Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer

Objective

Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A, a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer.

Methods

Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA.

Results

The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum-tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status.

Conclusions

This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer.     

Consensus guidelines on analgesia and sedation in dying intensive care unit patients

Background

Intensivists must provide enough analgesia and sedation to ensure dying patients receive good palliative care. However, if it is perceived that too much is given, they risk prosecution for committing euthanasia. The goal of this study is to develop consensus guidelines on analgesia and sedation in dying intensive care unit patients that help distinguish palliative care from euthanasia.

Methods

Using the Delphi technique, panelists rated levels of agreement with statements describing how analgesics and sedatives should be given to dying ICU patients and how palliative care should be distinguished from euthanasia. Participants were drawn from 3 panels: 1) Canadian Academic Adult Intensive Care Fellowship program directors and Intensive Care division chiefs (N = 9); 2) Deputy chief provincial coroners (N = 5); 3) Validation panel of Intensivists attending the Canadian Critical Care Trials Group meeting (N = 12).

Results

After three Delphi rounds, consensus was achieved on 16 statements encompassing the role of palliative care in the intensive care unit, the management of pain and suffering, current areas of controversy, and ways of improving palliative care in the ICU.

Conclusion

Consensus guidelines were developed to guide the administration of analgesics and sedatives to dying ICU patients and to help distinguish palliative care from euthanasia.     

Presence of mouse mammary tumour-like virus gene sequences may be associated with morphology of specific human breast cancer

BACKGROUND: Mouse mammary tumour virus (MMTV) has a proven role in breast carcinogenesis in wild mice and genetically susceptible in-bred mice. MMTV-like env gene sequences, which indicate the presence of a replication-competent MMTV-like virus, have been identified in some human breast cancers, but rarely in normal breast tissues. However, no evidence for a causal role of an MMTV-like virus in human breast cancer has emerged, although there are precedents for associations between specific histological characteristics of human cancers and the presence of oncogenic viruses. AIM: To investigate the possibility of an association between breast cancer and MMTV-like viruses. METHODS: Histological characteristics of invasive ductal human breast cancer specimens were compared with archival MMTV-associated mammary tumours from C3H experimental mice. The presence of MMTV-like env DNA sequences in the human breast cancer specimens was determined by polymerase chain reaction and confirmed by Southern hybridisation. RESULTS: MMTV-like env gene sequences were identified in 22 of 59 (37.3%) human breast cancer specimens. Seventeen of 43 (39.5%) invasive ductal carcinoma breast cancer specimens and 4 of 16 (25%) ductal carcinoma in situ specimens had some histological characteristics, which were similar to MMTV-associated mouse mammary tumours. However, these similarities were not associated with the presence or absence of MMTV-like gene sequences in the human breast tumour specimens. A significant (p = 0.05) correlation was found between the grade of the human breast cancer and similarity to the mouse mammary tumours. The lower the grade, the greater the similarity. CONCLUSION: Some human breast cancer specimens, in which MMTV-like env DNA sequences have been identified, were shown to have histological characteristics (morphology) similar to MMTV-associated mouse mammary tumours. These observations are compatible with, but not conclusive of, an association between the presence of MMTV-like env DNA sequences and some human breast cancers.     

Biogeography of the Sulfolobus islandicus pan-genome

Variation in gene content has been hypothesized to be the primary mode of adaptive evolution in microorganisms; however, very little is known about the spatial and temporal distribution of variable genes. Through population-scale comparative genomics of 7 Sulfolobus islandicus genomes from 3 locations, we demonstrate the biogeographical structure of the pan-genome of this species, with no evidence of gene flow between geographically isolated populations. The evolutionary independence of each population allowed us to assess genome dynamics over very recent evolutionary time, beginning ≈910,000 years ago. On this time scale, genome variation largely consists of recent strain-specific integration of mobile elements. Localized sectors of parallel gene loss are identified; however, the balance between the gain and loss of genetic material suggests that S. islandicus genomes acquire material slowly over time, primarily from closely related Sulfolobus species. Examination of the genome dynamics through population genomics in S. islandicus exposes the process of allopatric speciation in thermophilic Archaea and brings us closer to a generalized framework for understanding microbial genome evolution in a spatial context.     

