUNC) in a GENios reader (TECAN) at 30°C. Luminescence measurements were recorded every 30 min in parallel with optical density absorbance (A595) readings. The value of each reading (biofilm on various materials) was divided by the absorbance values to normalize the luminescence value of each sample to its cell density and to avoid dissimilarities caused by differences in growth rates. Fold induction above the non-specific luminescence background of the negative control was determined at the end of bacterial growth after approximately 15 hrs of growth. Fold induction in luminescence of each sample was normalized by the value of total fluorescence of live bacteria within the relevant biofilm as detected by CLSM.
Using DNA-microarray technology we identified the differentially expressed genes of S. mutans (Figure 1), reflecting the physiological state of biofilms formed on the different biomaterials tested. An empirical Bayesian method (B-test) was applied to test for differential expression in biofilms on various surfaces. Analysis of the microarray images revealed a total of 116, 93 and 44 genes on HA, composite, and Ti, respectively, in comparison with polystyrene surfaces, were differentially transcribed at a confidence level of P < 0.05 (Additional file 2, Tables S2-S4). For simplicity, the mostly differentially expressed genes were grouped into functional categories (Figure 2), (i.e., fulfilling the criteria B > 0 by B-test and more than 1.5-fold change), in biofilms formed on hydroxyapatite, titanium and composite vs. polystyrene surfaces. Eight selected genes were further analyzed by real time RT-PCR (Figure 3). Criteria for gene selection were either highly up-regulated or highly down-regulated genes, associated with virulence, and of known function rather than hypothetical genes. Among the most regulated ones were genes associated with stressful environmental conditions andsynthesis of molecular chaperones, in addition to cell wall associated proteins and adhesion-promoting genes. The real-time RT-PCR analysis confirmed only partially the expression ratios determined by microarray technique.
Figure 1. Differentially expressed genes in biofilms formed on different surfaces. Alignments of differentially expressed genes (P < 0.05) of S. mutans biofilms formed on hydroxyapatite, titanuim and composite (vs. polystyrene surfaces), showing the number of overlapping genes between the biofilms on different surfaces. Gene annotations are based on the genome information of S. mutans provided by TIGR.
Figure 2. functional categories of most differentially expressed genes. Most significant (B* > 0) differentially expressed genes of S. mutans, grouped in functional categories, in biofilms formed on hydroxyapatite (A), titanium (B) and composite (C) vs. polystyrene surfaces. Gene annotations are based on information provided by TIGR. *Bayesian test value, i.e. the probability for a gene to be really differentially expressed.
Figure 3. Expression of selected genes analyzed by RT-PCR. Comparison of RT-PCR expression values for selected genes of S. mutans, grown on different surfaces. SMU.81, SMU.82 (dnaK) and SMU.1954 (groEL) are stress-related genes; SMU.574c, SMU.609, and SMU.987 are associated with cell wall proteins. SMU.744 codes for FtsY, while SMU.618 codes for a hypothetical protein. The data are expressed as the means of at least two biologically independent experiments.
To evaluate the physiological state of the immobilized bacterial populations generated on the different tested surfaces, the biofilms were characterized by using CLSM. Biofilm depth analysis showed that the bacteria were able to construct more confluent and profound biofilms on HA surface compared to other tested surfaces (Figure 4). According to the CLSM images, relatively little biofilm growth of about 62-micron depth was observed on the polystyrene surface (Figure 4c), whereas the biofilm formed on the HA surface was notably deeper, up to 173-micron depth (Figure 4b). Moreover, the vitality of the bacteria grown on the HA surface was much greater than those cultured on the polystyrene surface (Figure 4).
Figure 4. Biofilms of S. mutans formed on different surfaces. Biofilms of S. mutans UA159 were grown on different surfaces in BHI, stained with LIVE/DEAD BacLight fluorescent dye and analyzed with CLSM. The panels show cross-section images of biofilms from polystyrene (A), Ti (B), HA (C) and composite (D) materials. Dead cells were stained red, and live cells were stained green.
To further determine the impact of the tested material surfaces on the physiology of the bacteria, we tested the secretion of AI-2 signal by S. mutans biofilms. As AI-2 reporter strain we used V. harveyi MM77, which does not produce endogenous AI-1 or AI-2. Thus, any increase of its luminescence above background level is due to exogenous AI present in the growth medium. The highest effect on the luminescence of the reporter strain was of the conditioned medium taken from biofilms grown on HA with normalized fold induction of ~100 per 10 million cells. Conditioned media from biofilms grown on composite and polystyrene had a similar effect on the luminescence resulting in normalized fold induction of ~40. The lowest effect on the reporter strain was of the conditioned medium taken from biofilm grown on titanium with normalized fold induction of only ~10 (Figure 5).
Figure 5. AI-2 signal secretion by S. mutans biofilms on different surfaces. Biofilms were grown on each material and the resulting conditioned media were exposed to V. harveyi MM77 for AI-2 bioassay. Fold induction in luminescence of each sample was calculated above background luminescence of the negative control (sample without addition of any conditioned medium) and was normalized by the value of total fluorescence of live bacteria within the relevant biofilm detected by CLSM.
Mechanisms governing biofilm formation have generated considerable interest in the general biofilm field and also in dental-related biofilms [30-35]. Oral biofilms vary in both structure and function but share general characteristics. In order to persist within the oral ecosystem, the bacteria need to adhere to either soft or hard tissues and to overcome local shear forces. Although it is well documented that saliva constituents coat biological surfaces in the oral cavity, the principal aim of this study was to examine a genetic adaptation of bacteria upon immobilization on non-biological surfaces. Our results indicate that bacteria can sense their non-biological substrate and express different genes accordingly, probably as part of the adjustment to a new micro-environment. It is likely that the stressful situation conducts the bacteria to enhance the factors of successful adjustment to certain surface by activation of expression of certain combination of genes. This could explain the fact that bacteria are able to adjust to any surface by manipulating their gene expression pattern. Differences in formed biofilm depths and viabilities among the different materials might be due to their surface properties. Therefore, it is reasonable to assume that bacterial profile of gene expression differs on various surfaces, allowing the species to adapt to the different types of micro-environment.
