Microbiota at teeth and implants in partially edentulous patients. A 10-year retrospective study.

OBJECTIVE To determine the microbiota at implants and adjacent teeth 10 years after placement of implants with a sandblasted and acid-etched surface. MATERIAL AND METHODS Plaque samples obtained from the deepest sites of 504 implants and of 493 adjacent teeth were analyzed for certain bacterial species associated with periodontitis, for staphylococci, for aerobic gram-negative rods, and for yeasts using nucleic acid-based methods. RESULTS Species known to be associated with periodontitis were detectable at 6.2-78.4% of the implants. Significantly higher counts at implants in comparison with teeth were assessed for Tannerella forsythia, Parvimonas micra, Fusobacterium nucleatum/necrophorum, and Campylobacter rectus. Higher counts of periodontopathogenic species were detectable at implants of current smokers than at those of non-smokers. In addition, those species were found in higher quantities at implants of subjects with periodontitis. The prevalence of Prevotella intermedia, Treponema denticola, C. rectus, and moreover of Staphylococcus warneri might be associated with peri-implant inflammation. Selected staphylococcal species (not Staphylococcus aureus), aerobic gram-negative rods, and yeasts were frequently detected, but with the exception of S. warneri, they did not show any association with periodontal or peri-implant diseases. CONCLUSIONS Smoking and periodontal disease are risk factors for colonization of periodontopathic bacteria at implants. Those bacterial species may play a potential role in peri-implant inflammation. The role of S. warneri needs further validation.

Absence of Hepatitis Delta Infection in a Large Rural HIV Cohort in Tanzania

OBJECTIVES The epidemiological and clinical determinants of hepatitis delta virus (HDV) infection in sub-Saharan Africa are ill-defined. We determined the prevalence of HDV infection in HIV/hepatitis B virus (HBV)-co-infected individuals in rural Tanzania. DESIGN AND METHODS We screened all hepatitis B virus (HBV)-infected adults under active follow-up in the Kilombero and Ulanga Antiretroviral Cohort (KIULARCO) for anti-HDV antibodies. In positive samples, we performed a second serological test and nucleic acid amplification. Demographic and clinical characteristics at initiation of antiretroviral therapy (ART) were compared between anti-HDV-negative and positive patients. RESULTS Among 222 HIV/HBV-coinfected patients on ART, 219 (98.6%) had a stored serum sample available and were included. Median age was 37 years, 55% were female, 46% had WHO stage III/IV HIV disease and median CD4 count was 179 cells/μL. The prevalence of anti-HDV positivity was 5.0% (95% confidence interval 2.8%-8.9%). There was no significant predictor of anti-HDV positivity. HDV could not be amplified in any of the anti-HDV-positive patients and the second serological test was negative in all of them. CONCLUSIONS We found no confirmed case of HDV infection among over 200 HIV/HBV-co-infected patients in Tanzania. As false-positive serology results are common, screening results should be confirmed with a second test.

Citrullination in the periodontium-a possible link between periodontitis and rheumatoid arthritis

OBJECTIVES The aim of the present study was to assess human and bacterial peptidylarginine deiminase (PAD) activity in the gingival crevicular fluid (GCF) in the context of serum levels of antibodies against citrullinated epitopes in rheumatoid arthritis and periodontitis. MATERIALS AND METHODS Human PAD and Porphyromonas gingivalis-derived enzyme (PPAD) activities were measured in the GCF of 52 rheumatoid arthritis (RA) patients (48 with periodontitis and 4 without) and 44 non-RA controls (28 with periodontitis and 16 without). Serum antibodies against citrullinated epitopes were measured by ELISA. Bacteria being associated with periodontitis were determined by nucleic-acid-based methods. RESULTS Citrullination was present in 26 (50 %) RA patients and 23 (48 %) controls. PAD and PPAD activities were detected in 36 (69 %) and 30 (58 %) RA patients, respectively, and in 30 (68 %) and 21 (50 %) controls, respectively. PPAD activity was higher in RA and non-RA patients with periodontitis than in those without (p = 0.038; p = 0.004), and was detected in 35 of 59 P. gingivalis-positive samples, and in 16 of 37 P. gingivalis-negative samples in association with high antibody levels against that species. CONCLUSIONS PAD and PPAD activities within the periodontium are elevated in RA and non-RA patients with periodontitis. PPAD secreted by P. gingivalis residing in epithelial cells may exert its citrullinating activity in distant regions of the periodontium or even distant tissues. CLINICAL RELEVANCE In periodontitis, the citrullination of proteins/peptides by human and bacterial peptidylarginine deiminases may generate antibodies after breaching immunotolerance in susceptible individuals.

