bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface and internal structure correlation: high‐voltage and scanning electron microscopies of wholemount alveolar walls of human lung
Authors: BASTACKY J.
Published: September 2010

We found that the high‐voltage electron microscope (HVEM) operating at 1–5 MeV was able to transilluminate and form a focused transmission image of whole‐mounts of alveolar walls from human lung, a tissue sufficiently thin to require no embedment and sectioning. Resultant micrographs resembled a composite of scanning and transmission electron microscope images: surface and internal structure of the alveolar wall were visualized in a single micrograph.

Although the scanning electron microscope extracts some subsurface information in the secondary electron mode, the HVEM produced better images of both surface and subsurface features.

Lungs were fixed, dehydrated, critical point dried, and metal coated as for conventional scanning electron microscopy, then individual alveolar walls were excised by hand and mounted on transmission electron microscope grids. Regions of the alveolar wall up to 10 μm thick were delineated with the high‐voltage electron microscope.

Cell surface characteristics were correlated with cell type as identified by underlying cell internal structure. Whole white blood cells within capillaries of the alveolar wall were identified by the configuration of their nuclei. Features of the nucleus and surface of alveolar type II cells were recorded simultaneously. Whole red blood cells were imaged within intact capillaries that branched and wove from one alveolar surface to the other.

HVEM analysis of excised alveolar septa allows definitive correlation of surface and underlying structures in single micrographs of broad portions of the alveolar wall and is an alternative to embedment, microtomy and serial section reconstruction for this uniquely thin tissue.
Direct‐view microscopy: optical sectioning strength for finite‐sized, multiple‐pinhole arrays
Authors: McCABE E. M. , FEWER D. T. , OTTEWILL A. C. , HEWLETT S. J. , HEGARTY J.
Published: September 2010

Direct‐view microscopes use multiple‐aperture arrays in the source and detector planes. We develop a theory for brightfleld and fluorescence direct‐view microscopy which allows us to determine the optical sectioning strength for finite‐sized, multiple‐pinhole arrays with an arbitrary distribution of apertures. We specialize to the cases of square, hexagonal and interleaving Archimedean spiral arrays and consider the implications of the array configuration on both the optical sectioning strength and the light budget.
The estimation of the Euler‐Poincare characteristic from observations on parallel sections
Authors: OHSER J. , NAGEL W.
Published: September 2010

An unbiased estimator of the Euler‐Poincaré characteristic (Euler number) of an arbitrary object or a random structure, respectively, is given. The estimator is based on joint observations of pairs of parallel section profiles. Thus the present paper extends the use of Sterio's disector from counting particles to determining the Euler number for a wide class of probes. The correctness of the given formulae is proved with mathematical strictness. Furthermore, the feasibility of the method is illustrated by an example from materials sciences.
An iterative algorithm for cell segmentation using short‐time Fourier transform
Authors: WD HAI‐SHAN , BARBA J. , GIL J.
Published: September 2010

In this paper, an iterative cell image segmentation algorithm using short‐time Fourier transform magnitude vectors as class features is presented. The cluster centroids of the magnitude vectors are obtained by the K‐means clustering method and used as representative class features. The initial image segmentation classifies only those image pixels whose surrounding closely matches a class centroid. The subsequent procedure iteratively classifies the remaining image pixels by combining their spatial distance from the regions already segmented and the similarities between their corresponding magnitude vectors and the cluster centroids. Experimental results of the proposed algorithm for segmenting real cell images are provided.
A focusing algorithm for high magnification cell imaging
Authors: WU HAI‐SHAN , BARBA J. , GIL J.
Published: September 2010

An algorithm to produce a uniformly focused image in digital acquisition of high magnification light microscopy images is presented. In very high magnification microscopic imaging the specimen surface cannot be considered ideally flat so that capturing a single image frame is usually not sufficient to capture an image that is focused everywhere. An image formation model for light microscopic images is presented, and based on this model an algorithm to construct a uniformly focused image is presented. The algorithm requires that multiple frames of the image at different focal planes be processed to combine their information to obtain an estimated of the desired image which is more completely focused than any of the individual frames. Experimental results show that the proposed algorithm is very effective in approximating the desired image in high magnification microscopic imaging and highly robust comparing to the gradient method.
Tracking differential interference contrast diffraction line images with nanometre sensitivity
Authors: Danuser G. , Tran P. T. , Salmon E. D.
Published: September 2010

This paper presents a computer vision framework for detecting and tracking diffraction images of linear structures in differential interference contrast (DIC) microscopy. The tracker can resolve image displacements of 1/10 of a pixel despite the weak and orientation‐dependent contrast in DIC, as well as the variable blur in such image data caused by vertical specimen movement. In our high numerical aperture, high magnification microscope set‐up, this resolution corresponds to 5 nm in object space. In video DIC similar resolution has been reported hitherto only for rotationally symmetric targets such as bead images. The tracker was developed for measuring deflections of clamped microtubules with a freely moving second end. By analysing the thermal fluctuations of such microtubules it was possible to derive their elasticity.  The paper describes a filtering scheme for the detection and localization of DIC diffraction line images which represent loci of microtubules. For tracking the movements of the extracted lines we adopted the sum of squared (brightness) differences algorithm from computer vision. The analysis of the fluctuation measurements demonstrates the high sensitivity of this tracking technique in quantifying positional and orientational changes. We derived that the theoretical limit in tracking displacements of such diffraction line images is 1.25 nm, four times below the experimentally verified sensitivity. This indicates that the proposed tracker is still suboptimal. Nevertheless, the tracking precision was sufficient to reveal subtle deviations in the distribution of microtubule deflection from free diffusion. They were induced by pivotal points and multiple positions of relaxation. Also, the results suggest that there were defects in the polymer structure which caused very small but significant bends in the microtubule axis.
Laser pressure catapulting followed by B actin gene identification in Japanese quail macrochromosomes and microchromosomes using teflon‐coated coverslip slides
Authors: MCNALLY L. R. , HENK W. G. , COOPER R. K.
Published: September 2010

