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"Conversion" has often been used to designate an event or period of discrete and intense change, especially in relation to an individual's belief and religious identity. The sources on Francis of Assisi's conversion show how pleasure and unpleasure converged in relation to material objects and the affects that they helped to create and sustain. Among others, Francis of Assisi's objects of conversion included lepers, cloth, fire, ice, and his own body. Instead of conversion as a discrete change in a predictable direction, taking the materials of Francis's conversion as the primary objects of the story animates a sense of perfect jouissance, suggesting that conversion entails realignments between pleasure and unpleasure—an experience Francis described, toward the end of his life, as "perfect joy." [ABSTRACT FROM AUTHOR]

The Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers. [ABSTRACT FROM AUTHOR]

Abstract: RT-PCR is the method of choice for detecting BCR–ABL in CML and ALL. The three predominant mRNA transcripts found are e1a2 (in ALL), e13a2, and e14a2 (in CML and ALL). However, a number of “atypical”BCR–ABL transcripts (e1a3, e13a3, e14a3, e19a2, e6a2, e8a2, etc.) resulting from chromosomal breakpoints outside ABL intron 1 or BCR intron 1, 13 or 14, respectively, have been reported. These atypical transcripts may escape detection when using methods that are optimized to detect just the typical ones. We present here a novel, fast, and reliable multiplex PCR for improved detection of typical and atypical BCR–ABL transcripts. [Copyright &y& Elsevier]

Aims: To determine the clinical efficacy of a 2-year continuous sub-antimicrobial dose doxycycline (SDD; 20 mg bid) in post-menopausal, osteopenic, oestrogen-deficient women on periodontal maintenance. Materials and Methods: One-hundred and twenty-eight subjects were randomized to SDD ( n=64) or placebo ( n=64). Clinical measurements were performed at posterior interproximal sites at baseline and every 6 months during this 2-year randomized, double-blind, placebo-controlled clinical trial with adjunctive, no-cost 3–4-month periodontal maintenance. Statistical analyses of secondary outcomes from this clinical trial used Generalized Estimating Equations in primarily intent-to-treat analyses. Results: For the placebo group, 3.4% of the sites showed improvement in clinical attachment levels (CAL) and 2.7% had progressive loss in CAL; for the SDD group, 5.0% of the sites showed an improvement in CAL and 2.2% had progressive loss in CAL. This difference (2.1% of sites) was more favourable in the SDD group than in the placebo [odds ratio (OR)=0.81%, 95% confidence interval (CI): 0.67–0.97, p=0.03] in these well-maintained patients, whereas probing depths, bleeding on probing and supragingival plaque did not differ significantly between groups ( p>0.2). However, in exploratory subgroup analysis of non-smokers, SDD showed reduced bleeding versus placebo (27% versus 33%; p=0.05). In protocol-adherent subjects, the odds of bleeding were 34% lower for SDD ( p=0.05). Conclusions: Analyses of secondary outcomes of this clinical trial indicated that SDD may be of benefit in reducing progressive attachment loss in post-menopausal females; additional research is needed to confirm these findings. Protocol registered at ClinicalTrials.gov. Identifier:NCT00066027 [ABSTRACT FROM AUTHOR]

There are many types of diseases associated with the oral cavity. As an adjunct to professional oral dental care, various drug delivery systems have been developed to improve the treatment or prevention of these diseases. In this manuscript, the current status of drug delivery strategies for these common orofacial diseases is reviewed. Biomineral-binding drug delivery and on-demand drug release are suggested to be considered in the future design of drug delivery systems for orofacial diseases. Apparently, this research field deserves much more attention from both pharmaceutical scientists and dental health professionals. [ABSTRACT FROM AUTHOR]

Reinhardt RA, Sanderfer VJ, Meinberg TA, Nummikoski P, Lee H-M, Marx DB: Local biochemical markers of bone turnover: relationship to subsequent density of healing alveolar bone defects. J Clin Periodontol 2004; 31: –. © Blackwell Munksgaard, 2004. This pilot study was designed to test whether biochemical markers of bone turnover in washes of periosteal or trabecular alveolar bone surfaces could be correlated with increases in bone density of an adjacent healing implant socket. Ten subjects had a canula inserted into the alveolar crest and sterile phosphate-buffered saline was washed over the periosteal and trabecular surfaces and collected. Surgical flaps were reflected, 5 mm diameter bone cores were removed from the bone wash site, and standardized radiographs were taken. The sites were allowed to heal for 12 weeks, and radiographs were repeated. Bone washes of the healing sites were also collected after 2 and 12 weeks. Washes were analysed for bone turnover markers osteocalcin (OC; radioimmunoassay) and C-terminal telopeptide of Type 1 collagen (ICTP; enzyme-linked immunosorbent assay (ELISA)), and blood component albumin (ALB; ELISA). Changes in bone density during healing were determined by radiographic absorptiometry. OC/ALB and ICTP/ALB ratios were higher for trabecular than periosteal washes at baseline ( p⩽0.01). Trabecular OC/ALB and ICTP/ALB were inversely correlated with increasing bone density of the healing bone core socket ( r=−0.72, p=0.03; Pearson's correlation coefficient). Biochemical markers of bone turnover in bone washes of specific alveolar bone sites may prove helpful in predicting how the bone density will increase around healing dental implants. [ABSTRACT FROM AUTHOR]