The importance of spatial scale in determining illusions of orientation

The twisted-cord illusion is a powerful demonstration of interaction between 1st-order (luminance-defined) and 2nd-order (contrast-defined) orientation processing. The perceived orientation of contrast-defined objects is pulled towards their 1st-order orientation content when the difference in orientation is small (Fraser effect), yet is pushed away from the 1st-order content at large orientation differences (Z?llner effect). Here we show that the relative spatial scale of carrier and envelope represents a decisive factor in determining the magnitude and direction of such interactions. We conclude that the perceived 2nd-order structure of a stimulus is biased by the properties of the 1st-order structure in a manner that depends on relative, rather than absolute spatial scale.     

Impact of the OSHA trench and excavation standard on fatal injury in the construction industry

In 1989 the US Occupational Safety & Health Administration revised the excavation and trenching standard. We examined fatal injuries from trench cave-in in the construction industry for five year periods before and after the revision in the 47 US states for which data were available for both periods. There was a 2-fold decline in the rate of fatal injury after revision of the standard, which substantially exceeded the decline in other causes of fatal injury in the construction industry during the same period. The decline was somewhat greater in large business firms but was evident in construction firms of all size classes. The fatality rate from trench cave-in in union construction workers was approximately half that of nonunion workers, but we were unable to determine whether this was best explained by union status, employment of union workers at larger construction firms, or both. This study provides evidence for the effectiveness of OSHA regulation in preventing fatal work injury.     

Preparation of a monoclonal antibody to citrullinated epitopes: its characterization and some applications to immunohistochemistry in human brain

Using hybridoma technology, an IgM monoclonal antibody (mAb), designated as F95, was developed against a deca-citrullinated peptide (DCP) consisting of 10 citrulline residues and a carboxyl Gly-Gly-Cys through which DCP was covalently linked to an activated carrier protein, keyhole limpet hemocyanin (KLH). Clones were selected on the basis of not reacting with human unmodified and noncitrullinated myelin basic protein (MBP), MBP-C1, but reacting well with human citrullinated MBP (MBP-C8). When tested by ELISA, this mAb demonstrated minimal reactivity with human MBP-C1, varying reactivity with the C2-C5 isomers of human MBP, moderate binding with guinea pig MBP-C8, and strong reactivity with human MBP-C8. By ELISA, mAb F95 was directed predominantly against citrulline, not MBP, as revealed by its binding to DCP linked with activated KLH, bovine serum albumin (BSA), or ovalbumin (OA), but not with KLH, BSA, or OA alone. Immunohistochemistry of normal human brain demonstrated that F95 stained central nervous system myelin and a subset of astrocytes. Given the citrulline-directed features of mAb F95, this immunohistochemical pattern suggests that certain astroglial filaments expressing glial fibrillary acidic protein also contain citrulline-bearing components. These potentially implicate citrullinated proteins, notably in astroglial filaments, in a variety of normal and pathological neurobiological processes.     

Progesterone receptors A and B differentially modulate corticotropin-releasing hormone gene expression through a cAMP regulatory element

Corticotrophin-releasing hormone (CRH) plays a major role in mechanisms controlling human pregnancy and parturition. Gene regulation by progesterone may be a key point in the control of placental CRH production. Studies in primary placental ceils show that antagonism of progesterone activity or production by RU486 or trilostane leads to an increase in CRH promoter activity. This effect can be reversed by the addition of progesterone. Overexpression of progesterone receptor A (PRA) or glucocorticoid receptor resulted in a decrease in CRH promoter activity following progesterone treatment, whereas an increase in promoter activity was observed with overexpressed PR-B.