Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer,Normal Colonic Mucosa,and Peripheral Blood Cells

Genome-wide DNA hypomethylation plays an important role in epigenomic and genomic instability and colorectal carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. In addition, LINE-1 hypomethylation in blood cells has been associated with colorectal adenoma risk, and LINE-1 hypomethylation in colorectal cancer is related with prognosis and linearly predicts shorter patient survival. However, no study has comprehensively evaluated the precision of sodium bisulfite conversion and PCR-pyrosequencing to measure LINE-1 methylation. Using 10 paraffin-embedded colon cancers, 5 matched normal colon mucosa, and 5 unrelated peripheral blood buffy coat leukocyte specimens, we enriched tumor DNA by macrodissection and laser capture microdissection. LINE-1 methylation was calculated as an average of 100 * C/(C + T) at 4 CpG sites after bisulfite-PCR-pyrosequencing. The LINE-1 methylation value in colon cancers varied, ranging approximately from 30 to 80. To measure assay precision, we performed bisulfite conversion on seven different DNA specimen aliquots and repeated PCR-pyrosequencing seven times. Run-to-run (between-run) SD ranged from 1.3 to 4.4 (median, 3.0) in macrodissected colon cancers; 1.1 to 10.5 (median, 3.8) in laser capture microdissection specimens; 1.3 to 2.5 (median, 1.9) in normal colon; and 1.5 to 3.4 (median, 1.9) in leukocyte DNA. In conclusion, bisulfite conversion and PCR-pyrosequencing assay can measure LINE-1 methylation in macrodissected colon cancer, normal colon, and blood DNA, and may be useful in clinical and research settings.Genome-wide DNA hypomethylation is considered to play an important role in genomic instability and carcinogenic processes.^{1,2,3,4,5,6,7} DNA methylation measured in LINE-1 (long interspersed nucleotide element-1, or L1) has been correlated with global DNA methylation,^8,9,10,11 and linearly predicts survival of colon cancer patients.¹² In addition, LINE-1 methylation levels in synchronous colorectal cancer pairs (ie, two or more primary colorectal cancers) are significantly correlated, suggesting the presence of a nonstochastic component in a variation of LINE-1 methylation levels in cancer.¹³ Nonetheless, utility of measuring LINE-1 or global DNA methylation levels in peripheral blood leukocytes remains controversial. One study has reported no significant association between LINE-1 methylation levels in blood leukocytes and risk of colorectal adenoma,¹⁴ whereas another study has shown an inverse association between global DNA methylation level in blood leukocytes and colorectal adenoma risk.¹⁵ Additional studies using precise LINE-1 methylation measurements are needed. Moreover, LINE-1 methylation measurement in peripheral blood can provide a useful way to evaluate the activity of DNA methylation inhibitors in solid tumor patients.¹⁶Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics,¹⁷ typically rely on conversion of unmethylated cytosine to uracil by sodium bisulfite. However, no previous study has evaluated the precision of bisulfite conversion to measure LINE-1 methylation. In addition, although pyrosequencing appears to be superior to combined bisulfite and restriction analysis or bisulfite/real-time PCR (MethyLight) in terms of precision of LINE-1 methylation measurement on plasma DNA,¹⁶ no study has comprehensively evaluated performance of bisulfite-pyrosequencing assay to measure LINE-1 methylation in paraffin-embedded tissue.In this study, we assessed the precision of sodium bisulfite conversion and PCR-pyrosequencing assay using 10 paraffin-embedded colon cancer specimens, 5 matched normal colon specimens, and 5 unrelated blood specimens. We have shown that LINE-1 pyrosequencing assay has good precision and can reliably measure LINE-1 methylation in paraffin-embedded colon cancer, normal colon tissue, and peripheral blood cells.     

Antifungal protein PAF severely affects the integrity of the plasma membrane of Aspergillus nidulans and induces an apoptosis-like phenotype

The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the beta-lactam producer Penicillium chrysogenum. PAF inhibits the growth of various important plant and zoopathogenic filamentous fungi. Previous studies revealed the active internalization of the antifungal protein and the induction of multifactorial detrimental effects, which finally resulted in morphological changes and growth inhibition in target fungi. In the present study, we offer detailed insights into the mechanism of action of PAF and give evidence for the induction of a programmed cell death-like phenotype. We proved the hyperpolarization of the plasma membrane in PAF-treated Aspergillus nidulans hyphae by using the aminonaphtylethenylpyridinium dye di-8-ANEPPS. The exposure of phosphatidylserine on the surface of A. nidulans protoplasts by Annexin V staining and the detection of DNA strand breaks by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) gave evidence for a PAF-induced apoptotic-like mechanism in A. nidulans. The localization of reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate and the abnormal cellular ultrastructure analyzed by transmission electron microscopy suggested that ROS-elicited membrane damage and the disintegration of mitochondria played a major role in the cytotoxicity of PAF. Finally, the reduced PAF sensitivity of A. nidulans strain FGSC1053, which carries a dominant-interfering mutation in fadA, supported our assumption that G-protein signaling was involved in PAF-mediated toxicity.     