Laser microdissection of individual mammalian chromosomes (> 2 µm) has been achieved though the use of a microscope slide coated with a polyethylene naphthalate (PEN) membrane. Although these slides have proved sufficient for larger chromosomes, they are insufficient for small chromosomes (< 1 µm). We have developed a new type of slide which allows laser microdissection of single Japanese quail microchromosomes (0.5 µm) and macrochromosomes (3–4 µm). To test the usefulness of these slides, a Japanese quail single nucleus, a macrochromosome, and a microchromosome were collected with Laser pressure catapulting, the B‐actin gene was PCR amplified, and sequenced. The resulting PCR product was confirmed by nucleotide sequencing to be B‐actin. These newly developed slides were shown to facilitate the laser microdissection of both Japanese quail macrochromosomes and microchromosomes.
Analysis of spatial cross‐correlations in multi‐constituent volume data
Authors: RACK A. , HELFEN L. , BAUMBACH T. , KIRSTE S. , BANHART J. , SCHLADITZ K. , OHSER J.
Published: September 2010

We investigate spatial cross‐correlations between two constituents, both belonging to the same microstructure. These investigations are based on two approaches: one via the measurement of the cross‐correlation function and the other uses the spatial distances between the constituents. The cross‐correlation function can be measured using the fast Fourier transform, whereas the distances are determined via the Euclidean distance transform. The characteristics are derived from volume images obtained by synchrotron microtomography. As an example we consider pore formation in metallic foams, knowledge of which is important to control the foam production process. For this example, we discuss the spatial cross‐correlation between the pore space and the blowing agent particles in detail.
A quantitative morphological analysis of nanostructured ceria–silica composite catalysts
Authors: MOREAUD M. , JEULIN D. , THOREL A. , CHANE‐CHING J. Y.
Published: September 2010

This study aims at examining the morphology of different catalysts, which are based on a dispersion of ceria nanoparticles embedded in a high surface area mesoporous silica framework. In order to fully describe the mesostructured composite material, we propose here a quantitative description of the microstructure based on a quantitative analysis of micrographs that were obtained via high‐resolution transmission electron microscopy. We have therefore developed an automatic image analysis process in order to automatically and efficiently extract all the components of the catalyst images. A statistical and a morphological analysis of the spatial arrangement of the components of the catalyst are also presented. The study shows clear differences between the materials analysed in terms of the spatial arrangement and the total surface area of the ceria phase emerging into the pores, parameters of prime importance for the catalytic properties. Thus, the silica–ceria nanostructured composite materials, displaying large surface area up to 300 m2 g−1 are shown to exhibit highly rugged surfaces resulting from ceria nanoparticles emerging in the pores.
Canny optimization technique for electron microscope image colourization
Authors: SIM K.S. , TSO C.P. , TING H.Y.
Published: September 2010

Images of scanning electron microscope are usually in the monochrome mode. A simple and user‐friendly approach is proposed to improve the mechanical contrast of the scanning electron microscope grey images. Also, most colourization techniques involve image segmentation or region tracking, which tend to degrade the image with fuzzy or complex region boundaries. A technique is proposed, which is a hybrid between the Canny edge detection technique and the optimization technique. Compared with existing methods, the new Canny optimization technique gives satisfactory results for scanning electron microscope images.
The structure of 1D CuI crystals inside SWNTs
Authors: KISELEV N.A. , ZAKALYUKIN R.M. , ZHIGALINA O.M. , GROBERT N. , KUMSKOV A.S. , GRIGORIEV YU. V. , CHERNYSHEVA M.V. , ELISEEV A.A. , KRESTININ A.V. , TRETYAKOV YU.D. , FREITAG B. , HUTCHISON J.L.
Published: September 2010

Nanocomposites consisting of one‐dimensional CuI crystals inside single‐walled carbon nanotubes were obtained using the capillary technique. high‐resolution transmission electron microscopy investigations of the atomic structure of the encapsulated 1D CuI crystals revealed two types of 1D CuI crystals with growth direction <001> and relative to the bulk hexagonal CuI structure. Atomic structure models were proposed based on the high‐resolution transmission electron microscopy images. According to the proposed models and image simulations, the main contrast in the 1D crystal images arises from the iodine atoms whereas copper atoms, with lower atomic number giving lower contrast, are thought to be statistically distributed.
Decrease in laser ablation threshold for epithelial tissue microsurgery in a living Drosophila embryo during dorsal closure
Authors: THAYIL A.K.N , PEREIRA A. , MATHEW M. , ARTIGAS D. , BLANCO E. MARTÍN , LOZA‐ALVAREZ P.
Published: September 2010

