Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.     

Virulence studies on chromosomal α-toxin and Θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as Θ-toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α-toxin or phospholipase C. The resultant mutants failed to produce detectable Θ-toxin or α-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α-toxin in gas gangrene or clostridial myonecrosis.     

Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.     

A mixed-valence Cu(I)-Cu(II) and metal-metal bond containing coordination polymer obtained from an in situ oxidation reaction route

A new mixed-valence copper coordination polymer with copper-copper metal bonds in a two-dimensional network was generated from an in situ oxidation reaction route under hydrothermal conditions. The synthesis of this coordination polymer demonstrated that the novel compounds that may not be accessible using the known methods could be synthesized via an oxidation reaction route. The reaction conditions are mild enough to keep the building blocks intact during the oxidation and self-assembly process under hydrothermal conditions.     

Sizes of granular vesicles in the rat median eminence,with special reference to the zona granulosa

Summary A quantitative evaluation has been made of the sizes of neuronal granular vesicles (GV) in different (sub-)zones of the rat median eminence. On the basis of the frequency distribution of the mean sizes of the GV in the individual nerve profiles, at least 4 tentative categories of nerve profiles may be distinguished. However, available data do not permit characterization of nerve fibre categories in terms of neurohormone and/or neurotransmitter storage. From dorsal to ventral the palisade zone shows a decrease in mean size of GV; furthermore, the relative number of nerve profiles characterized by small GV (mean size smaller than 112 nm) increases. In contrast, the GV in the zona granulosa, that part of the external zone of the median eminence ventral to the palisade zone, are of relatively large size. Qualitative observations indicate that the zona granulosa may be regarded as an area for both storage and release of biologically active substances. We are grateful to Mrs. R.M.Y. Hartsteen for technical assistance, to Miss P.C. Delfos and Mr. W. van den Oudenalder for photographic assistance and to Prof. J. Moll for helpful criticism.     

Opposite Contributions of Trunk and Leg Fat Mass with Plasma Lipase Activities: The Hoorn Study

Objective: Lipoprotein lipase (LPL) and hepatic lipase (HL) are essential in hydrolysis of triglyceride‐rich lipoproteins. LPL activity is negatively, whereas HL activity is positively, associated with total body fat. We determined the associations of trunk and leg fat mass with plasma LPL and HL activities in a cross‐sectional study. Research Methods and Procedures: LPL and HL activities were determined in post‐heparin plasma in a sample of 197 men and 209 women, 60 to 87 years of age. A total body DXA scan was performed to determine trunk and leg fat mass. Results: In women, but not in men, trunk fat mass was negatively associated with LPL activity, whereas leg fat mass was positively associated, after mutual adjustment and adjustment for age. Standardized βs (95% confidence interval) for trunk and leg fat mass were ?0.24 (?0.41; ?0.08) and 0.14 (?0.02; 0.31), respectively (interaction by sex, p = 0.03). Larger trunk fat mass was associated with higher HL activity in men [0.48 (0.28; 0.68)] and women [0.40 (0.24; 0.56)]. A negative association of leg fat mass and HL activity was observed in men, although not statistically significant [?0.13 (?0.33; 0.06)], and in women [?0.28 (?0.38; ?0.18)]. Discussion: Abdominal fat is associated with unfavorable and femoral fat with favorable LPL and HL activities in plasma.