AbstractBackground, aims: The purpose of this 2-year longitudinal clinical study was to determine the impact of smoking on alveolar bone height and density changes in postmenopausal females. Methods: 59 postmenopausal women completed this study, including 38 non-smokers and 21 smokers. All subjects had a history of periodontitis, participated in 3- to 4-month periodontal maintenance programs and were within 5 years of menopause at the study outset. 4 vertical bite-wing radiographs of posterior sextants were taken at baseline and 2-year visits. Radiographs were evaluated using computer-assisted densitometric image analysis (CADIA); changes in interproximal alveolar bone density and changes in alveolar bone height were determined. Relative clinical attachment levels (RCAL) and presence/absence of plaque and bleeding on probing were recorded. Results: Smokers exhibited a higher frequency of alveolar bone height loss (p< 0.05) and crestal (p<0.03) and subcrestal (p<0.02) density loss relative to non-smokers. Smokers exhibited a trend (p<0.08) toward a higher frequency of >=2.0 mm RCAL loss over the 2-year period. Plaque and bleeding on probing did not differ between smokers and non-smokers. A significant interaction, determined by repeated measures ANOVA, was noted between systemic bone mineral density (BMD) at the lumbar spine and smoking on alveolar bone density change (p<0.05). Only non-smoking patients with normal BMD realized a mean net gain in alveolar bone density; osteoporotic/osteopenic subjects (n=25) and smokers lost alveolar bone density. Conclusion: Postmenopausal female smokers were more likely to lose alveolar bone height and density than non-smokers with a similar periodontitis, plaque and gingival bleeding experience. In addition, both smoking and osteoporosis/osteopenia provided a negative influence on alveolar bone. [ABSTRACT FROM AUTHOR]

Intracrevicular antimicrobial therapy is consistent with the site-specific nature of periodontitis. Considerable research has focused on the use of nonresorbable fibers. However, a bioerodible system is desirable. The purpose of this study was to assess tetracycline release and safety following a single application of a syringable 35% tetracycline hydrochloride in a lactic-glycolic acid gel. 31 generally healthy adult volunteers (mean age = 59 years) were enrolled in and completed this randomized, double-blind eight day study. 2, 6-10 mm non-adjacent interproximal pockets that bled on pocket probing were chosen as experimental sites in each subject. 1 experimental site and the surrounding gingival crevice received small particle size tetracycline in gel while the other site received larger particle size tetracycline in gel. Gingival crevicular fluid (GCF) was collected prior to treatment and 15 min, 1, 2, 3, 4 and 8 days post-treatment. GCF tetracyline concentrations were determined by agar diffusion bioassay and GCF volume measurements. 61% and 71% of sites had ≥ 100 μg/ml tetracycline 3 days following application of large (mean concentration = 430 ±92 μg/ml) and small particle gels (mean concentration = 418 ±70 μg/ml), respectively. 37% and 55% of sites had measurable tetracycline 8 days after placement of large (mean concentration = 86 ±31 μg/ml) and small particle gels (mean concentration = 293 ±79 μg/ml), respectively. The most common adverse event was "bitter taste" (10% of subjects). Based upon the reduction in probing depths and % of sites bleeding on probing at 8 days relative to pretreatment, and the absence of any serious adverse events, it is concluded that these bioerodible gels are safe, and since the bacteriostatic range for most putative periodontopathogens is in the 2-10 μg/ml range, the tetracycline levels observed at days 3 and 8 likely represent significant antimicrobial efficacy. [ABSTRACT FROM AUTHOR]

Controlled local delivery of antibiotics has been shown to reduce periodontopathic micro-organisms with minimal side-effects. Clinical studies in our laboratory have shown that 25% tetracycline HCl delivered from poly(D,L-lactide/glycolide) film strips (25 TTC-PLGA) released therapeutic concentrations of tetracycline for 10 days. The present pilot study compared the intracrevicular delivery of 25% tetracycline HCl incorporated in these biodegradable film strips to scaling and root planing (SRP) in 10 adult periodontitis patients, who in spite of therapy and regular supportive periodontal treatment (SPT), continued to possess 5 bleeding periodontal pockets at least 5 mm deep. Sites were randomly selected to receive the following treatments: (1) 25 TTC-PLGA, (2) control strips without TTC (PLGA), (3) SRP, and (4) untreated control. Film-strip retention was augmented with a suture/cement technique, followed by strip removal after 2 weeks. Clinical parameters and subgingival bacterial morphotypes (darkfield analysis) were evaluated over time (0, 2.4, 8, 12, 26 weeks). Results indicated that, compared to baseline, 25 TTC-PLGA film strips caused significant (p ≤ 0.01): (1) probing depth reduction for 26 weeks, (2) a clinical attachment level gain for 12 weeks, (3) lower %s of spirochetes for 4 weeks and motile rods for 8 weeks (p ≤ 0.05), and (4) an accompanying increase in cocci for 4 weeks. In the scaled and root planed sites, probing depth was the only finding that demonstrated a significant change from baseline (p ≤ 0.01). Controls and PLGA showed isolated reductions in probing depth and % of motile organisms. From these findings, applications of intracrevicular 25 TTC-PLGA, when compared to scaling and root planing, appears to have an enhanced antibacterial effect and a similar clinical effect in SPT patients. The results of this study indicate further investigation of 25 TTC-PLGA film strips should be undertaken using more subjects and sophisticated microbiological and clinical measurements. [ABSTRACT FROM AUTHOR]