Most recent global transcription and proteomic profiling has revealed several aspects of the physiological adaptations that S. mutans undergoes following attachment to and growth on surfaces [21,36-38]. Nevertheless, only a few comprehensive studies have compared the influence of surface materials on the gene expression of immobilized bacteria adhering to different dental biomaterials. It is conceivable that the chemistry of the surface on which the biofilm is formed would affect the properties of the biofilm. Recent gene expression profiling showed marked differences in gene responses of bone cells on smooth and rough titanium surfaces [39]. Additional studies demonstrated that the biodegradation of composite resins differentially impacts the growth and gene expression of S. mutans [40]. In addition, biomaterial surface chemistry affected biofilm formation, and polyethylene oxide significantly inhibited S. epidermidis biofilm formation in vitro [41]. In the current study, we have shown that gene expression differs in S. mutans biofilms formed on different surfaces, therefore likely changing the physiology and virulence of the immobilized bacteria. Our CLSM biofilm depth analysis shows that the bacteria were able to construct more confluent and thick biofilms on a hydroxyapatite surface compared to the other surfaces tested.
AI-2 is a furanone borate diester that is synthesized in many bacteria by the LuxS protein and detected in Vibrio harveyi by a periplasmic protein called LuxP. It was proposed to function as a universal quorum-sensing signal for interaction between different bacterial species [42]. It has been previously shown that the AI-2 level decreased in chemostat-grown E. coli cultures exposed to different stresses [43]. In addition, QS is likely involved in stress gene regulation in Porphyromonas gingivalis [44]. The consequences of these data may provide the potential link between the type of surface, QS and stress regulation in biofilm-grown bacteria. This might suggest that the attachment of bacteria to a particular surface may have altered the level of AI-2 signaling in the generated biofilm to overcome stressful conditions. Consistent with this hypothesis is that the levels of AI-2 in biofilms from various tested surfaces were found to be different (Figure 5). The stressful situation during the transition to a new surface apparently induces the bacteria to enhance the QS process to overcome the challenge by activating stress-related as well as biofilm-associated genes at the same time. Although small peptides termed competence stimulating peptides (CSP) are the main QS signaling molecules in S. mutans [45], It was shown that AI-2produced by S. mutans play a role in biofilm formation [27] and analogues of the AI-2 may affect biofilm formation of S. mutans [46]. Moreover, secretion of AI-2 of S. mutans is related to protein synthesis, stress, and cell division [28]. Accordingly, production of different amounts of AI-2 by S. mutans on the different surfaces could contribute to adaptation of the immobilized bacteria and their acclimation to the new micro-environment. The highest level of AI-2 was detected in the conditioned medium taken from biofilms grown on HA. This result is in consistence with the biofilm depth analysis showing that the bacteria were able to construct more confluent and profound biofilms on HA surface. However, the lowest amount of AI-2 was found in Ti biofilms, while bacteria still formed relatively confluent biofilm on this substrate. The differences between the AI-2 levels and biofilm thickness could be explained by alternative mechanisms of biofilm development which enable the bacteria to bypath AI-2 requirement to form confluent biofilm. It is apparent that AI-2, especially in gram positive bacteria, is not solely responsible for biofilm control and it may have other physiological effects on the immobilized bacteria.
The use of the array-based approach enabled us to study the complex interplay of the entire S. mutans genome simultaneously. We examined the pattern of gene expression as a reflection of the bacteria's physiological state influenced by biofilm formation on several representative types of dental materials. Differences in expression of the various genes provide an indication as to their function in biofilm formation, and may help to understand the different physiological pathways associated with this process. A substantial number of differentially expressed genes, such as SMU.574c, SMU.609, and SMU.987, are associated with cell wall proteins. SMU.987 encodes a cell wall-associated protein precursor WapA, a major surface protein [47], which modulates adherence and biofilm formation in S. mutans. Previous studies demonstrated that levels of wapA in S. mutans were significantly increased in the biofilm phase [48], whereas inactivation of wapA resulted in a reduction in cell aggregation and adhesion to smooth surfaces [49]. The wapA mutants have reduced cell chain length, a less sticky cell surface, and unstructured biofilm architecture compared to the wild-type [50]. The differential expression of those genes coding for cell wall associated proteins indicates their role in activation of initial biofilm formation and adjustment of the bacteria to various surfaces.
Additional differentially expressed gene SMU.618 which was found to be most significantly upregulated in biofilm formed on composite is annotated as hypothetical protein with unknown function. SMU.744, encoding the membrane-associated receptor protein FtsY, the third universally conserved element of the signal recognition particle (SRP) translocation pathway [51], was also found among the differentially expressed genes. SRP was first identified in mammalian cells, and later in bacteria, and it was further shown that components of the SRP pathway are universally conserved in all three domains of life [52]. The SRP pathway delivers membrane and secretory proteins to the cytoplasmic membrane or endoplasmic reticulum [53]. S. mutans remained viable but physiologically impaired and sensitive to environmental stress when ftsY and other genes of the SRP elements were inactivated [51]. The high regulation of FtsY in biofilms grown on different types of surface indicates that the SRP system is crucial for bacterial survival in the transition of bacteria from polystyrene to the other surfaces tested.
Our microarray data also show that stress-related genes, including SMU.81, SMU.82 (dnaK) and SMU.1954 (groEL), were differentially regulated within biofilms of S. mutans formed on the surfaces. It is known that these genes are intimately involved in the clearance of misfolded aggregates and premature polypeptides produced during stress. This result indicates that there is a firm correlation between the transition of bacteria from one type of surface to another and the stress response. One possible explanation of these differences could be because of the environmental stress encountered by the biofilm bacteria during the transition to dental surfaces rather than to the polystyrene. The challenge of stressful situations during the transition and adjustment to a new surface induces the bacteria to switch on surface dependent gene expression for successful adjustment to certain surface.
Interestingly, a minority of the differentially expressed genes showed more than 2.5-fold change between the different surfaces. However, even small changes in mRNA levels could have the biological potential to affect bacterial metabolism and physiology. Relatively small changes in the level ofexpression of one gene can be amplified through regulatory networks. and result in significant phenotypic alteration [54]It is noticeable that biofilm formation on different surfaces does not radically alter the transcriptome. However, closer assessment reveals that these changes in gene expression have the potential to profoundly affect cellular physiology, which adapts the bacteria in the biofilm formed on various surfaces. It should be remarked also that real-time RT-PCR results did not fully agree with the microarray data for selected genes. The most prominent differences between the array and RT-PCR approaches are probably due to the inherent technical variability of the microarray technique. Another reason for the residual variation between the two techniques could be associated with the incorporation of labeling compounds only for the microarray technique and the intrinsic dependence on the enzyme used for labeling [55].