In this work, we use a two‐photon fluorescence microscope for combined imaging and laser tissue ablation of a living Drosophila Melanogaster embryo. By using tightly focused near‐infrared femtosecond pulses at MHz repetition rate and of sub‐nanojoule energy we are able to produce microsurgery on the epithelial tissue within a Drosophila embryo at the final stages of its embryonic development. Ablation was performed on labelled and unlabelled embryos during and after dorsal closure. We observed that ablation of GFP‐labelled tissue required lower energy deposition than unlabelled tissue ensuring that the tissue ablation is mediated by multiphoton absorption of Green Fluorescent Protein (GFP). In addition, the energy deposition to produce ablation is further decreased during dorsal closure. These results show the presence of additional tensile forces on the tissue during dorsal closure. Furthermore, an increased activity of actin near the laser wounds was observed as the tissue heals.
Application of channel electron multipliers in an electron detector for low‐voltage scanning electron microscopy
Authors: HEJNA J.
Published: September 2010

An electron detector containing channel electron multipliers was built and tested in the range of low‐voltage scanning electron microscopy as a detector of topographic contrast. The detector can detect backscattered electrons or the sum of backscattered electrons and secondary electrons, with different amount of secondary electrons. As a backscattered electron detector it collects backscattered electrons emitted in a specific range of take‐off angles and in a large range of azimuth angles enabling to obtain large solid collection angle and high collection efficiency. Two arrangements with different channel electron multipliers were studied theoretically with the use of the Monte Carlo method and one of them was built and tested experimentally. To shorten breaks in operation, a vacuum box preventing channel electron multipliers from an exposure to air during specimen exchanges was built and placed in the microscope chamber. The box is opened during microscope observations and is moved to the side of the scanning electron microscope chamber and closed during air admission and evacuation cycles enabling storing channel electron multipliers under vacuum for the whole time. Experimental tests of the detector included assessment of the type of detected electrons (secondary or backscattered), checking the tilt contrast, imaging the spatial collection efficiency, measuring the noise coefficient and recording images of different specimens.
The chord length transform and the segmentation of crossing fibres
Authors: SANDAU K. , OHSER J.
Published: September 2010

Segmentation of crossing fibres is a complex problem of image processing. In the present paper, various solutions are presented basing on tools of morphological image processing. Two new image transforms are introduced – the lineal distance transform and the chord length transform. Both transforms are applied to two‐dimensional images and their results are three‐dimensional images. Thus, the segmentation problem originally formulated for crossing fibres observed in a two‐dimensional image can be reformulated as a segmentation problem in a three‐dimensional image. This can be solved by a segmentation in the three‐dimensional image. Algorithms for the lineal distance transform and the chord length transform are given and their use in image analysis is demonstrated. Furthermore, the chord length distribution function of the foreground of a binary image can efficiently be estimated via the chord length transform.
Usefulness and economic evaluation of ADSL‐based live interactive teledermatology in areas with shortage of dermatologists
Authors: Dekio Itaru , Hanada Eisuke , Chinuki Yuko , Akaki Tatsuya , Kitani Mitsuhiro , Shiraishi Yuko , Kaneko Sakae , Furumura Minao , Morita Eishin
Published: September 2010

Background  To overcome the problem of maldistribution of dermatologists in rural areas, live interactive teleconsultation systems are being used in some countries. However, these systems are not in common use because few evaluations on their efficiency and economic viability were reported.

Methods  We constructed an easy‐to‐use asymmetric digital subscriber line (ADSL)‐based live interactive teleconsultation system and conducted 150 trial sessions between two rural hospitals and Shimane University Hospital. The clinical usefulness and economic advantages of this system were evaluated using data obtained from the trials.

Results  The system efficiently captured images at a resolution sufficient for specialized consultations: follicular openings were visible in the images obtained from a distance of 2 m. This system is more advantageous than a conventional clinic if the following condition is fulfilled: y≤ 6.00 x−3.86 [x, time required for one‐way travel (h); y, time required for consultation (h)]. Our two lines in trial fulfilled this condition.

Conclusions  Asymmetric digital subscriber line‐based live interactive teleconsultation technology is beneficial in many rural hospitals that do not have a dermatologist.
Muco‐cutaneous manifestations in 178 renal transplant recipients
Authors: Khosravi Masoud , Golchai Javad , Mokhtari Gholamreza
Published: September 2010

Khosravi M, Golchai J, Mokhtari G. Muco‐cutaneous manifestations in 178 renal transplant recipients. Clin Transplant 2011: 25: 395–400. © 2010 John Wiley & Sons A/S.

Abstract:  Introduction:  Mucosal membrane and skin can be affected by immunosuppressive drug(s) and immunosuppression itself. The spectrum of muco‐cutaneous lesions can range from malignancy at one end to infection, iatrogenic lesions, and esthetic effects on the other end.

Method:  In Razi Hospital of Guilan University of Medical Sciences, a cross‐sectional study for the detection of muco‐cutaneous lesions in 178 renal transplant recipients (RTRs) was conducted from the years 2001 to 2006. Biopsy and skin scraping according to the type of skin lesions were performed.

Results:  A total of 31 RTRs (25%) had normal skin. Iatrogenic lesions were the most common (70%) followed by infectious lesions (57%), and miscellaneous skin lesions were exhibited by 26% of the patients. Among the iatrogenic skin lesions, gingival hyperplasia was the most common lesion (48%), followed by hypertrichosis and acne. Malignant lesions (biopsy proven) were recorded in seven patients (5%). Four patients were found to have Kaposi’s sarcoma, and three patients were identified with basal cell carcinoma.