Menopause and oophorectomy without estrogen therapy (ED) have been associated with increased production of bone-active cytokines by peripheral blood mononuclear cells. The current study extended evaluation to gingival crevicular fluid (GCF) levels of interleukin (IL)-1ß and IL-6 in such subjects compared to premenopausal and postmenopausal estrogen-treated females (ES). 13 ED and 13 ES Caucasians with a history of moderate-severe adult periodontitis provided GCF from 1-3 clinically identical sites each (5 6 mm probing depth, 5-7 mm clinical attachment loss, bleeding on probing). 30 s GCF samples were obtained and evaluated for IL-1ß and IL-6 levels using two-site enzyme-linked immunosorbent assays (ELISAs). The frequency of GCI IL-1ß-positive subjects was elevated in ED versus ES (92% versus 23%; P < 0.0004, analysis). IL-6 was detected more frequently in ED subjects (23% versus 8%; not signfiicant): however, the frequency of IL-6 detection was low in both coups due to short sampling times. These data support the concept that clinical conditions causing low estrogen environments allow increased local production of the bone-active cytokine IL- 1ß, and perhaps IL-6. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-1 alpha and beta are cytokines which can mediate inflammatory, bone resorbing, and reparative effects in the periodontium, but few longitudinal data exist exploring their role following periodontal therapy. This study examined gingival crevicular fluid (GCF) concentrations of IL-1 alpha and IL-1 beta at sites which shallow sulci (SS) or inflamed moderate/advanced pockets (M/AP) before and 6 months after treatment with closed scaling/root planning (SC/RP) or papillary flap debridement (PFD), all in the same subject (n = 14 patients). No significant differences were noted in IL-1 alpha or beta concentrations (determined with two-site enzyme-linked immunosorbent assays) between SS and M/AP sites at baseline. While both therapies improved clinical parameters of periodontal disease, IL-1 alpha concentration increased significantly (p≤0.05) in M/AP-PFD sites 6 months after treatment, but were unchanged in other groups. IL-1 beta concentrations were numerically lower after therapy. These data suggest that surgical wound healing in an inflamed, plaque-infected site (M/AP-PFD) results in prolonged production of IL-1, which may be a reflection of the extent of tissue trauma and delayed would healing. In spite of increased IL-1 levels, these sites demonstrated significant short-terms improvement in clinical attachment level (+ 1.8 mm, p &les;0.001) postoperatively. [ABSTRACT FROM AUTHOR]

Selected gingival bacteria and cytokine profiles associated with patients who did not respond to conventional periodontal therapy (refractory) were evaluated. 10 subjects with a high incidence of post-active treatment clinical attachment loss (>2% sites/year lost &Ge; 3 mm) were compared to 10 age-, race-, and supragingival plaque-matched patients with low post-treatment clinical attachment loss (<0.5% sites/year) relative to the following paramenters at 2 sites/patient with the deepest probing depths: (1) presence of 3 selected periodontal pathogens (Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Eikenella corrodens) in subgingival plaque as determined by selective culturing, and (2) gingival crevicular fluid (GCF) levels of 3 cytokines associated with bone resorption (IL-1 alpha, IL-1 beta, IL-6) as determined by two -site ELISA. Results indicated no significant differences in any clinical measurement (except incidence of clinical attachment loss), in the presence of any bacterial pathogen, or in GCF cytokine levels between refractory subject sites versus stable subject sites. However, when sites production the greatest total GCF cytokine/patient were compared sites from refractory patients produced significantly more IL-6 (30.1±4.0 versus 15.4±2.8nM, p<0.01). The subgingival presence of each of the 3 bacterial pathogens was associated with elevated GCF IL-1 concentrations. These data suggest that gingival IL-1 and IL-6 production is different in response to local and systemic factors associated with periodontitis, and that Il-6 play a rôle in the identification and mechanisms of refractory periodontitis. [ABSTRACT FROM AUTHOR]