Prognosis factors and outcome of community-acquired pneumonia needing mechanical ventilation

PURPOSE: To evaluate the variables associated with mortality of patients with community-acquired pneumonia who require mechanical ventilation and to determine the attributable morbidity and intensive care unit (ICU) mortality of community-acquired pneumonia. MATERIAL AND METHODS: Retrospective cohort study carried out in 361 ICUs from 20 countries including 124 patients who required mechanical ventilation on the first day of admission to the hospital due to acute respiratory failure secondary to severe community-acquired pneumonia. To assess the factors associated with outcome, a forward stepwise logistic regression analysis was performed, and to determine the attributable mortality of community-acquired pneumonia, a matched study design was used. RESULTS: We found 3 independent variables significantly associated with death in patients with community-acquired pneumonia requiring mechanical ventilation: simplified acute physiological score greater than 45 (odds ratio, 5.5 [95% confidence interval, 1.7-12.3]), shock (odds ratio, 5.7 [95% confidence interval, 1.7-10.1]), and acute renal failure (odds ratio, 3.0 [95% confidence interval, 1.1-4.0]). There was no statistically significant difference in ICU mortality among patients with or without community-acquired pneumonia (32% vs 35%; P=.59). CONCLUSIONS: Community-acquired pneumonia needing mechanical ventilation is not a disease associated with higher mortality. The main determinants of patient outcome were initial severity of illness and the development of shock and/or acute renal failure.     

Violence in the care of adult persons with intellectual disabilities

Background. Violence, for example physical, psychological, financial and sexual abuse and neglect, exists and is an under‐reported problem in caring situations involving adult persons with intellectual disabilities and their caregivers, where both parties can be seen as victims and perpetrators. Aims and objectives. To investigate violent situations involving Swedish adult persons with intellectual disabilities and their caregivers in group‐dwellings. Design. A total population‐based survey. Methods. A questionnaire, including violence towards adults with intellectual disabilities and violence towards staff members during 1 year, was sent to all staff members (n = 164) from 17 care settings for adults with intellectual disabilities with a response rate of 74%. Results. Thirty‐five per cent of 122 respondents admitted they had been implicated in or witnessed a violent incident towards an adult person with intellectual disabilities and 14% of the staff members admitted they themselves had been the perpetrators. Sixty‐one per cent of the staff members described various situations when they were exposed to violence from an adult person with intellectual disabilities. Physical violence was most frequently reported. Most of the aggression occurred in helping situations when persons with intellectual disabilities did not co‐operate or when both actors reacted with violence. The violent situations led the staff members to feel powerless and inadequate. In order to cope they discussed with each other or with the manager. Conclusions. Violence seems to be accepted as a natural part of the daily care for adult persons with intellectual disabilities. Most of the violence is physical and psychological and occurs in close helping situations. Relevance to clinical practice. Supportive interventions, i.e. supervision for the staff members and training of communication skills individually or in group for the adults with intellectual disabilities.     

Age-related alterations in the protein expression profile of C57BL/6J mouse pituitaries

The aim of our study was to monitor the protein expression profile in pituitary glands of healthy C57BL/6J mice during aging. Pituitary glands of 4-week old (immature), 3-month old (mature), and >25-month old mice were analysed by proteomic tools such as two-dimensional electrophoresis and N-terminal micro-sequencing. A change was detected in the expression of growth hormone after sexual maturation. Our particular interest, however, was directed against up-regulated proteins in the old pituitary glands, which are proposed to be involved in the process of neuroendocrine aging.

Among these proteins, the expression of glutathione-S-transferase (GST) and apolipoprotein A-1 were increased in old pituitaries. Furthermore, ubiquitin carboxyl-terminal hydrolase (UCH-L1) was significantly up-regulated in senescent C57BL/6J mouse pituitaries. Since only the rat homologue was known, we isolated and analysed the mouse UCH-L1 sequence. Since GST is involved in antioxidative defence and UCH-L1 is part of the ubiquitin/proteasome system, which is responsible for the removal of damaged proteins, these results suggest increased oxidative burden and an increased activity of the ubiquitin system.     

Somatostatin receptor 1–5; expression profiles during rat development

Background

Somatostatin acts through five receptor subtypes (SSTRs 1–5). We aimed to investigate SSTRs mRNA expression and protein distribution in whole rat embryos, with special emphasis on the pancreas.

Material and methods

Rat embryos were collected on embryonal days 10, 11, 12, 14, 15, 17, 19, 21, and at birth. Presence of SSTRs was investigated with RT-PCR techniques and immunohistochemistry.

Results

There was no SSTR5 mRNA expression in the whole rat embryos. All SSTR1–5 proteins were observed at embryonal day 10, but the localization varied between the different subtypes. From day 11 to birth SSTRs protein presence increased with time in major structures such as skin and cartilage. It remained similar over time in the heart and liver. In the fetal pancreas mRNA expression of SSTR2 and 4 was detected at day 14, and there was an increase up to birth. Only SSTR1 protein co-localized to a higher extent with the islet hormones studied. SSTR2 was present in all islet endocrine cells except for β-cells. In contrast, the immunostaining for SSTR3–4 was co-localized with insulin and PP, and, finally, SSTR5 with glucagon and pancreatic polypeptide. In mRNA isolated from whole rat embryos SSTR1-2 and SSTR4 expression showed a peak at day 14, while SSTR3 mRNA was not present until day 15.

Conclusion

The present data suggest a role for SSTRs during the development of the rat embryo. Subsequent functional studies may elucidate regulatory roles of specific SSTRs for the growth and differentiation of the pancreas as well as other organs.