Conclusion:  Our results showed that muco‐cutaneous lesions are crucial problems with RTRs. Attending physicians must pay close attention to skin care regularly and consider reduction of immunosuppression to a safe level, and patient must have self‐checkups.
Chemical characterization and biologic properties of lipopolysaccharide from Bacteroides gingivalis strains W50, W83, and ATCC 33277
Authors: Bramanti Thomas E. , Wong Guie G. , Weintraub Susan T. , Holt Stanley C.
Published: September 2010

The chemistry and selected biological activity of lipopolysaccharide (LPS) from Bacteroides gingivalis strains W50, W83, and ATCC 33277 were compared, as well as the role of this molecule as a mediator of selected inflammatory responses. Chemically, the LPSs consisted of 47–58% Lipid A, 5–10% carbohydrate, 0.05% 3‐deoxy 2‐octulosonic acid, 0.3% heptose, 3.8–5.2% hexosamine, and 2% phosphate. Rhamnose represented the dominant sugar (26–36%), with lesser amounts of glucose (18–34%), galactose (18–25%), mannose (9–12%), glucosamine (7–11%), and galaclosamine (2–5%). The major fatty acids were: 13‐methyl‐tetradecanoate (42–45%), 3‐OH‐heptadecanoate (21–23%), hexadecanoate (16–19%), and 12‐methyl‐tetradecanoate (6–8%). SDS‐PAGE and sodium deoxy‐cholate‐PAGE revealed the LPS to be a smooth chemotype. Differences in migration patterns between the virulent and avirulent strain LPSs also occurred. C3H/HeN macrophages (Mo) exposed to 1 μg/ml of LPS released 3.2–4.2 ng of prostaglandin E (PGE)/ml of supernatant, representing 236–278% of control. Interleukin‐1 (IL‐1) activity in C3H/HeN and C3H/HeJ M0 exposed to 50 /*g of LPS/ml was 382–724% and 270–300% of control, respectively; similar Mo exposed to 10 μg of LPS/ml released 1.6–2.0 ng and 0.3–0.5 ng of tumor necrosis factor (TNF)/ml of supernatant, respectively. Maximum TNF release in C3H/HeN M0 occurred in response to 50 μg of LPS/ml, and was sustained for up to 96 hours. These results suggest that LPS from the B. gingivalis strains stimulate cytokine production from M0 which, in turn, may play a role in orchestrating the inflammatory response for the development of periodontal diseases.
Effects of periodontopathic bacteria on IL‐1 and IL‐1 inhibitor production by human polymorphonuclear neutrophils
Authors: Yamazaki1 Kazuhisa , Polak Boris , Bird Philip S. , Gemmell Erica , Hara Kohji , Seymour Gregory J.
Published: September 2010

The effect of heat‐killed periodontopathic bacteria on the production of interleu‐kin‐1 (IL‐1) and an IL‐1 inhibitor by human polymorphonuclear neutrophils (PMN's) was examined. Peripheral blood was obtained from 18 healthy volunteers and the PMN's were separated using dextran sedimentation and Ficoll‐Paque density gradient centrifugation. The PMN's (5 ×105cells/well) were cultured in serum‐free media with or without heat‐killed periodontopathic bacteria. Four gram‐negative periodontopathic bacteria were used;Bacteroides gingivalisFDC 381,Bacteroides forsythusFDC 338,Actinobacillus actinomycetemcomitansY4 andFusohacterium nucleatumFDC 263. The non‐oralFusobaclerium mortifenintATCC 25557 was used as a control organism. IL‐1 activity was assayed using thymocyte proliferation. The non‐oral organismF. mortiferumstimulated IL‐1 production by PMN's, in contrast none of the periodontopathic bacteria stimulated IL‐1 release although the bacteria themselves had an IL‐1 enhancing effect. Following fractionation of the periodontopathic bacteria stimulated PMN supernatants, an IL‐I inhibitory fraction was identified. These results may illustrate a further mechanism by which periodontopathic bacteria may evade the protective effect of PMN's and may also suggest a regulatory role for PMN's in chronic inflammatory periodonlal disease.
Effect of Actinobacillus actinomycetemcomitans, Wolinella recta and Bacteroides gingivalis on the viability of retinoic acid‐induced and dimethyl sulfoxide‐induced HL‐60 cells
Authors: Armitage Gary C. , Holt Stanley C.
Published: September 2010

We studied the interactions between viable and heat‐killed, opsonized and unopsonized periodontopathic bacteria with both uninduced and induced HL‐60 promyelocytic leukemia cells. The cells were induced to differentiate into granulocyte‐like cells by incubation with retinoic acid (RA) or dimethyl sulfoxide (DMSO). When unopsonized, Wolinella recta ATCC 33228 significantly suppressed the net Proliferation of uninduced HL‐60 cells, Actinobacillus actinomycetemcomitans strain Y4 was markedly lethal to the cells, and Bacteroides gingivalis ATCC 33277 had no effect. Unopsonized and opsonized A. actinomycetemcomitans and W. recta had equally potent lethal effects on induced HL‐60 cells. Unopsonized B. gingivalis Was not lethal to the induced cells in the dose used (100 bacteria/HL‐60 cell), but opsonized B. gingivalis was lethal, especially in the first 24 h. The killing effects of A. actinomycetemcomitans and W. recta were largely eliminated if they were heated (56°C, 30 min) before being added to the induced HL‐60 cells. RA‐induced HL‐60 cells were more sensitive to the lethal effects of A. actinomycetemcomitans and W. recta than were DMSO‐induced cells. The results suggest that the HL‐60 cell line may be a useful model for studying granulocyte‐bacteria interactions.
Effect of iron limitation on Bacteroides gingivalis
Authors: Barua1 P. K. , Dyer D. W. , Neiders M. E.
Published: September 2010