In vitro tests in our laboratory have shown that 40% tetracycline HCl in a white petrolatum carrier (TTC-WP) has potential as a sustained release, auto- dissipating system. The present study tested subgingival placement of TTC-WP via syringe in viva Quadrants (2 diseased sites in each) in 9 patients with moderate/severe periodontitis were randomly assigned to receive the following treatments: (1) TTC-WP: (2) WP only; (3) scaling and root planing: (4) untreated control. TTC release into gingival crevicular fluid (GCF) over time (baseline, 8, 24, 72 and 168 h) was measured using an agar diffusion bioassay. Clinical parameters and subgingival bacterial morphotypes (darkfield analysis) were also evaluated over time (baseline, 2, 4, 8, 12 weeks). Results indicated that TTC-WP was easily placed into periodontal pockets and biologically effective TTC was released into GCF for at least 3 days (mean concentration= 115.8±43.1 μg/ml at 3 days). TTC-WP reduced probing pocket depths and bleeding on probing relative to baseline measurements for 8-12 weeks post-treatment. and reduced %s of motile rods and spirochetes, with an accompanying increase in cocci, for 2-8 weeks. Similar effects were noted in the scaled and root-planed sites. but for a longer duration. TTC-WP and WP were clinically dissipated after 2 weeks and no adverse tissue reactions were observed. From these findings, subgingival TTC-WP cannot replace scaling and root planing therapy, but has characteristics useful in subgingival plaque control. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate clinical, microbiological, and gingival crevicular fluid (GCF) profiles in periodontitis-resistant and periodontitis-susceptible subjects during 4 weeks of experimental gingivitis. Experimental groups of similar ages were defined as gingivitis controls (GC; n=10) and history of rapidly progressive periodontitis (RPP; n=10), respectively. Prior to baseline, all subjects achieved good plaque control (plaque index (PlI)&ap;0) and gingival health (gingival index (GI)=0), and had probing depths ≤4 mm on experimental teeth. For 4 weeks after baseline, oral hygiene around maxillary 2nd premolar and 1st molar teeth was inhibited by a plaque guard. The plaque guard was removed weekly for GCF sampling to determine interleukin (IL)-1β and prostaglandin (PG)E2 amounts by ELISAs. In addition, PlI, GI, probing depth, and gingival recession measurements were made. Subgingival plaque darkfield microscopy and DNA probe analysis also were performed. Results indicated that clinical signs of inflammation, microbiological patterns and GCF profiles progressed similarly in both groups. However, plaque accumulated more rapidly in the susceptible subjects. PlI in RPP at 4 weeks was 2.1±0.1 compared to 1.5±0.2 in GC, with an incidence of PlI>1 of 100% versus 50%, respectively (logistic regression; p<0.0001). Hence, the clinical, microbiological and host factors selected for this study were unrelated to previous susceptibility to periodontitis when evaluated in the experimental gingivitis model. However, the increased rate of plaque accumulation, following thorough plaque removal, in RPP patients suggests a potential factor in disease recurrence in these susceptible subjects. [ABSTRACT FROM AUTHOR]

Rhinoviruses (RVs) are major instigators of acute exacerbations of asthma, COPD, and other respiratory diseases. RVs are categorized into three species (RV-A, RV-B, and RV-C), which comprise more than 160 serotypes, making it difficult to develop an effective vaccine. Currently, no effective treatment for RV infection is available. Pulmonary surfactant is an extracellular complex of lipids and proteins that plays a central role in regulating innate immunity in the lung. The minor pulmonary surfactant lipids, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI), are potent regulators of inflammatory processes and exert antiviral activity against respiratory syncytial virus (RSV) and influenza A viruses (IAV). In the current study, we examined the potencies of POPG and PI against rhinovirus A16 (RV-A16) in primary human airway epithelial cells (AECs) differentiated at an air–liquid interface (ALI). After AECs were infected with RV-A16, PI reduced the viral RNA copy number by 70% and downregulated (55–75%) the expression of antiviral (MDA5, IRF7, and IFN-lambda) and CXCL11 chemokine genes. In contrast, POPG only slightly decreased MDA5 (24%) and IRF7 (11%) gene expression but did not inhibit IFN-lambda gene expression or RV-A16 replication in AECs. However, both POPG and PI inhibited (50–80%) IL6 gene expression and protein secretion and CXCL11 protein secretion. PI treatment dramatically attenuated global gene expression changes induced by RV-A16 infection alone in AECs. The observed inhibitory effects were indirect and resulted mainly from the inhibition of virus replication. Cell-type enrichment analysis of viral-regulated genes opposed by PI treatment revealed the PI-inhibited viral induction of goblet cell metaplasia and the virus-induced downregulation of ciliated, club, and ionocyte cell types. Notably, the PI treatment also altered the ability of RV-A16 to regulate the expression of some phosphatidylinositol 4-kinase (PI4K); acyl-CoA-binding, domain-containing (ACBD); and low-density lipoprotein receptor (LDLR) genes that play critical roles in the formation and functioning of replication organelles (ROs) required for RV replication in host cells. These data suggest PI can be used as a potent, non-toxic, antiviral agent for RV infection prophylaxis and treatment. [ABSTRACT FROM AUTHOR]

Background: Histone posttranslational modifications (PTMs) represent a focal point of chromatin regulation. The genome-wide and locus-specific distribution and the presence of distinct histone PTMs is most commonly examined with the application of histone PTM-specific antibodies. In spite of their central role in chromatin research, polyclonal antibodies suffer from disadvantages like batch-to-batch variability and insufficient documentation of their quality and specificity. Results: To mitigate some of the pitfalls of using polyclonal antibodies against H3K4me3, we successfully validated the application of a recombinant TAF3 PHD domain as anti-H3K4me3 affinity reagent in peptide array, western blot and ChIP-like experiments coupled with qPCR and deep sequencing. Conclusions: The successful addition of the TAF3 PHD domain to the growing catalog of recombinant affinity reagents for histone PTMs could help to improve the reproducibility, interpretation and cross-laboratory validation of chromatin data. [ABSTRACT FROM AUTHOR]