A New Family of High-Affinity Transporters for Adenine, Cytosine, and Purine Derivatives in Arabidopsis

In many organisms, including plants, nucleic acid bases and derivatives such as caffeine are transported across the plasma membrane. Cytokinins, important hormones structurally related to adenine, are produced mainly in root apices, from where they are translocated to shoots to control a multitude of physiological processes. Complementation of a yeast mutant deficient in adenine uptake (fcy2) with an Arabidopsis cDNA expression library enabled the identification of a gene, AtPUP1 (for Arabidopsis thaliana purine permease1), belonging to a large gene family (AtPUP1 to AtPUP15) encoding a new class of small, integral membrane proteins. AtPUP1 transports adenine and cytosine with high affinity. Uptake is energy dependent, occurs against a concentration gradient, and is sensitive to protonophores, potentially indicating secondary active transport. Competition studies show that purine derivatives (e.g., hypoxanthine), phytohormones (e.g., zeatin and kinetin), and alkaloids (e.g., caffeine) are potent inhibitors of adenine and cytosine uptake. Inhibition by cytokinins is competitive (competitive inhibition constant Ki = 20 to 35 μM), indicating that cytokinins are transported by this system. AtPUP1 is expressed in all organs except roots, indicating that the gene encodes an uptake system for root-derived nucleic acid base derivatives in shoots or that it exports nucleic acid base analogs from shoots by way of the phloem. The other family members may have different affinities for nucleic acid bases, perhaps functioning as transporters for nucleosides, nucleotides, and their derivatives.

Mechanism of methylation gene reactivation in human cancer cells

Epigenetic silencing of tumor suppressor genes by DNA hypermethylation at promoter regions is a common event in carcinogenesis and tumor progression. Abrogation of methylation and reversal of epigenetic silencing is a very potent way in cancer treatment. However, the reactivation mechanisms are poorly understood. In this study, we first developed a cell line model system named YB5, derived from SW48 cancer cell line, which bears one copy of stably integrated EGFP gene on Chromosome 1p31.1 region. The GFP gene expression is transcriptionally silenced due to the hypermethylated promoter CMV. However, the GFP expression can be restored using demethylating agent 5-aza-2' deoxycytidine (DAC), and detected by FACS and fluorescent microscopy. Using this system, we observed the heterogeneous reactivation induced by DAC treatment. After flow sorting, GFP negative cells exhibited similar level of incomplete demethylation compared to GFP positive cells on repetitive LINE1 element, tumor suppressor genes such as P16, CDH13, and RASSF1a, and CMV promoter as well. However, the local chromatin of CMV-GFP locus altered to an open structure marked by high H3 lysine 9 acetylation and low H3 lysine 27 tri-methylation in GFP positive cells, while the GFP negative cells retained mostly the original repressive marks. Thus, we concluded that DAC induced DNA hypomethylation alone does not directly determine the level of re-expression, and the resetting of the local chromatin structure under hypomethylation environment is required for gene reactivation. Besides, a lentivirus vector-based shRNA screening was performed using the YB5 system. Although it is the rare chance that vector lands in the neighboring region of GFP, we found that the exogenous vector DNA inserted into the upstream region of GFP gene locus led to the promoter demethylation and reactivated the silenced GFP gene. Thus, epigenetic state can be affected by changing of the adjacent nucleic acid sequences. Further, this hypermethylation silenced system was utilized for epigenetic drug screening. We have found that DAC combined with carboplatin would enhance the GFP% yield and increase expression of other tumor suppressor genes than DAC alone, and this synergistic effect may be related to DNA repair process. In summary, these studies reveal that reversing of methylation silencing requires coordinated alterations of DNA methylation, chromatin structure, and local microenvironment. ^

Consistent increase in dimethyl sulfide (DMS) in response to high CO2 in five shipboard bioassays from contrasting NW European waters