By evaluating gene expression patterns in S. mutans following immobilization on different surfaces, we demonstrated that biofilm development is accompanied by significant transcriptional changes (Tables S1-3). However, the existence of a surface-dependent universal biofilm gene-expression pattern is still questionable. Nonetheless, our results suggest that genes associated with stressful environmental conditions and the synthesis of molecular chaperones, as well as cell wall-associated proteins and adhesion-promoting genes, seem to be responsible for biofilm generation on different surfaces. Biofilm formation as a complex developmental process is characterized by intricate interplay of gene expression pattern; hence, the bacteria have very sophisticated ways to be better adjusted to particular surface by manipulating their gene expression pattern. We have tested only representatives of dental surfaces natural (HA), implant (Ti) and restorative material (composite), it is conceivable that biofilm formation accompanied by gene and signal changes would occur also on other types of dental surfaces.
Transcriptional profiling revealed broadly based changes in the patterns of gene expression during biofilm development of S. mutans on different solid surfaces, which manifest the physiological state of bacteria influenced by the type of attachment substance. Moreover, the stressful circumstances of adjustment to a particular surface may stimulate the bacteria to enhance intercellular signaling and surface dependent biofilm formation.
AHL: acyl homoserine lactone; AI-2: autoinducer-2; BHI: brain heart infusion; CLSM: confocal laser scanning microscope; EPS: extracellular polysaccharides; QS: quorum sensing; TIGR: The Institute for Genomic Research; HA: Hydroxyapatite; Ti: Titanium disks.
Authors' contributions
MS planned and carried out the experiments, performed DNA-microarrays and real time RT-PCR analyses and wrote the original manuscript. AT assisted in biofilms generation, RNA extraction, RT-PCR and CLSM experiments. RA helped in set up and performing the AI-2 assay experiments. DS conceived the study and oversaw its execution; he also revised the manuscript critically for important intellectual content. MS and DS integrated all of the data throughout the study and crafted the final manuscript. All authors read and approved the final manuscript.
Microarrays were provided by the NIDCR through the PFGRC at TIGR. This study was partially supported by the Norton-Ross Foundation of IADR. We are grateful to Dr. Miriam Kott-Gutkowski for her excellent technical assistance.
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Bioinformatics 2005, 21(9):2067-2075. PubMed Abstract | Publisher Full Text
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J Bacteriol 2008, 190(1):401-415. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
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Chu F, Kearns DB, McLoon A, Chai Y, Kolter R, Losick R: A novel regulatory protein governing biofilm formation in Bacillus subtilis.
Mol Microbiol 2008, 68(5):1117-1127. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kearns DB: Division of labour during Bacillus subtilis biofilm formation.
Mol Microbiol 2008, 67(2):229-231. PubMed Abstract | Publisher Full Text
Bayles KW: The biological role of death and lysis in biofilm development.
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Kolter R, Greenberg EP: Microbial sciences: the superficial life of microbes.
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O'Toole GA, Stewart PS: Biofilms strike back.
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Klein MI, Duarte S, Xiao J, Mitra S, Foster TH, Koo H: Structural and molecular basis of the role of starch and sucrose in Streptococcus mutans biofilm development.
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text:
Rated 4 out of 5 by 845 reviewers.
Rated 5 out of 5 by Isie Ms. I. Wills I enjoyed my stay. The check-in was prompt, my room was ready when I arrived. The location was great and I will stay at this hotel if I visit this city again. August 19, 2014
Rated 1 out of 5 by Andreakh Re-grout the bathroom in Room 636 Good day, I stayed at the Sheraton Boston in Room 636, 08.14-17.2014, while attending a conference. Please consider re-grouting the bathroom in Room 636. There appears to be some type of mold/dirt where the tile meets the top of the tub, along the right corner and halfway down the top right side of the tub. The appearance of the "black stuff" does not bode well for what is touted as a luxury hotel. Thank you for the opportunity to share my comments. August 18, 2014
Rated 2 out of 5 by Rm2502 Rm 2502 The bathroom didn't drain properly; so the entire bathroom floor was covered in water after each bath. I doubt this begun upon my arrival so why place a guest there? I couldn't return to the bathroom after a bath, and when dressed up because I'd be standing in water. I don't know how quickly the mess was cleaned up because I left the room each time I called for clean up but the hotel staff was quite polite. August 18, 2014
Rated 5 out of 5 by AJRSummer2014 Boston was Awesome Sheraton Boston stay couldn't have been any nicer. Absolutely NO complaints - nothing but friendly, helpful experiences with all the staff ! Would stay again anytime & will recommend to others. August 17, 2014
Rated 1 out of 5 by NeverAgain456 Did not get room we booked We booked a double room with 2 double beds for me my wife and teenage son 5 weeks prior to arrival. After a long day of travel and visiting colleges in the area we checked in only to find out the room we booked was not available. Tired and hungry it seemed we had no choice but to accept the free valet parking as a token and a cot put in our room for our son to sleep on. After unpacking we grabbed a quick bite to eat in the lounge area and ordered some apps to hold us over until dinner. My son and I ordered sliders, horrible, stale bread and small dry burgers that my son said seemed like they were just microwaved. My wife had a salad which she said was fine. $72 dollars poorer ( with tip, service was good ) we left to rest up and get ready for dinner. After dinner at one of the local restaurants the COT was left in our room, unfortunately for us to figure out how to unpack and set up. 15 minutes later after a frustrating call they were unpacking it and setting up for us. Nothing like coming back from a nice dinner to have hotel staff in our room while we watched them unpack and set up a cot we never wanted in the first place because we did not get the room we booked. Room were dated, carpeting stained, terrible view from our 19th floor room. Overall this was my first and last stay at a Sheraton Hotel. Very disappointing with the only consolation given was free valet parking. August 16, 2014