The identification of body fluids is an essential tool for clarifying the course of events at a criminal site. The analytical problem is the fact that the biological material has been very often exposed to detrimental exogenous influences. Thereby, the molecular substrates used for the identification of the traces may become degraded. So far, most protocols utilize cell specific proteins or RNAs. Instead of measuring these more sensitive compounds this paper describes the application of the differential DNA-methylation. As a result of two genome wide screenings with the Illumina HumanMethylation BeadChips 27 and 450k we identified 150 candidate loci revealing differential methylation with regard to the body fluids venous blood, menstrual blood, vaginal fluid, saliva and sperm. Among them we selected 9 loci as the most promising markers. For the final determination of the methylation degree we applied the SNuPE-method. Because the degree of methylation might be modified by various endogenous and exogenous factors, we tested each marker with approximately 100 samples of each target fluid in a validation study. The stability of the detection procedure is proved in various simulated forensic surroundings according to standardized conditions. We studied the potential influence of 12 relatively common tumors on the methylation of the 9 markers. For this purpose the target fluids of 34 patients have been analysed. Only the cervix carcinoma might have an remarkable effect because impairing the signal of both vaginal markers. Using the Illumina MiSeq device we tested the potential influence of cis acting sequence variants on the methylation degree of the 9 markers in the specific body fluid DNA of 50 individuals. For 4 marker loci we observed such an influence either by sole SNPs or haplotypes. The identification of each target fluid is possible in arbitrary mixtures with the remaining four body fluids. The sensitivity of the individual body fluid tests is in the same range as for the forensic STR-analysis. It is the first forensic body fluid protocol which considers the exogenic and endogenic parameters potentially interfering with the true results. [ABSTRACT FROM AUTHOR]

Background: The majority of patients with JAK2 V617F-negative essential thrombocythemia or primary myelofibrosis harbor mutations involving the calreticulin ( CALR) gene. These mutations are located in CALR exon 9 and lead to a frameshift with subsequent alteration of the CALR protein C-terminus. They have emerged as valuable molecular markers for the diagnosis of clonal myeloproliferative diseases. Although a variety of CALR mutations have been described, two mutations, denoted type 1 and type 2, account for around 85 % of cases. The type 1 mutation encompasses a 52 bp deletion and the type 2 mutation a 5 bp TTGTC insertion. Methods: This work describes the development and testing of quantitative real-time PCRs (qPCRs) for detecting these two mutations. Results: The final type 1 CALR qPCR displayed a sensitivity of <0.1 % mutant alleles and the type 2 CALR qPCR had a sensitivity of <0.01 % mutant alleles. Additionally, two new CALR mutations are reported. Conclusion: These sensitive and specific qPCRs should be helpful in establishing the diagnosis and in monitoring minimal residual disease in patients during or after therapy. [ABSTRACT FROM AUTHOR]

Summary: Large quantities of organic matter are continuously deposited, and (a)biotic gradients intersect in the soil–rhizosphere, where biodegradation contributes to the global cycles of elements. The betaproteobacterial genus Aromatoleum comprises cosmopolitan, facultative denitrifying degradation specialists. Aromatoleum aromaticum. pCyN1 stands out for anaerobically decomposing plant‐derived monoterpenes in addition to monoaromatic hydrocarbons, polar aromatics and aliphatics. The catabolic network's structure and flexibility in A. aromaticum pCyN1 were studied across 34 growth conditions by superimposing proteome profiles onto the manually annotated 4.37 Mbp genome. Strain pCyN1 employs three fundamentally different enzymes for C–H‐bond cleavage at the methyl groups of p‐cymene/4‐ethyltoluene, toluene and p‐cresol respectively. Regulation of degradation modules displayed substrate specificities ranging from narrow (toluene and cyclohexane carboxylate) via medium‐wide (one module shared by p‐cymene, 4‐ethyltoluene, α‐phellandrene, α‐terpinene, γ‐terpinene and limonene) to broad (central benzoyl‐CoA pathway serving 16 aromatic substrates). Remarkably, three variants of ATP‐dependent (class I) benzoyl‐CoA reductase and four different β‐oxidation routes establish a degradation hub that accommodates the substrate diversity. The respiratory system displayed several conspicuous profiles, e.g. the presence of nitrous oxide reductase under oxic and of low‐affinity oxidase under anoxic conditions. Overall, nutritional versatility in conjunction with network regulation endow A. aromaticum pCyN1 with broad adaptability. [ABSTRACT FROM AUTHOR]

The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/ DNA fibers. However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNA fiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition of a catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylation rate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experiments indicate that stable protein/ DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experiments with substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzyme concentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stable protein/ DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μM concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understanding the mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has been implicated in changing Dnmt3a processivity. [ABSTRACT FROM AUTHOR]