This study was undertaken to describe the effects of iron limitation on Bacteroides gingivalis. Four strains of B. gingivalis were grown in brain heart infusion broth, substituting protoporphyrin IX for hemin. Culture with protoporphyrin IX resulted in a loss of a 28 kDa membrane protein, but no decrease in growth. Iron‐restricted cultural conditions for the growth of B. gingivalis were achieved using α,α′‐dipyridyl, a ferrous iron chelator, at concentrations from 12.5 μM to 300 μM. Total suppression of bacterial growth for strain A7A1‐28 and strain 381 was achieved at 200 μM α,α′‐dipyridyl. At 300 μM α,α'‐dipyridyl, strain W50 and Bowden 18/10 showed 100% and 80% suppression of growth, respectively. The ferric iron chelator Desferal did not show suppression of growth in concentrations up to 500 μM. The dipyridyl inhibition of cell growth for strain A7A1‐28 could be reversed by adding excess ferrous ammonium sulphate but not by ferric nitrate. Iron regulation of proteolytic enzymes could not be demonstrated. Two new membrane proteins 42 kDa and 24 kDa are expressed with iron limitation, and the 45 kDa membrane protein was decreased with iron limitation.
Effect of female sex hormones on Campylobacter rectus and human gingival fibroblasts
Authors: Yokoyama M. , Hinode D. , Masuda K. , Yoshioka M. , Grenier D.
Published: September 2010

Recent studies have suggested a relationship between maternal Campylobacter rectus infections and preterm low birth weight. The purpose of this study was to investigate the effect of female sex hormones, estradiol and progesterone, on C. rectus and human gingival fibroblasts (HGF). The growth of C. rectus was significantly enhanced by incorporating either estradiol or progesterone in the culture medium. The production of vascular endothelial growth factor (VEGF), interleukin (IL)‐6 and IL‐8 by HGF increased following stimulation with estradiol or progesterone, at concentrations comparable to those present in the plasma of pregnant women. In addition, a significantly higher secretion of VEGF by HGF treated with the combination of C. rectus and estradiol was observed in comparison with a treatment with C. rectus alone. Stimulation of HGF with VEGF resulted in production of IL‐6 and IL‐8 in a dose‐dependent manner. The capacity of female sex hormones to enhance both C. rectus growth and VEGF, IL‐6, and IL‐8 production by HGF has the potential to contribute to periodontal disease progression during pregnancy.
Effect of mixed mutans streptococci colonization on caries development
Authors: Seki M. , Yamashita Y. , Shibata Y. , Torigoe H. , Tsuda H. , Maeno M.
Published: September 2010

Objective:  To evaluate the clinical importance of mixed mutans streptococci colonization in predicting caries in preschool children.

Methods:  Caries prevalence was examined twice, with a 6‐month interval, in 410 preschool children aged 3–4 years at baseline. A commercial strip method was used to evaluate the mutans streptococci score in plaque collected from eight selected interdental spaces and in saliva. Mutans streptococci typing polymerase chain reaction (PCR) assays (Streptococcus sobrinus and Streptococcus mutans, including serotypes c, e, and f) were performed using colonies on the strips as template.

Results:  Twenty variables were examined in a univariate analysis to predict caries development: questionnaire variables, results of clinical examination, mutans streptococci scores, and PCR detection of S. sobrinus and S. mutans (including serotypes c, e, and f). Sixteen variables showed statistically significant associations (P < 0.04) in the univariate analysis. However, when entered into a logistic regression, only five variables remained significant (P < 0.05): caries experience at baseline; mixed colonization of S. sobrinus and S. mutans including S. mutans serotypes; high plaque mutans streptococci score; habitual use of sweet drinks; and nonuse of fluoride toothpaste.

Conclusion:  ‘Mixed mutans streptococci colonization’ is a novel measure correlated with caries development in their primary dentition.
Characterization ofSerratia marcescensisolates from subgingival biofilm, extraoral infections and environment by prodigiosin production, serotyping, and genotyping
Authors: Barbosa F. C. B. , Irino K. , Carbonell G. V. , Mayer M. P. A.
Published: September 2010

Introduction: Serratia marcescensis widely distributed in nature, and has emerged in the last years as an important nosocomial pathogen. The organism may also be found in subgingival biofilm in periodontitis patients. This study aimed to verify the subgingival prevalence ofS. marcescensin different periodontal conditions and to evaluate whether the oral cavity would harbor strains similar to those causing infectious diseases.

Methods: The subgingival occurrence ofS. marcescenswas determined in 334 subjects. The phenotypic and genotypic diversity of 23 isolates from subgingival biofilm, 22 from extra‐oral infections and 10 environmental strains, was compared by prodigiosin production, O and H serotyping and genotyping using polymorphic GC‐rich repetitive sequences–polymerase chain reaction.