Rated 1 out of 5 by Andreakh Hellish Check-In Experience Hi, I checked-in at the Sheraton in Boston, MA on Thursday (08.14.2014). I arrived at approx. 1:30pm and waited on a line for 20-30 minutes waiting to check-in. Admittedly, I attempted to check-in early. The room I reserved and partially paid for in advance was in the queue to be cleaned. My reservationist, "Alex", checked-me in, and asked if I would not mind waiting until the room was cleaned. I agreed (because I arrived early), gave Alex my cell number, and was told I would receive a call when the room was ready. I decided to check-in my bag at the baggage desk while waiting for the call re: my room being ready. At approx. 3:45pm, I returned to the check-in desk and asked what was the status of the room cleaning. I was told that some additional time would be needed to clean the block of rooms that was reserved for a convention that I planned to attend. At approx. 6:15, I returned to the check-in desk and again asked what was the status of the room cleaning. This time I was told by a manager, Ryan "?", that still more time would be needed to clean the room. At approx. 6:20pm, I spoke to Ryan again, while Alex was present, and expressed displeasure at having to wait 3.5 hours for a room that should have been available at 3pm. The hotel had my reservation (and credit card number), and it knew I planned to arrive. Ryan typed some information while at the check-in desk and found a room for me; although it needed to be cleaned because a duvet cover was missing from one of the double beds. I agreed to accept the room knowing that housekeeping needed additional time to place the duvet cover on the bed. Before Ryan gave me the room keys, I asked whether the room had Wi-Fi. Ryan confirmed that the room had Wi-Fi, at no cost. After waiting approx. 30 minutes to pick-up my bag from the baggage desk, I arrived in the room which had a stellar view of the hvac system of a neighboring building. Ten minutes thereafter, I attempted to access the Internet via Wi-Fi. Without having success, I called guest services and requested assistance. The associate put me on hold while she tried to resolve my log-in issue. After being place on hold without any communication from the associate, I hung up and re-dialed guest services. The same associate answered the phone and she was able to transfer my call to another associate who told me to attempt to log-in again after 30 seconds. My subsequent attempt to log-in was successful. And, as soon as I logged in, I received a call from the Sheraton (617.236.2000). The caller told me that my room was ready. I told the caller I was already in a room at the Sheraton hotel and asked what was the "new" room assignment? I asked because I wondered whether the "new" room had a view that differed from the neighboring building's HVAC system. The caller was not able to provide me with the "new" room number because doing so would be against hotel policy. As a convention attendee and due to circumstances beyond my control, I need to stay at the Sheraton Boston (MA) through the weekend. At approx. $250/night, I am absolutely disgusted with the level of service provided thus far. And to add insult to my injury, while on the elevator riding up to my room, I overheard two other guests smile with glee about the food coupons and room discount they received from one of the customer service managers. I can't seem to shake the aura of Charlie Brown. Stay tuned for the rest of my saga at the Sheraton Boston (MA)! August 14, 2014
Rated 3 out of 5 by foxylol why bother with concierge lounge we were put in a terrible room to start, despite the fact that we would be there for 5 nights! crowded and miserable view. we switched to a better room. the "lounge" was sad, not stocked with any meaningful amenities, -- really pointless, low end breakfast, very low end cocktail hour. room service was fine. good breakfast. good cocktail lounge downstairs. we should be reimbursed for the pretense of access to exec lounge. August 13, 2014
Rated 5 out of 5 by JSDSWSLaw Great Hotel Very close to the convention center, amazing views of the city, and relaxing atmosphere. August 12, 2014
Rated 3 out of 5 by danmarino13 junior suit selection you gave me a Christian Science overlooking side junior suite. But, I was a long staying guest, you could have giving me the other side overlooking much interesting things ,boston. August 12, 2014
Rated 4 out of 5 by EdCos716 Great location I enjoy staying in this hotel as it provides good location and service. What keeps from staying is usually price. I would stay more often if price was competitive August 10, 2014
Rated 5 out of 5 by mbq102 Great Great customer service at check in; comfortable room. August 9, 2014
Rated 4 out of 5 by 3soxfans This hotel has a great location for Red Sox games. This hotel has a great location when attending Fenway Park events. Our room met our needs but we expected a different room configuration (separate sleeping/sitting area). The check-in/check-out process was very slow and while the front desk had 8 workstations, only two were manned despite the long line and wait time. August 9, 2014
Rated 5 out of 5 by Mountain1 Great location Traveled for business and was able to explore the city and play. Great location and staff were accommodating! Great view of the ST. Charles river. Room was comfortable. August 8, 2014
Rated 2 out of 5 by Unhappyguest99 This hotel needs customer compassion In the fine print, I must have missed that making a green choice means that I automatically decline housekeeping for my entire stay. When speaking to the checkout clerk, she offered me a $5 voucher or a Starbucks coffee to go. I declined and decided to just go, gladly. Poor customer empathy and a bad process for serving their guests. August 8, 2014
Rated 5 out of 5 by maw1 Perfect Location for conference Please, if you are thinking of renting a car to get around - DON"T. Everything you need is right at your foot tips and definitely within walking distance. There is an ample option of various types of entertainment, restaurants, Whole Food stores, healthy eating, and places to indulge, like the Chocolate Bar. The staff is most accommodating and there is a serene feeling of safety. For a few extra dollars suggest the upgrade that includes continental breakfast and dinner (appetizers) all located in a lounge that can accommodate your small business meetings. I absolutely loved my stay, slept extremely well- usually I am in a hurry to get home, but every morning I woke up in an ergonomic bed with light sheets to a beautiful view of the Boston skyline. So yeah, I loved my stay August 7, 2014
Rated 5 out of 5 by HappyRob Recent Stay at Sheraton/Prudential Builiding Our experience was nothing but positive. From the moment we arrived until check-out we felt welcomed and supported. Thanks for making this a special place to stay in Boston. August 7, 2014
Rated 4 out of 5 by Idespisenicknames Convenient to everything nice but... The hotel was in a great location. Right next to the convention center, and in the heart of back bay. The staff was courteous and efficient especially the cleaning staff. I would give this place an excellent rating if it weren't for the extremely loud air conditioning. It sounded like an old generator on it's last legs. Thankfully I didn't need it much during my stay but it was so loud that it was unpleasant. August 6, 2014
Rated 5 out of 5 by Douglas73 Great location Pillows are extremely soft. Made sleeping difficult. Room was phenomenal otherwise. Food service in the club was mediocre. We enjoyed the beverage service. August 5, 2014
Rated 5 out of 5 by Hockeyops Boston in July Customer Service was exceptional. The staff from the front desk to the Club Room were fantastic. One of the ladies in the Club Room knew us by name by day 2 and really made a difference. The front counter staff and valet were great and very friendly. We wanted for nothing. August 5, 2014
Rated 5 out of 5 by Wayne757 Comfortable, accesible, gracious The lobby was an inviting place to meet colleagues and friends. Our room was comfortable and the bed was heavenly. The WiFi worked like a charm. We liked the convenience of being connected to the Hynes convention center with easy access to Boylston and Huntington streets. Oh, the Logan Airport Express is fabulous. Pnly $5 and it whisked me to the airport in 25 minutes. August 5, 2014
Rated 4 out of 5 by Woodsie Club Floor The appetizers were not delivered on time at 5:30pm. A lot of people were waiting as we were attending the Red Sox Game and had to wait 10-15 minutes for them to set up the area and than people starting towards the food without waiting in line. August 5, 2014
Rated 5 out of 5 by Kelly03 This hotel has clean, updated rooms. The staff was very friendly and the rooms were clean and updated nicely. Great location and proximity to shopping, restaurants and Fenway Park. August 5, 2014