The evolution of new genes can ensue through either gene duplication and the neofunctionalization of one of the copies or the formation of a de novo gene from hitherto nonfunctional, neutrally evolving intergenic or intronic genomic sequences. Only very rarely are entire genes created de novo. Mostly, nonfunctional sequences are coopted as novel parts of existing genes, such as in the process of exonization whereby introns become exons through changes in splicing. Here, we report a case in which a novel nonprotein coding RNA evolved by intron-sequence recruitment into its structure. cDNAs derived from rat brain small RNAs, revealed a novel small nucleolar RNA (snoRNA) originating from one of the Snord115 copies in the rat Prader–Willi syndrome locus. We suggest that a single-point substitution in the Snord115 region led to the expression of a longer snoRNA variant, designated as L-Snord115. Cell culture and footprinting experiments confirmed that a single nucleotide substitution at Snord115 position 67 destabilized the kink-turn motif within the canonical snoRNA, while distal intronic sequences provided an alternate D-box region. The exapted sequence displays putative base pairing to 28S rRNA and mRNA targets. [ABSTRACT FROM AUTHOR]

The small RNAs associated with the protein Hfq constitute one of the largest classes of post-transcriptional regulators known to date. Most previously investigated members of this class are encoded by conserved free-standing genes. Here, deep sequencing of Hfq-bound transcripts from multiple stages of growth of Salmonella typhimurium revealed a plethora of new small RNA species from within mRNA loci, including DapZ, which overlaps with the 3? region of the biosynthetic gene, dapB. Synthesis of the DapZ small RNA is independent of DapB protein synthesis, and is controlled by HilD, the master regulator of Salmonella invasion genes. DapZ carries a short G/U-rich domain similar to that of the globally acting GcvB small RNA, and uses GcvB-like seed pairing to repress translation of the major ABC transporters, DppA and OppA. This exemplifies double functional output from an mRNA locus by the production of both a protein and an Hfq-dependent trans-acting RNA. Our atlas of Hfq targets suggests that the 3? regions of mRNA genes constitute a rich reservoir that provides the Hfq network with new regulatory small RNAs. [ABSTRACT FROM AUTHOR]

Background: The Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam. Results: A normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra high-throughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring. Conclusions: The transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress. [ABSTRACT FROM AUTHOR]

Bdellovibrio bacteriovorus HDI00 is an obligate predatory bacterium that attacks and invades Gram-negative bacteria. The predator requires living bacteria to survive as growth and replication take place inside the bacterial prey. It is possible to isolate mutants that grow and replicate outside prey bacteria. Such mutants are designated host or prey independent, and their nutritional requirements vary. Some mutants are saprophytic and require prey extracts for extracellular growth, whereas other mutants grow axenically, which denotes the formation of colonies on complete medium in the absence of any prey components. The initial events leading to prey-independent growth are still under debate, and several genes may be involved. We selected new mutants by three different methods: spontaneous mutation, transposon mutagenesis, and targeted gene knockout. By all approaches we isolated mutants of the hit (host interaction) locus. As the relevance of this locus for the development of prey independence has been questioned, we performed whole-genome sequencing of five prey-independent mutants. Three mutants were saprophytic, and two mutants could grow axenically. Whole-genome analysis revealed that the mutation of a small open reading frame of the hit locus is sufficient for the conversion from predatory to saprophytic growth. Complementation experiments were performed by introduction of a plasmid carrying the wild-type hit gene into saprophytic mutants, and predatory growth could b restored. Whole-genome sequencing of two axenic mutants demonstrated that in addition to the hit mutation the colony formation on complete medium was shown to be influenced by the mutations of two genes involved in RNA processing. Complementation experiments with a wild-type gene encoding an RNA helicase, RhIB, abolished the ability to form colonies on complete medium, indicating that stability of RNA influences axenic growth. [ABSTRACT FROM AUTHOR]

Background: The Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam. Results: A normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra highthroughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring. Conclusions: The transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress. [ABSTRACT FROM AUTHOR]

Background: Bisulfite sequencing is a popular method to analyze DNA methylation patterns at high resolution. A region of interest is targeted by PCR and about 20-50 subcloned DNA molecules are usually analyzed, to determine the methylation status at single CpG sites and molecule resolution. Results: The BISMA (Bisulfite Sequencing DNA Methylation Analysis) software for analysis of primary bisulfite sequencing data implements sequencing data extraction and enhanced data processing, quality controls, analysis and presentation of the methylation state. It uses an improved strategy for detection of clonal molecules and accurate CpG site detection and it supports for the first time analysis of repetitive sequences. Conclusions: BISMA works highly automated but still provides the user full control over all steps of the analysis. The BISMA software is freely available as an online tool for academic purposes for the analysis of bisulfite sequencing data from both unique and repetitive sequences http://biochem.jacobs-university.de/BDPC/BISMA/. [ABSTRACT FROM AUTHOR]