Results: S. marcescenswas found more frequently in severe periodontitis patients (4.1%) than in gingivitis (3.2%) and healthy subjects (2.5%), but these differences were not statistically significant. Analysis of serotype distribution, prodigiosin production, and genotyping revealed that environmental strains were markedly different from most human isolates, either oral or extraoral.

Conclusion: These data suggest thatS. marcescensisolates from subgingival biofilm are not just contaminants from the environment, but that the oral cavity may act as a reservoir of strains able to promote human infections. However, further studies are needed to elucidate the role of this bacterium in the pathogenesis of periodontal diseases.
Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets
Authors: de Lillo A. , Ashley F. P. , Palmer R. M. , Munson M. A. , Kyriacou L. , Weightman A. J. , Wade W. G.
Published: September 2010

Introduction:  Molecular ecological analysis based on 16S rRNA gene sequence analysis is well established for the characterisation of complex bacterial communities. However, ‘universal’ PCR primers can introduce biases into the analysis of the species composition of clone libraries because of mismatches between the primer and target organism sequences. In this study, three universal primer sets were compared for the analysis of the microflora in subgingival plaque.

Methods:  Three subgingival plaque samples were collected from two subjects with localised severe chronic periodontitis. Half of each sample was cultured while DNA was extracted from the remaining half and 16S rDNA amplified with universal primer pairs 27F, 1525R (A); 27F, 1492R (B) and 530F, 1525R (C). Amplified genes were cloned, sequenced and identified by comparison with 16S rRNA databases.

Results:  137 taxa were identified among 177 isolates and 417 clones sequenced. Of these, 86 were detected only by the molecular technique whereas 26 were found only by culture. Sequences from 81 taxa did not correspond to those of named species and of these, 38 were not represented in the nucleotide databases. 16S RNA genes for these 38 taxa were sequenced and deposited with GenBank.

Conclusion:  The use of three sets of universal primers allowed the identification of 38 novel bacterial phylotypes. There were marked differences in the composition of the libraries generated by the different primer sets. A combination of molecular and cultural techniques is recommended to maximise the coverage of detection of bacterial taxa in oral samples.
Stimulation of Fusobacterium nucleatum biofilm formation by Porphyromonas gingivalis
Authors: Saito Y. , Fujii R. , Nakagawa K.‐I. , Kuramitsu H. K. , Okuda K. , Ishihara K.
Published: September 2010

Background/aims:  Bacterial infection is a major cause of periapical periodontitis. Eradication of these microorganisms from apical lesions is essential to the success of endodontic treatment. The aim of this study was to clarify the molecular interaction between Fusobacterium nucleatum, Porphyromonas gingivalis and other microorganisms associated with periapical periodontitis.

Methods:  Microorganisms isolated from periapical lesions were inoculated into type‐I collagen‐coated polystyrene microtiter plates and maintained at 37°C under anaerobic conditions for 2 days, after which, the quantity of organized biofilm on the plates was evaluated by crystal violet staining. Growth enhancement via soluble factor was evaluated by separated coculture using a 0.4‐μm membrane filter.

Results:  F. nucleatum exhibited strong adherence to type‐I collagen‐coated polystyrene microplates. Biofilm formation by F. nucleatum was significantly enhanced by P. gingivalis. It was complemented by compartmentalized coculture with P. gingivalis. Enhancement of biofilm formation by P. gingivalis was only slightly reduced by inactivation of its autoinducer‐2‐producing gene luxS.

Conclusion:  The results suggest that P. gingivalis enhances biofilm formation by F. nucleatum by releasing diffusible signaling molecules other than autoinducer‐2.
Bacterial composition and red fluorescence of plaque in relation to primary and secondary caries next to composite: an in situ study
Authors: Thomas R. Z. , Van Der Mei H. C. , Van Der Veen M. H. , De Soet J. J. , Huysmans M. C. D. N. J. M.
Published: September 2010

Background/hypothesis:  Secondary caries has been suggested as the main reason for restoration replacement. We hypothesized that more caries‐associated bacteria are found on composite resin restoration material, compared to sound tooth tissue.

Methods:  Both restored and unrestored dentin and enamel samples were placed in a full denture of eight subjects for 20 weeks. The microbiological composition of approximal plaque and the association between caries‐associated bacteria and red autofluorescence of dental plaque was studied. Every 4 weeks the specimens were microradiographed using transversal wavelength independent microradiography (T‐WIM). After 1 and 20 weeks red fluorescence pictures and plaque samples were taken. Samples were cultured for total anaerobic counts, mutans streptococci, lactobacilli, candida and Actinomyces odontolyticus.

Results:  Lesion depth in the dentin and enamel was positively associated with lactobacilli, and lesion depth in dentin was positively associated with A. odontolyticus, whereas no association was found between mutans streptococci and lesion depth. The red‐fluorescent bacteria A. odontolyticus and lactobacilli did not correlate with red‐fluorescent plaque, indicating that red fluorescence is probably not caused by a single species of these bacteria. After 20 weeks, a higher proportion of combined mutans streptococci and lactobacilli was found on restored tissue compared to non‐restored tissue (P = 0.04).