Rated 5 out of 5 by KyeB12 First time to Boston This was my first trip to Boston, and I really appreciated how easy it was to get to and from the hotel to the airport. The staff was excellent when it came to getting help whether it was towels I needed in my room, or information about getting around town. During the conference I attended they really made our stay a memorable experience, and the cleanliness of the hotel was grade A. The room rate was another plus. Altogether, this hotel is one of the best I've ever stayed in. August 5, 2014
Rated 5 out of 5 by richied always a winner only one nite but great room...great view easy walk to fenway wonderful breakfast August 4, 2014
Rated 5 out of 5 by BeerCoffeePhotography Great Hotel in a Great Location I had the privilege of staying at the Boston Sheraton for the last three months. The staff at this location is amazing and they truly value SPG members. I can not count the number of times they had fruit/ chocolate / etc. waiting for me when I walked into my room. The hotel is located at the Prudential Center so it is connected to a lot of restaurants, bars, other hotels, etc. Plus, it is steps away from Newbury St. Finally, the rooms are nice. They are your standard Sheraton rooms. However, the view from the higher floors of the hotel are amazing! The only thing to note is the bathrooms are on the smaller side but still manageable. You wont go wrong staying at this hotel! August 4, 2014
Rated 5 out of 5 by Davids80 Great room with a view Was able to upgrade to mini-suite and absolutely loved it. Great view of Boston skyline and Fenway Park. Housekeeping did lovely job everyday. Hotel connects to mall and Hynes Convention Center. Great overall experience. August 4, 2014
Rated 4 out of 5 by DocProf High End The hotel essentially offered amenities to match the price, but my room was noisy. The food in the Apropos restaurant was significantly over-priced. August 4, 2014
Rated 4 out of 5 by Steve1954 Mixed Feelings I wish I knew that the parking would be so expensive. (Hotel staff did not inform us.) I assumed we would pay $40, the posted daily rate. It actually cost $70! Ouch! The parking attendant said validation was only "good" for a few hours. Again, no one told us. Because of this, the experience left me with a bad final impression. Not good. Steve S August 4, 2014
Rated 5 out of 5 by Gus4 This hotel meets high expectations Although this is a busy hotel because of the capability to host conferences/conventions, the service is very good. Bell men very helpful and friendly. August 3, 2014
Rated 5 out of 5 by Roar great location great location for shopping and dining. excellent place for business meetings or even conventions. great staff. August 2, 2014


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Paul Brownlie, a restricted patient at the Rohallion Secure Care Clinic in Perth, went missing from the outing in the city on Wednesday.
The 33-year-old contacted authorities in Edinburgh after an appeal was made over his whereabouts yesterday.
A Government spokesman had said Mr Brownlie was not a "high risk" but urged the public not to approach him.
He has now returned to the Rohallion clinic.


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Get up and running with the essential features in Cubase Pro 8, the venerable DAW that produces cutting-edge sounds.
Get up and running with the cutting-edge DAW Cubase 7 in this quick one-hour class.
"As a teacher, I respect the way that your tutorials are hands-on and interactive. They are quick and to-the-point, and the teachers you have chosen are patient and concise." —Joshua M.
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text:
Wisconsin Senior
242 at East-West
PROS: A high motor player that has an explosive first step off the edge. Has good closing burst on the ball and gives chase in pursuit. Has the speed to run with backs and tight ends. Shows athleticism and potential to move in space. Can make the stop in the open field.
CONS: Not asked to play much in space, and not natural there. Lacks a good counter move as a pass rusher and doesn't change direction well. Engulfed at the point of attack by blockers and tries to run around too many blocks. Doesn't disengage well once blockers get their hands on him.
OVERVIEW: Tore his ACL on the first day of the Senior Bowl practices, and subsequently dropped about 20 pounds between then and the Combine as he measured at 242 at the East-West Shrine game. A two-year starter at defensive end that had a productive senior year with 24.5 tackles for loss and 12 sacks.
NFL FORECAST: Schofield's injury will disrupt his development as he's not likely to contribute much as a rookie. But before his injury, he looked like he could be a solid fit as a linebacker in a 3-4 scheme. He's not experienced working in coverage and space, but flashed good ability there the few times he did. His first step and motor means that he should be an effective edge rusher, although I'm not sure he has the pass rush moves and instincts to be a top guy there. More than likely, he'll be a guy that can give you 6-8 sacks per year. Before the injury, I would have thought he could impact right away as a situational pass rusher, and then by his second year be a capable starter once he gets used to playing in space. But now with the injury, that progress could be delayed by a year.
ATL FORECAST: He won't contribute much as a rookie because of the injury, and more than likely would spend the year on the PUP or IR. But he could compete with Nicholas for a starting spot in his second year. Because he is a good athlete with good speed, he has better potentail than Nicholas on the edge, especially in coverage matching up with backs and tight ends. He could push Nicholas for the starting SAM job and eventually win it. But more than likely he's a long-term option because of his inability to contribute much in his first two years as he's recovering from his injury and learning how to be an NFL linebacker. But he could be an upgrade.
VALUE: Before the injury, he had the potential to be a Top 50 pick. But with the injury, he's probably shouldn't go off the board before the fourth round because he won't contribute as a rookie at all.
1-poor, 2-weak, 3-above average, 4-very good, 5-elite
O'Brien Schofield, OLB, Wisconsin
Scouting reports of outside and inside linebackers in the 2010 draft.
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Duchesne Drew, the Star Tribune's Managing Editor for Operations, will lead the Foundation's Leadership Programs, Community Innovation and Communications teams, and oversee efforts to build stronger leadership networks across the region.
Our commitment to education will expand in 2015 as we make new investments in multiple Major Initiatives.
What's it take to change history? Rebuilders Kevin Killer and Anna Takes the Shield.
Two leaders with experience in nonprofit administration, constituent advocacy and community engagement will join the Bush Foundation as 2015 Ron McKinley Philanthropy Fellows.
The sixth cohort of Native Nation Rebuilders unites the passions of 30 leaders who will work together and individually for a brighter future for their people built on self-determination.
Meet the 2014 Bush Prize Winners.
A small selection of the many accomplishments of Bush Fellows and Rebuilders.
The bushCONNECT Team recently selected its recruitment and programming partners for bushCON 2015
Beacon Interfaith Housing Collaborative is the recipient of the Archibald Bush Legacy Grant a one-time grant of $200,000 to support a nonprofit organization that embodies Archibald Bush's legacy of working to strengthen the civic, social and cultural vitality of Saint Paul.
The Bush Foundation is coming to town! Come chat with us.
Twelve organizations that help create the environment for the education initiatives of others to succeed are the first recipients of the Bush Foundation's Education Ecosystem Grants.
We're looking for a public relations consultant to help us spread the word about the Bush Prize and our Community Innovation Grant program.