Bulk mRNA was used to explore the transcriptional activity of bacterial communities in oxic versus anoxic paddy soil. Two microbial cDNA libraries were constructed from composite samples using semi-randomly primed RT-PCR. cDNAs averaged 500–600 bp in length and were treated as expressed sequence tags (ESTs). Clustering analysis of 805 random cDNAs resulted in 179 and 155 different ESTs for the oxic and anoxic zones respectively. Using an E-value threshold of e−10, a total of 218 different ESTs could be assigned byblastx, while 116 ESTs were predicted novel. Both the proportion and significance of the EST assignments increased with cDNA length. Taxonomic assignment was more powerful in discriminating between the aerobic and anaerobic bacterial communities than functional inference, as most ESTs in both oxygen zones were putative indicators of similar housekeeping functions, in particular ABC-type transporters. A few ESTs were putative indicators for community function in a biogeochemical context, such as β-oxidation of long-chain fatty acids specifically in the oxic zone. Expressed sequence tags assigned to Alpha- and Betaproteobacteria were predominantly found in the oxic zone, while those affiliated with Deltaproteobacteria were more frequently detected in the anoxic zone. At the genus level, multiple assignments to Bradyrhizobium and Geobacter were unique to the oxic and anoxic zones respectively. The phylum-level affiliations of 93 16S rRNA sequences corresponded well with two taxonomically distinct EST patterns. Expressed sequence tags affiliated with Acidobacteria and Chloroflexi were frequently detected in both oxygen zones. In summary, the soil metatranscriptome is accessible for global analysis and such studies have great potential in elucidating the taxonomic and functional status of soil bacterial communities, but study significance depends on the number and length of cDNAs being randomly analysed. [ABSTRACT FROM AUTHOR]

We characterized two overlapping sugar beet ( Beta vulgaris) bacterial artificial chromosome (BAC) clones representing different haplotypes. A total of 254 kbp of the genomic sequence was determined, of which the two BACs share 92 kbp. Eleven of 15 genes discovered in the sequenced interval locate to the overlap region. The haplotypes differ in exons by 1% (nucleotide level) and in non-coding regions by 9% (6% mismatches, 3% gaps; alignable regions only). Large indels or high sequence divergence comprised 11% of either sequence. Of such indels, 68 and 45%, respectively, could be attributed to haplotype-specific integration of transposable elements. We identified novel repeat candidates by comparing the two BAC sequences to a set of genomic sugar beet sequences. Synteny was found with Arabidopsis chromosome 1 (At1), At2 and At4, Medicago chromosome 7, Vitis chromosome 15 and paralogous regions on poplar chromosomes II and XIV. [ABSTRACT FROM AUTHOR]

Abstract: Summary: Antibody titers to P. gingivalis are increased in patients with rheumatoid arthritis and are associated with disease-specific autoimmunity. Background: Periodontitis (PD) has been implicated as a risk factor for rheumatoid arthritis (RA). We sought to characterize antibody titers to P. gingivalis (a pathogen in PD) in subjects with RA, PD, and in healthy controls and to examine their relationship with disease autoantibodies. Methods: P. gingivalis antibody was measured in subjects with RA (n =78), PD (n =39), and in controls (n =40). Group frequencies of bacterial titer elevations were compared using the Chi-square test and antibody titers were compared using non-parametric tests. Correlations of P. gingivalis titer with C-reactive protein (CRP), antibody to cyclic citrullinated peptide (anti-CCP), and rheumatoid factor (RF) were examined in those with RA while CRP and autoantibody concentrations were compared based on seropositivity to P. gingivalis. Results: Antibody titers to P. gingivalis were highest in PD, lowest in controls, and intermediate in RA (p =0.0003). Elevations in P. gingivalis (titer≥800) were more common in RA and PD (67% and 77%, respectively) than in controls (40%) (p =0.002). In RA, there were significant correlations with P. gingivalis titer with CRP, anti-CCP-IgM, and -IgG-2. CRP (p =0.006), anti-CCP-IgM (p =0.01) and -IgG2 (p =0.04) concentrations were higher in RA cases with P. gingivalis titers ≥800 compared to cases with titers <800. Conclusion: Antibodies to P. gingivalis are more common in RA subjects than controls, although lower than that in PD. Associations of P. gingivalis titers with RA-related autoantibody and CRP concentrations suggests that infection with this organism plays a role in disease risk and progression in RA. [Copyright &y& Elsevier]

Abstract: Objective: Osteogenic protein-1 (OP-1) has been shown to stimulate undifferentiated cells to produce mineralized tissue. Adipose tissue is a rich source of undifferentiated cells for tissue engineering purposes. The purpose of this study was to investigate the effect of OP-1 on osteogenic differentiation of adipose-derived stem cells and the production of bony tissue in vitro. Design: Adipose-derived stem cells (ADSCs) were isolated from inguinal fat pads of adult mice. Following cell expansion the cells were plated in 8-well chambered slides. The cells received one of four treatments: Group 1 cells were maintained in control medium, Group 2 cells were cultured in a common osteogenic medium, Group 3 cells were cultured in osteogenic medium supplemented with 250ng/mL of OP-1, and Group 4 cells were cultured with 250ng/mL of OP-1 added to control medium. Osteogenic differentiation of ADSCs was determined by estimating the number and size of mineralized nodules, and the amount of extracellular osteopontin secreted into cell culture medium. Mineralized nodule production was assessed at day 21 with von Kossa staining. Extracellular osteopontin release was measured after 8 and 21 days by enzyme-linked immunosorbant assay (ELISA). ANOVA/Tukey tests were used to identify differences among the four treatment groups for mineralized nodule production and osteopontin release (p ≤0.05). Results: Deposition of calcified nodules and osteopontin secretion was significantly greater for cell cultures incubated with OP-1 (p ≤0.05). At day 21, no significant differences in osteopontin secretion were noted among groups incubated with osteogenic nutrients and/or OP-1 (p >0.05), which were significantly higher than the group incubated in cell growth medium only (p ≤0.05). No significant differences in osteopontin secretion were noted between 8 and 21 days for any group (p >0.05). Linear regression analysis demonstrated a linear relationship was present between the presence of calcified nodules and the amount of osteopontin released (p ≤0.05). Conclusions: OP-1 is a powerful inducer of osteogenic differentiation of adult adipose-derived stem cells. [Copyright &y& Elsevier]