Conclusion:  The higher proportion of caries‐associated bacteria on restored tissue indicates that the ecology on the surface of primary lesions differs from that on lesions next to composite, and that secondary caries next to composite may differ from the primary caries process.
Effects of antibodies against a fusion protein consisting of parts of cell surface protein antigen and glucosyltransferase of Streptococcus sobrinus on cell adhesion of mutans streptococci
Authors: Kawato T. , Yamashita Y. , Katono T. , Kimura A. , Maeno M.
Published: September 2010

Background/aims:  The cell surface protein antigen (PAg) and glucosyltransferases (GTFs) produced by Streptococcus sobrinus are considered to be major colonization factors of the organism.

Methods:  We constructed a fusion gene encoding a protein composed of the alanine‐rich region of PAg (PAgA) and the glucan‐binding domain (GB) of GTF‐I, which catalyzes the synthesis of water‐insoluble glucan in S. sobrinus. The fusion protein PAgA‐GB was purified from cell extracts of Escherichia coli harboring the fusion gene, and antibodies against the fusion protein were prepared in rabbits.

Results:  In the presence of sucrose, the antibody against PAgA‐GB significantly inhibited the adhesion of both S. sobrinus MT8145 and Streptococcus mutans Xc to saliva‐coated hydroxyapatite beads, and the inhibitory effect on S. sobrinus was stronger than that on S. mutans. In the absence of sucrose, the antibody against PAgA‐GB significantly inhibited the adhesion of both S. sobrinus and S. mutans, however the inhibitory effect on S. sobrinus was unexpectedly weaker than that on S. mutans. A similar result was observed with the antibody against the intact recombinant PAg protein (rPAg), while the same antibody reacted more strongly against S. sobrinus than against S. mutans cells.

Conclusion:  Taken together, these results show that the antibody against S. sobrinus GTF‐I may be useful for effective inhibition of the sucrose‐dependent adhesion of S. sobrinus. However, PAg of S. sobrinus may not function primarily as a receptor for acquired pellicles, and other cell surface proteins may be involved in the sucrose‐independent adhesion of S. sobrinus.
The stage of native biofilm formation determines the gene expression of human β‐defensin‐2, psoriasin, ribonuclease 7 and inflammatory mediators: a novel approach for stimulation of keratinocytes with in situ formed biofilms
Authors: Eberhard J. , Menzel N. , Dommisch H. , Winter J. , Jepsen S. , Mutters R.
Published: September 2010

Background/aims:  Antimicrobial peptides such as human β‐defensin‐2 (hBD‐2), psoriasin (PSO), and ribonuclease 7 (RNase 7) play an important role in innate immunity. The aim of the present study was to test the hypothesis that epithelial cells show a differential gene expression pattern of antimicrobial peptides (hBD‐2, PSO, RNase 7) and inflammatory mediators such as interleukin‐8 (IL‐8) and 5‐lipoxygenase (5‐LO) in response to different stages of naturally formed biofilms.

Methods:  Epithelial cells were cultured from biopsies obtained from five healthy individuals. Native bacterial biofilms were taken from the same subjects that donated the gingival biopsies. To obtain different stages of biofilm formation, polymer disks were attached to prostheses and carried intraorally for 1, 3, 5, and 9 days. The expression of genes for hBD‐2, PSO, RNase 7, 5‐LO, and IL‐8 was examined using semi‐quantitative reverse transcription–polymerase chain reaction. The bacterial composition of the individual biofilms was defined using a microarray system (Parocheck®), which showed the presence of 20 different bacterial species that are associated with plaque formation.

Results:  The expression of the messenger RNAs of hBD‐2, RNase 7, and 5‐LO was upregulated as a result of the exposure to early biofilm stages, whereas the gene expression of IL‐8 was increased in response to matured biofilms. Inter‐individual differences in the innate immune response were observed.

Conclusion:  The results of the present study showed a time‐dependent messenger RNA expression of antimicrobial peptides (hBD‐2, RNase 7), 5‐LO, and IL‐8 in oral epithelial cells responding to different stages of biofilm formation.
Biological properties of the native and synthetic lipid A of Porphyromonas gingivalis lipopolysaccharide
Authors: Kumada H. , Haishima Y. , Watanabe K. , Hasegawa C. , Tsuchiya T. , Tanamoto K. , Umemoto T.
Published: September 2010

Introduction and methods:  A pentaacyl and diphosphoryl lipid A molecule found in the lipid A isolated from Porphyromonas gingivalis lipopolysaccharide (LPS) was chemically synthesized, and its characteristics were evaluated to reconfirm its interesting bioactivities including low endotoxicity and activity against LPS‐unresponsive C3H/HeJ mouse cells.

Results:  The synthesized P. gingivalis lipid A (synthetic Pg‐LA) exhibited strong activities almost equivalent to those of Escherichia coli‐type synthetic lipid A (compound 506) in all assays on LPS‐responsive mice, and cells. LPS and native lipid A of P. gingivalis displayed overall endotoxic activities, but its potency was reduced in comparison to the synthetic analogs. In the assays using C3H/HeJ mouse cells, the LPS and native lipid A significantly stimulated splenocytes to cause mitosis, and peritoneal macrophages to induce tumor necrosis factor‐α and interleukin‐6 production. However, synthetic Pg‐LA and compound 506 showed no activity on the LPS‐unresponsive cells. Inhibition assays using some inhibitors including anti‐human Toll‐like receptor 2 (TLR2) and TLR4/MD‐2 complex monoclonal antibodies showed that the biological activity of synthetic Pg‐LA was mediated only through the TLR4 signaling pathway, which might act as a receptor for LPS, whereas TLR2, possibly together with CD14, was associated with the signaling cascade for LPS and native lipid A of P. gingivalis, in addition to the TLR4 pathway.