As we gear up for b magazine 2015, we're accepting proposals for a creative director.
The Bush Foundation announced today the inaugural recipients of its Leadership Network Grants. These 12 organizations will work to develop leaders who have the skills and cultural agility to provide effective leadership in diverse communities.
The Bush Foundation, through its Community Innovation Grant program, will award nearly $5 million to 34 organizations pursuing breakthrough solutions to community problems – from poverty to human trafficking to economic development.
Think bigger. Think differently.
© 2015 Bush Foundation


text:
Kick-Ass 2 Issue 1
Well, it's about time. After an excruciatingly long wait (This is my sarcasm by the way), Balls to the Wall is finally here, and it's just what you'd expect it to be. Violent, foul-mouthed, and quite enjoyable. I'm a big fan of Mark Millar and John Romita Jr., so the chance for more of their goodness is always a plus, but unlike the masses, I wasn't clamoring for this book so soon. Regardless, it's here and it's here to stay so it's time to kick some more @$$ with Balls to the Wall.
Essentially, this comic takes place months after Kick-Ass volume 1 and shows Kick-Ass training with Hit-Girl, Hit-Girl getting scolded by Marcus, and Kick-Ass reminiscing about the events of the past few months where his life has turned to cr@p. Flashback to earlier, closer to Kick-Ass volume 1, and we see Kick-Ass dealing with school, teaming up with Doctor Gravity (Who I love by the way), and and then meeting some people who will surely have an impact on the future of this comic.
Mark Millar seems slightly off here. I don't know if it's my growing older and more aware of things, but this comic just didn't read completely naturally to me. The first scene with Hit-Girl training Kick-Ass just dropped too many pop culture references for it to feel authentic. People don't talk like that, sorry. However, bringing in nicer superhero characters for Kick-Ass to work with was nice. Doctor Gravity just seems like some normal guy trying to escape his life and his dialogue when Kick-Ass meets up with him is great. Plus, the ending, loved it and can't wait to see it play out.
Now, when I saw that this was coming out while John Romita Jr. was still working on Avengers, I was interested in seeing how the art would work. As it is, John Romita Jr. supplies rough breakdowns that are then inked and given a wash by Tom Palmer. Dean White then colors it all. Seeing this, it looks fine. In fact, while this isn't the truest John Romita Jr. art, it may be the most accessible to those who don't like his art, because it's much smoother and more natural than his other work. I was very pleased with it, and his designs for the new superheroes we encounter are simple and likable, just like I'd want them to be.
Overall, this is a good start to the second volume. I'm really looking forward to seeing where this goes from here and hopefully this book will be as ridiculously fun as volume one. 4 out of 5 stars, not quite kick-ass yet, but I know it'll get there.


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Report: Red Sox Still 'in the Mix' for Josh Hamilton, With Other Teams Reticent to Offer Long-Term Deal
Originally written on NESN.com | Last updated 12/11/12
The longer Josh Hamilton stays on the market, the better the chances are of him landing in Boston. Hamilton, who went into the offseason looking for a deal of at least six years, has found few teams willing to give him an offer past three or four years. That means the Red Sox, who are reportedly interested in Hamilton to the tune of three years and as much as $25 million a year (his desired range), are still "in the mix," according to Gordon Edes of ESPN. With other teams unwilling to give Hamilton the longer deal he desires, the Red Sox could have a chance at the slugger, according to Edes. The Rangers, with whom Hamilton has played since 2008, appear to be the frontrunner. Hamilton has reportedly told the team that it will have the chance to match any offer he gets. But the latest reports show Texas offering Hamilton just four years, meaning he may be willing to look toward other interested clubs, such as Boston or Seattle, to get a deal he prefers. Hamilton is the biggest free agent on the market this offseason. He drove in 128 runs with 43 home runs on a .285 batting average last year, the third time in the last five years he's had 30 or more home runs and 100 or more RBIs.
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WunderPhotos Upload
desert beetle on mountain
Uploaded by: wunderwinter7
Friday April 15, 2011
sharjah, United Arab Emirates
Caption: i was climbing a mountain in the desert and suddenly i saw a beetle riding another beetle,s back!
Image Description:
Manufacturer: Canon
Model: Canon PowerShot A480
Orientation: top - left
x-Resolution: 180.00
y-Resolution: 180.00
Resolution Unit: Inch
YCbCr Positioning: co-sited
Compression: JPEG compression
Exposure Time: 1/1250 sec.
FNumber: f/3.0
ISO Speed Ratings: 80
Exif Version: Exif Version 2.21
Components Configuration: Y Cb Cr -
Compressed Bits per Pixel: 3.00
Shutter speed: 10.28 EV (APEX: 35, 1/1244 sec.)
Aperture: 3.16 EV (f/3.0)
Exposure Bias: 0.00 EV
MaxApertureValue: 3.16 EV (f/3.0)
Metering Mode: Pattern
Flash: Flash did not fire, auto mode.
Focal Length: 6.6 mm
User Comment:
FlashPixVersion: FlashPix Version 1.0
Color Space: sRGB
PixelXDimension: 3648
PixelYDimension: 2736
Focal Plane x-Resolution: 15136.93
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Sensing Method: One-chip color area sensor
File Source: DSC
Custom Rendered: Normal process
Exposure Mode: Auto exposure
White Balance: Auto white balance
Digital Zoom Ratio: 1.00
Scene Capture Type: Portrait
InteroperabilityIndex: R98
InteroperabilityVersion: 0100
RelatedImageWidth: 3648
RelatedImageLength: 2736
April 15, 2011 Photo Series
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David Hunter: Unless you've been there, you don't know
When I was a fairly young police officer, I had a memorable experience one year as I attended the mandatory 40 hours of annual in-service training required by the state of Tennessee for a police officer to remain qualified to practice law enforcement.
Returning from lunch one afternoon, we found a blackboard on which the following was printed: "The Rules of Gun-fighting." Nobody said anything, but several of us began to look around with raised eyebrows.
A few minutes later, the instructor of that afternoon's class came in and took his place at the podium. Before he could start, a captain with 25 or 30 years on the job raised his hand. Appearing somewhat annoyed, the instructor, who was outranked by the captain and had almost zero street time, nodded and said, "Yes, Captain?"
"The blackboard says, 'The Rules of Gun-fighting,' but everyone who has ever been in a real gunfight knows that there ain't no rules." Those of us who had actually been in gunfights nodded in agreement and there were a few chuckles. The instructor had never been in a gunfight and everyone present knew it.
The last couple of weeks, as I've watched talking heads on 24-hour news outlets and their unpaid allies on social media, such as Facebook, dissect and critique the tactics of the various law enforcement agencies involved in the frantic search for the Boston Marathon bombers, I noticed there were few veteran street cops among them.