The ubiquitous marine trace gas dimethyl sulfide (DMS) comprises the greatest natural source of sulfur to the atmosphere and is a key player in atmospheric chemistry and climate. We explore the short-term response of DMS production and cycling and that of its algal precursor dimethyl sulfoniopropionate (DMSP) to elevated carbon dioxide (CO2) and ocean acidification (OA) in five 96 h shipboard bioassay experiments. Experiments were performed in June and July 2011, using water collected from contrasting sites in NW European waters (Outer Hebrides, Irish Sea, Bay of Biscay, North Sea). Concentrations of DMS and DMSP, alongside rates of DMSP synthesis and DMS production and consumption, were determined during all experiments for ambient CO2 and three high-CO2 treatments (550, 750, 1000 µatm). In general, the response to OA throughout this region showed little variation, despite encompassing a range of biological and biogeochemical conditions. We observed consistent and marked increases in DMS concentrations relative to ambient controls (110% (28-223%) at 550 µatm, 153% (56-295%) at 750 µatm and 225% (79-413%) at 1000 µatm), and decreases in DMSP concentrations (28% (18-40%) at 550 µatm, 44% (18-64%) at 750 µatm and 52% (24-72%) at 1000 µatm). Significant decreases in DMSP synthesis rate constants (µDMSP /d) and DMSP production rates (nmol/d) were observed in two experiments (7-90% decrease), whilst the response under high CO2 from the remaining experiments was generally indistinguishable from ambient controls. Rates of bacterial DMS gross consumption and production gave weak and inconsistent responses to high CO2. The variables and rates we report increase our understanding of the processes behind the response to OA. This could provide the opportunity to improve upon mesocosm-derived empirical modelling relationships and to move towards a mechanistic approach for predicting future DMS concentrations.

Benthic respiration and physical oceanography on two contrasting continental margins in the western Indian Ocean: the Yemen-Somali upwelling region and the margin off Kenya

During the Netherlands Indian Ocean Project (NIOP, 1992-1993) sediment community oxygen consumption (SCOC) was measured on two continental margins in the Indian Ocean with different productivity: the productive upwelling region off Yemen-Somalia and the supposedly less productive Kenyan margin, which lacks upwelling. The two margins also differ in terms of river input (Kenya) and the more severe oxygen minimum in the Arabian Sea. Simultaneously with SCOC, distributions of benthic biomass and phytodetritus were studied. Our expectation was that benthic processes in the upwelling margin of the Arabian Sea would be relatively enhanced as a result of the higher productivity. On the Kenyan margin, SCOC (range 1-36 mmol/m**2/d) showed a clear decrease with increasing water depth, and little temporal variation was detected between June and December. Highest SCOC values of this study were recorded at 50 m depth off Kenya, with a maximum of 36 mmol/m**2/d in the northernmost part. On the margin off Yemen-Somalia, SCOC was on average lower and showed little downslope variation, 1.8-5.7 mmol/m**2/d, notably during upwelling, when the zone between 70 and 1700 m was covered with low O2 water (10-50 µM). After cessation of upwelling, SCOC at 60 m depth off Yemen increased from 5.7 to 17.6 mmol/m**2/d concurrently with an increase of the near-bottom O2 concentration (from 11 to 153 µM), suggesting a close coupling between SCOC and O2 concentration. This was demonstrated in shipboard cores in which the O2 concentration in the overlying water was raised after the cores were first incubated under in situ conditions (17 µM O2). This induced an immediate and pronounced increase of SCOC. Conversely, at deeper stations permanently within the oxygen minimum zone (OMZ), SCOC showed little variation between monsoon periods. Hence, organic carbon degradation in sediments on a large part of the Yemen slope appears hampered by the oxygen deficiency of the overlying water. Macrofauna biomass and the pooled biomass of smaller organisms, estimated by the nucleic acid content of the sediment, had comparable ranges in the two areas in spite of more severe suboxic conditions in the Arabian Sea. At the Kenyan shelf, benthic fauna (macro- and meiofauna) largely followed the spatial pattern of SCOC, i.e. high values on the northern shelf-upper slope and a downslope decrease. On the Yemen-Somali margin the macrofauna distribution was more erratic. Nucleic acids displayed no clear downslope trend on either margin owing to depressed values in the OMZ, perhaps because of adverse effects of low O2 on small organisms (meiofauna and microbes). Phytodetritus distributions were different on the two margins. Whereas pigment levels decreased downslope along the Kenya margin, the upper slope off Yemen (800 m) had a distinct accumulation of mainly refractory carotenoid pigments, suggesting preservation under low 02. Because the accumulations of Corg and pigments on the Yemen slope overlap only partly, we infer a selective deposition and preservation of labile particles on the upper slope, whereas refractory material undergoes further transport downslope.