Conclusion:  These results suggested that the moderated and reduced biological activity of P. gingivalis LPS and native lipid A, including their activity on C3H/HeJ mouse cells via the TLR2‐mediated pathway, may be mediated by bioactive contaminants or low acylated molecules present in the native preparations having multiple lipid A moieties.
Identification of the genes associated with a virulent strain of Porphyromonas gingivalis using the subtractive hybridization technique
Authors: Tachibana‐Ono M. , Yoshida A. , Kataoka S. , Ansai T. , Shintani Y. , Takahashi Y. , Toyoshima K. , Takehara T.
Published: September 2010

Background/aims:  Porphyromonas gingivalis, a major etiological organism implicated in periodontal disease, can be classified into virulent and avirulent strains. Our aim was to identify a gene for the virulence of P. gingivalis.

Methods:  The subtractive hybridization technique was employed to identify the genes specific to P. gingivalis W83, a virulent strain. In this study, P. gingivalis W83 was used as the tester strain, and P. gingivalis ATCC 33277 was the driver strain. The prevalence of W83‐specific genes was determined by Southern blot analysis of several P. gingivalis strains.

Results:  We obtained 575 colonies using the subtractive hybridization technique. From among these, 26 DNA fragments were subjected to a homology search using the BLAST program. Compared with strain ATCC 33277, strain W83 contained 12 unique clones. The specificities of the isolated DNA fragments were analyzed among four P. gingivalis strains by Southern blot analysis. Five genes showed specificity for strain W83 compared with strain ATCC 33277. All five genes were also identified in strain W50.

Conclusions:  The subtractive hybridization technique was effective in screening the two strains for specific DNA sequences, some of which might be responsible for determining virulence. The results suggested that several genes specific to strain W83 were associated with its virulence. Further analysis of these DNA fragments will provide important information on the pathogenesis of virulent P. gingivalis strains.
Correlations of oral bacterial arginine and urea catabolism with caries experience
Authors: Nascimento M. M. , Gordan V.V. , Garvan C. W. , Browngardt C. M. , Burne R. A.
Published: September 2010

Background/aim:  Alkali generation by oral bacteria plays a key role in plaque pH homeostasis and may be a major impediment to the development of dental caries. To determine if the capacity of oral samples to produce ammonia from arginine or urea was related to caries experience, the arginine deiminase system (ADS) and urease activity in saliva and dental plaque samples were measured in 45 adult subjects.

Methods:  The subjects were divided into three groups according to caries status; 13 caries‐free (CF) individuals (decayed, missing, and filled teeth = 0); 21 caries‐active (CA) individuals (decayed teeth ≥ 4); and 11 caries‐experienced (CE) individuals (decayed teeth = 0; missing and filled teeth > 0). Real‐time polymerase chain reaction was used to quantify the proportion of certain acid‐ or alkali‐producing organisms in the samples.

Results:  The amount of ammonia generated from the test substrates by plaque samples was generally higher than that produced by salivary samples in all groups. Significantly higher levels of salivary ADS activity and plaque urease activity were observed in CF subjects compared to CA subjects (P = 0.0004 and P = 0.014, respectively). The proportions of Streptococcus mutans from saliva and dental plaque of CA subjects were significantly higher than those from the CF group (P = 0.0153 and P = 0.0009, respectively). In the CA group, there was an inverse relationship between urease activity and the levels of S. mutans (P < 0.0001).

Conclusion:  This study supports the theory that increased caries risk is associated with reduced alkali‐generating capacity of the bacteria colonizing the oral cavity; providing compelling evidence to further our understanding of oral alkali‐generation in health and disease.
Invasion of human coronary artery endothelial cells by Streptococcus mutans OMZ175
Authors: Abranches J. , Zeng L. , Bélanger M. , Rodrigues P. H. , Simpson‐Haidaris P. J. , Akin D. , Dunn Jr W. A. , Progulske‐Fox A. , Burne R. A.
Published: September 2010

Introduction:  Dissemination of oral bacteria into the bloodstream has been associated with eating, oral hygiene, and dental procedures; including tooth extraction, endodontic treatment, and periodontal surgery. Recently, studies identified Streptococcus mutans, the primary etiological agent of dental caries, as the most prevalent bacterial species found in clinical samples from patients who underwent heart valve and atheromatous plaque surgery.

Methods:  By using antibiotic protection assays, we tested the capacity of 14 strains of S. mutans to invade primary human coronary artery endothelial cells (HCAEC).

Results:  Serotype e strain B14 and serotype f strain OMZ175 of S. mutans were able to efficiently invade HCAEC. Among the tested strains, serotype f S. mutans OMZ175 was the most invasive, whereas strains of serotype c S. mutans, the most prevalent serotype in dental plaque, were not invasive. Based on its high invasion rate, we further investigated the invasive properties of serotype f OMZ175. Using transmission electron microscopy and antibiotic protection assays we demonstrate that S. mutans OMZ175 is capable of attaching to the HCAEC surface, entering the cells and surviving in HCAEC for at least 29 h.

Discussion:  Our findings highlight a potential role for S. mutans in the pathogenesis of certain cardiovascular diseases.