Generally, when someone tried to pin down a real police officer who had actually worked the streets about what some viewed as tactical errors, the answers were along the lines of, "Well, I wasn't there, so I can't really say what I would have done under the same circumstances."
That is the only type of answer possible when a man or woman who has actually been in street combat is asked to critique actions that happened when real bullets — and in the Boston Marathon case, explosive devices — were flying at the officers.
"When the first shot is fired," an instructor at Fort Jackson once said when I was going through Army basic training, "the plan falls apart." It's true in any combat situation. When the first shot is fired, there's a flood of adrenaline, time becomes distorted, tunnel vision sets in and the primal urge is to avoid being shot or blown up while returning fire.
It was true when a drill instructor named Cordle said it in 1965 and just as true in the mid-1980s when one of my police mentors, the street-savvy Capt. Bill Wilson, pointed it out to an inexperienced instructor.
As for all the false information that was broadcast during the hunt for the bombers, some of it came from people monitoring police channels without understanding that cops under fire are not striving for technical detail but warning their colleagues of impending danger. Some of the false information, of course, was plain bad reporting. Everyone within blocks of a violent situation becomes a source of rumors, and a journalist who uses a rumor without verification risks embarrassment when true facts emerge.
So, to all the armchair strategists on Facebook and talk radio shows explaining what the cops should have done rather than what they did, all you are proving is that you have never been where they were.
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Program receives provisional accreditation in earliest possible timeframe
For the first time in nearly 50 years, a Tennessee law program has received accreditation from the American Bar Association (ABA). The ABA's Council of the Section of Legal Education and Admissions to the Bar informed Belmont University College of Law it had been granted provisional accreditation at a meeting this past weekend. The milestone was achieved in the earliest possible timeline allowed by accreditation guidelines.
Belmont University Provost Dr. Thomas Burns said, "We are extremely pleased by this recognition of the legal education program that Dean Jeff Kinsler and the faculty of the Belmont College of Law have developed. The granting of provisional accreditation by the ABA validates the outstanding work being done by our administration, faculty and staff to develop a law program of the highest quality focused on preparing practice-ready attorneys."
Under ABA rules, provisionally accredited law schools are entitled to all rights of fully accredited law schools. In particular, graduates of provisionally accredited law schools are entitled to the same recognition accorded to graduates of fully accredited law schools. A law school must be provisionally accredited for at least two years before applying for full accreditation. To grant provisional accreditation, the ABA reviews numerous factors including curriculum, facilities, library, admissions and faculty.
College of Law Dean Jeff Kinsler said, "Although a new law school, Belmont has become a sought after destination for law study evidenced by the quality of students enrolling in the program and the talented faculty teaching in the program."
Jaz Boon, a charter class member who will graduate in May 2014, noted, "ABA accreditation validates the hard work of the Belmont Law administration, faculty, staff and students in building a great program. Receiving ABA approval strengthens the momentum our law program has garnered and allows us to become an even greater asset to the state of Tennessee and beyond."
On October 7, 2009, Belmont University announced its intention to open a College of Law and the charter class began course work in the fall of 2011. It is now the first law school in Nashville to be accredited by the ABA since 1925. The College of Law provides a natural extension of the university's mission and vision, which emphasize challenging academics, a service-minded approach, real-world experience and community leadership.
The College of Law is housed in a new building, the Randall and Sadie Baskin Center, which opened in August 2012 and includes a state-of-the-art Law Library. The College of Law is integrated into Belmont's main campus and offers specialties in areas that complement the university's nationally-known undergraduate program offerings, including healthcare and entertainment/music business.
Belmont University
1900 Belmont Boulevard
Nashville, Tennessee 37212
About Belmont UniversityRanked No. 5 in the Regional Universities South category and named for the seventh consecutive year as one of the top "Up-and-Comer" universities by U.S. News & World Report, Belmont University consists of approximately 7,300 students who come from every state and more than 25 countries. Committed to being a leader among teaching universities, Belmont brings together the best of liberal arts and professional education in a Christian community of learning and service. The University's purpose is to help students explore their passions and develop their talents to meet the world's needs, With more than 80 areas of undergraduate study, 22 master's programs and five doctoral degrees, there is no limit to the ways Belmont University can expand an individual's horizon.
For more information visit www.belmont.edu


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Recent readings of the IOW reveal: Oxygen-rich saltwater from the North Sea has entered the Central Baltic Sea and there, for the first time since 2003, has displaced hydrogen sulfide in the deep water.
After a long period of oxygen depletion and hydrogen sulfide formation in the deep water of the Central Baltic Sea (since 2003), saltwater being rich in oxygen has reached these submarine areas once again, as the results of the most recent measuring campaign of the Leibniz Institute for Baltic Sea Research in Rostock-Warnemünde now reveal.
This slightly improves the living conditions of higher organisms in these regions which were often referred to as "dead zones".
The last time a similar event has been observed was in November / December 2011. Back then, the deep water of the Southern Baltic Sea was not ventilated further than the Gdansk Basin. The recently measured inflowing salt water succeeded in progressing farther to the Northeast up to the Central Gotland Basin.
There,the bottom-near water layer in depths between 200 and 240 m was provided with oxygen while toxic hydrogen sulfide was removed. At the time of the measuring campaign, this inflow however did not yet reach the Northern part the Gotland Basin.
According to the Warnemünde scientists, two long-lasting phases of westerly winds in February and March 2014 have triggered these processes. The gauge data from February 3 – 20 and March 8 – 19 show minor wind-induced inflow events with estimated volumina of approximately 141 km³ and 203 km³, with the March event carrying the major volumes into the Baltic.
The scientific cruise from July 19 – 30, 2014 was one of five regular measurement campaigns per year which the IOW is conducting to monitor the state of the marine environment of the Baltic Sea. The campains follow a fixed station network reaching as far as to the Gotland Basin. Within the German EEZ, this is done on behalf of the Federal Maritime and Hydrographic Agency (BSH) which is responsible for the performance of the duties that the Federal Republic of Germany agreed upon in the Helsinki Convention.
The gained data are used as a foundation for regular assessments of the state of the Baltic Sea, both on national and international level, as well as for numerous other scientific publications. Besides, they provide the scientific basis for further measures planned to protect or restore the ecosystem of the Baltic Sea.