Abundance of bacteria and virus during the METEOR cruise M87/1

In temperate, subpolar and polar marine systems, the classical perception that bacteria are carbon limited by end of winter and respond in activity and abundance to the production of new carbon during the diatom spring bloom and post bloom. Contrary to this view, we here document an strong increase in bacterial abundance and activity (latter measured by increasing high nuclei acid (HNA) to low nuclei acid (LNA) bacteria ratio) during the winter-spring transition, where phytoplankton smaller than 10 µm dominate. Further DNA-virus were enumerated and revealed the virus to bacteria ratio (VBR) to be decreasing during winter-spring transition, indicating that the virus did not increase in number accordingly to bacteria. During repeated visits to stations in the deep Icelandic and the Norwegian Basins and the shallow Shetland Shelf (26 March to 29 April 2012), we investigated the abundance of bacteria and the succession of HNA:LNA bacteria and VBR. Water samples were collected from CTD rosette .10 L Niskin bottles and fixed in glutaraldehyde (final conc. 5%), flash frozen in liquid Nitrogen and stored at -80°C until analysis.

Composition of bacterioplankton in the North Atlantic subtropical gyre

Subtropical oceanic gyres are the most extensive biomes on Earth where SAR11 and Prochlorococcus bacterioplankton numerically dominate the surface waters depleted in inorganic macronutrients as well as in dissolved organic matter. In such nutrient poor conditions bacterioplankton could become photoheterotrophic. We assessed the photoheterotrophy of the key microbial taxa in the North Atlantic oligotrophic gyre and adjacent regions. The experimental work was performed on board the Royal Research Ship James Cook (cruise no. JC53, October-November 2010) as part of the Atlantic Meridional Transect programme, and on board the Royal Research Ship Discovery (cruise no. D369, August-September 2011). At each station, samples were collected from 20m depth with a sampling rosette of 20-l Niskin bottles mounted on aconductivity-temperature-depth profiler. Samples were collected in 1 l thermos flasks (washed with10% v/v HCl) in the dark and processed immediately. Depth of 20m was chosen because it represents the mixed layer and it was the shallowest depth unaffected by the ship's movement, including thrusting, that could artificially affect microbial metabolism in nutrient-depleted stratified surfacewaters. Molecular identification of flow-sorted cells CARD-FISH was performed on flow-sorted cells to identify the groups for which uptake rates were measured. High nucleic acid-containing bacteria, based on SYBR Green DNA staining, that had virtually undetectable chlorophyll autofluorescence, were phylogenetically affiliated with Prochlorococcus,in agreement with our previously reported results (Zubkov et al., 2007; doi:10.1111/j.1462-2920.2007.01324.x).

Detection and characterization of carcinoma cells in the blood

A highly sensitive assay combining immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis has been developed to detect, enumerate, and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood. Peripheral blood (10–20 ml) from 30 patients with carcinoma of the breast, from 3 patients with prostate cancer, and from 13 controls was examined by flow cytometry for the presence of circulating epithelial cells defined as nucleic acid+, CD45−, and cytokeratin+. Highly significant differences in the number of circulating epithelial cells were found between normal controls and patients with cancer including 17 with organ-confined disease. To determine whether the circulating epithelial cells in the cancer patients were neoplastic cells, cytospin preparations were made after immunomagnetic enrichment and were analyzed. Epithelial cells from patients with breast cancer generally stained with mAbs against cytokeratin and 3 of 5 for mucin-1. In contrast, no cells that stained for these antigens were observed in the blood from normal controls. The morphology of the stained cells was consistent with that of neoplastic cells. Of 8 patients with breast cancer followed for 1–10 months, there was a good correlation between changes in the level of tumor cells in the blood with both treatment with chemotherapy and clinical status. The present assay may be helpful in early detection, in monitoring disease, and in prognostication.