Background: The goldfish (Carassius auratus) uses steroids and prostaglandins as pheromone cues at different stages of the reproductive cycle to facilitate spawning synchronization. Steroid progestin pheromone binding has been detected in goldfish olfactory membranes but the receptors responsible for this specific binding remain unknown. In order to shed some light on the olfactory epithelium transcriptome and search for possible receptor candidates a large set of EST from this tissue were analysed and compared to and combined with a similar zebrafish (Danio rerio) resource. Results: We generated 4,797 high quality sequences from a normalized cDNA library of the goldfish olfactory epithelium, which were clustered in 3,879 unique sequences, grouped in 668 contigs and 3,211 singletons. BLASTX searches produced 3,243 significant (E-value < e-10) hits and Gene Ontology (GO) analysis annotated a further 1,223 of these genes (37.7%). Comparative analysis with zebrafish olfactory epithelium ESTs revealed 1,088 identical unigenes. The transcriptome size of both species was estimated at about 16,400 unigenes, based on the proportion of genes identified involved in Glucose Metabolic Process. Of 124 G-protein coupled receptors identified in the olfactory epithelium of both species, 56 were olfactory receptors. Beta and gamma membrane progestin receptors were also isolated by subcloning of RT-PCR products from both species and an olfactory epithelium specific splice form identified. Conclusion: The high similarity between the goldfish and zebrafish olfactory systems allowed the creation of a 'cyprinid' olfactory epithelium library estimated to represent circa 70% of the transcriptome. These results are an important resource for the identification of components of signalling pathways involved in olfaction as well as putative targets for pharmacological and histochemical studies. The possible function of the receptors identified in the olfactory system is described. Moreover, the role of olfactory epithelium specific isoforms of classical membrane progestin receptor genes as candidates for preovulatory pheromone sensing is discussed. [ABSTRACT FROM AUTHOR]

Abstract: A biomineral-binding alendronate-β-cyclodextrin conjugate (ALN-β-CD, Fig. 1) was developed as a novel drug delivery system. “Click” chemistry was used in conjugation of alendronate (ALN) to β-cyclodextrin (β-CD). The delivery system shows very strong binding to hydroxyapatite (HA, main component of tooth enamel). Its ability in forming molecular inclusion complex with dexamethsone (Dex, model drug) was investigated independently with phase solubility experiments, isothermal titration calorimetry (ITC), Job plot and 1H NMR. The stoichiometry of ALN-β-CD/Dex molecular complex was determined as 1:1, and the binding constant of the complex obtained from ITC study is 3.8×103 M−1, which is similar to the binding constant of β-CD/Dex. In vitro data indicate that the ALN-β-CD/Dex complex bound to HA could gradually release Dex upon repeated extraction with phosphate buffer saline (PBS). This novel drug delivery system may have great potential in improving treatment of diseases in the oral cavity. [Copyright &y& Elsevier]

Major parts of the virulence-associated vrl locus known from the gammaproteobacterium Dichelobacter nodosus, the causative agent of ovine footrot, were analyzed in the genome of the sulfate-reducing deltaproteobacterium Desulfococcus multivorans. In the genome of D. multivorans 13 of the 19 vrl genes described for D. nodosus are present and highly conserved with respect to gene sequence and order. The vrl locus and its flanking regions suggest a bacteriophage-mediated transfer into the genome of D. multivorans. Comparative analysis of the deduced Vrl proteins reveals a wide distribution of parts of the virulence-associated vrl locus in distantly related bacteria. Horizontal transfer is suggested as driving mechanism for the circulation of the vrl genes in bacteria. Except for the vrlBMN genes D. multivorans and Desulfovibrio desulfuricans G20 together contain all vrl genes displaying a high degree of similarity. For D. multivorans it could be shown that guanine plus cytosine (GC) content, GC skew, di-, tri- or tetranucleotide distribution did not differ between the vrl locus and its flanking sequences. This could be a hint that the vrl locus originated from a related organism or at least a genome with similar characteristics. The conspicuous high degree of conservation of the analyzed vrl genes may result from a recent transfer event or reflect a function of the vrl genes, which is still unknown and not necessarily disease associated. The latter is supported by the evidence for expression of the vrl genes in D. multivorans, which has not been described as pathogen or to be associated to any disease pattern before. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]