Dr. Günther Nausch, Sektion Meereschemie, Arbeitsgruppe Allgemeine Meereschemie (Nährstoffanalytik), IOW
(Tel.: 0381 / 5197 332, Email: email@example.com)
Dr. Michael Naumann, Sektion Physikalische Ozeanographie und Messtechnik, IOW (Tel.: 0381 / 5197 267, Email: firstname.lastname@example.com)
Nils Ehrenberg, Öffentlichkeitsarbeit, IOW
(Tel.: 0381 / 5197 106, Email: firstname.lastname@example.com)
Background information: salt water inflows
The water body of the Baltic Sea is permanently stratified with brackish surface water fed by the constant freshwater discharge of numerous rivers. This surface layer is in constant exchange with the atmosphere. It is well provided with oxygen by wind mixing, temperature-induced convection processes and the biological production. From a depth of approximately 70 m on, in areas which are no longer influenced by wind mixing, the Baltic Sea shows its close connection to the North Sea: it is here that the salty North Sea water concentrates which periodically flows into the Baltic Sea via the Danish Belt Sea, Being saltier than the Baltic Sea water, it is also heavier, thus it is flowing along the bottom of the Baltic Sea into the deep basins. Both water bodies do not mix but to a minor degree which causes a permanent stratification. Solid particles like dead organic matter can easily pass this boundary whereas gases dissolved in the water are hold back efficiently. The oxygen content of the deep water therefore constantly decreases as the oxygen is consumed during the decomposition of the deposited organic particles. When the oxygen is entirely depleted, toxic hydrogen sulfide forms. An improvement of this state can only be reached by the lateral supply with large amounts of North Sea water which has been in contact with the atmosphere and therefore is rich in oxygen.
Submarine sills in the Western Baltic Sea hamper this horizontal water exchange. Only under specific meteorological conditions, the salt water can pass these natural impediments to supply the eastern/central parts of the Baltic Sea with oxygen. These sills are the so called Darß Sill, an extended sandy plain between the Danish Island of Moen and the peninsula of Fischland-Darß-Zingst with water depths of 18 – 19 m, and the Drogden Sill being positioned in the Oere Sound between the Danish island Zealand and the Swedish mainland with water depths of only 8 – 9 m.
Since 2003, no major salt water inflow has occured, which has led to the fact that in the deep areas of the central Baltic Sea (>90 m) all oxygen has been consumed and an increased formation of hydrogen sulfide went on. Thus, the biological living conditions for higher organisms have strongly been limited in these areas, which therefore were often referred to as "dead zones" in the media.
Link to images in high resolution:
Further material on demand
The IOW is a member of the Leibniz Association to which 89 research institutes and scientific infrastructure facilities for research currently belong. The focus of the Leibniz Institutes ranges from Natural, Engineering and Environmental Science to Economic, Social, and Space Sciences and to the humanities. The institutes are jointly financed at the state and national levels. The Leibniz Institutes employ a total of 17.200 people, of whom 8.200 are scientists, of which 3.300 are junior scientists. The total budget of the Institutes is more than 1.5 billion Euros. Third-party funds amount to approximately € 330 million per year. www.leibniz-gemeinschaft.de
Dr. Barbara Hentzsch | idw - Informationsdienst Wissenschaft
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SOUTH KINGSTOWN ŌĆō Green signs exclaiming ŌĆśvote yesŌĆÖ rattled throughout the town council chamber Wednesday night as the downtown Wakefield community poured into the town hall to support PhilŌĆÖs Restaurant owners Carl and Kenneth Tetzner and the Contemporary Theater CompanyŌĆÖs plans to revitalize Main Street. Despite the flood of support for the plans, the zoning board postponed their decision until Tuesday.
ŌĆ£I thought this would be the night we got an answer. We all did,ŌĆØ Carl Tetzner said after the board meeting.
SOUTH KINGSTOWN ŌĆō The town council meeting was caught in a stalemate Monday night as union leaders repeatedly asked for apologies from Councilman Jim OŌĆÖNeill, who remained steadfast in his silent refusal to give into what they wanted.
ŌĆ£I spoke at the last town council meeting. I expect an apology for the way I was treated and comments I received from a certain town council member,ŌĆØ Deborah Bergner, resident and member of the Democratic Town Committee said. ŌĆ£I was using my right as a citizen and taxpayer and was treated poorly.ŌĆØ
NARRAGANSETT ŌĆö Auditor General Dennis E. Hoyle released his audit report on Daniel E. Doyle, Jr.ŌĆÖs failure to repay funds to the both the University of Rhode Island and the state. Doyle, who has served at the Executive Director for the Institute of International Sport at URI since 1986, was audited after concerns were expressed to the Auditor GeneralŌĆÖs office about the unfinished state of the instituteŌĆÖs residential and coaching center, for which a state grant of $575,000 was given in 2007.
By LINDSAY OLIVIER
NORTH KINGSTOWN ŌĆō For the better part of a couple months now, North Kingstown school officials and town administrators have debated two key questions: WhoŌĆÖs going to pay for a new roof at Davisville Middle School and what account or bond it will come from?
Monday evening came the first glance at a potential solution.


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Enamelled bronzes from Roman Britain have turned up all over the Roman world. This poses an interesting question: were Celtic artists making tourist knick knacks for Roman soldiers to take back home? Leading expert Ernst Künzl puts a British 'souvenir' into context.
{mosimage}Ancient public opinion regarded Britain as an island of metal. 'Britain produces gold, silver, and other metals,' declared Tacitus around AD 100. Philostratus, in the 3rd century AD, specifically referred to enamelled metalwork, reporting that it was a typical product of the people near Oceanus – that is, the north-west coasts of the empire, including the British Isles.
{mosimage}We can identify metalworkers in Roman Britain who have come from the Continent and produce metal artefacts in a 'Roman' style. On the other hand, we also find in Britain artists working in a strong late La Tène tradition, and still trained in the lore of Celtic Britain's metalworking craft.
There was a strong Celtic element in the decoration of some extraordinary enamelled vessels found in Britain, France, and even Spain. All involve non-figural decoration in late La Tène style, where Celtic ornament has been created geometrically using compasses.
{mosimage}The Romans seem to have invented a species of commodity still very much in vogue today: the tourist souvenir. A spectacular variety of the Roman souvenir industry seems to have developed on Hadrian's wall in the 2nd century. Soldiers could buy paterae decorated in colourful enamel that depicted aspects of the Wall. Some, like the Rudge Cup and a patera from Amiens in France, combined a stylised look at the Wall with a list of military place-names.
For the full article, see Current Archaeology 222.
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Hi , I'm looking fot this texture. If anybody has seen it please could you send me the link ? Thanks
link removed
Thread moved to product Suggestions as it is a request for, not an offer of, a freebie.
The image has also been removed as it appeared to be part of the texture, rather than an image - if you are trying to identify a texture set you need to apply it to a figure and post a render of the figure (without nudity).
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