Multistage carcinogenesis and the fraction at risk

Abstract.: Multistage carcinogenesis models describe the evolution of the cells in an individual's organ from a normal stage to a pre-neoplastic stage to a neoplastic stage. The triggers for the passage from one stage to the next one are presumed to be genetic alterations, which are not only governed by purely random events but also by individual environmental and genetic factors. We generalize existing models of carcinogenesis to populations composed of heterogeneous individuals, thus taking the environmental and genetic variability into accoun

Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE)

<p>Abstract</p> <p>Background</p> <p>Rapid means to discover and enumerate unknown mutations in the exons of human genes on a pangenomic scale are needed to discover the genes carrying inherited risk for common diseases or the genes in which somatic mutations are required for clonal diseases such as atherosclerosis and cancers. The method of constant denaturing capillary electrophoresis (CDCE) permitted sensitive detection and enumeration of unknown point mutations but labor-intensive optimization procedures for each exonic sequence made it impractical for application at a pangenomic scale.</p> <p>Results</p> <p>A variant denaturing capillary electrophoresis protocol, cycling temperature capillary electrophoresis (CTCE), has eliminated the need for the laboratory optimization of separation conditions for each target sequence. Here are reported the separation of wild type mutant homoduplexes from wild type/mutant heteroduplexes for 27 randomly chosen target sequences without any laboratory optimization steps. Calculation of the equilibrium melting map of each target sequence attached to a high melting domain (clamp) was sufficient to design the analyte sequence and predict the expected degree of resolution.</p> <p>Conclusion</p> <p>CTCE provides practical means for economical pangenomic detection and enumeration of point mutations in large-scale human case/control cohort studies. We estimate that the combined reagent, instrumentation and labor costs for scanning the ~250,000 exons and splice sites of the ~25,000 human protein-coding genes using automated CTCE instruments in 100 case cohorts of 10,000 individuals each are now less than U.S. $500 million, less than U.S.$500 per person.</p

Seasonal and Spatial Variation of the Bacterial Mutagenicity of Fine Organic Aerosol in Southern California

The bacterial mutagenicity of a set of 1993 urban particulate air pollution samples is examined using the Salmonella typhimurium TM677 forward mutation assay. Ambient fine particulate samples were collected for 24 hr every sixth day throughout 1993 at four urban sites, including Long Beach, central Los Angeles, Azusa, and Rubidoux, California, and at an upwind background site on San Nicolas Island. Long Beach and central Los Angeles are congested urban areas where air quality is dominated by fresh emissions from air pollution sources; Azusa and Rubidoux are located farther downwind and receive transported air pollutants plus increased quantities of the products of atmospheric chemical reactions. Fine aerosol samples from Long Beach and Los Angeles show a pronounced seasonal variation in bacterial mutagenicity per cubic meter of ambient air, with maximum in the winter and a minimum in the summer. The downwind smog receptor site at Rubidoux shows peak mutagenicity (with postmitochondrial supernatant but no peak without postmitochondrial supernatant) during the September-October periods when direct transport from upwind sources can be expected. At most sites the mutagenicity per microgram of organic carbon from the aerosol is not obviously higher during the summer photochemical smog period than during the colder months. Significant spatial variation in bacterial mutagenicity is observed: mutagenicity per cubic meter of ambient air, on average, is more than an order of magnitude lower at San Nicolas Island than within the urban area. The highest mutagenicity values per microgram of organics supplied to the assay are found at the most congested urban sites at central Los Angeles and Long Beach. The highest annual average values of mutagenicity per cubic meter of air sampled occur at central Los Angeles. These findings stress the importance of proximity to sources of direct emissions of bacterial mutagens and imply that if important mutagen-forming atmospheric reactions occur, they likely occur in the winter and spring seasons as well as the photochemically more active summer and early fall periods

Multistage carcinogenesis and the fraction at risk

Multistage carcinogenesis models describe the evolution of the cells in an individual's organ from a normal stage to a pre-neoplastic stage to a neoplastic stage. The triggers for the passage from one stage to the next one are presumed to be genetic alterations, which are not only governed by purely random events but also by individual environmental and genetic factors. We generalize existing models of carcinogenesis to populations composed of heterogeneous individuals, thus taking the environmental and genetic variability into account

Human fetal/tumor metakaryotic stem cells: pangenomic homologous pairing and telomeric end-joining of chromatids

Metakaryotic cells and syncytia with large, hollow, bell-shaped nuclei demonstrate symmetrical and asymmetrical amitotic nuclear fissions in microanatomical positions and numbers expected of stem cell lineages in tissues of all three primordial germ layers and their derived tumors. Using fluorescence in situ hybridization, mononuclear metakaryotic interphase cells have been found with only 23 centromeric and 23 telomeric staining regions. Syncytial bell-shaped nuclei found approximately during weeks 5–12 of human gestation display 23 centromeric and either 23 or 46 telomeric staining regions. These images suggest that (1) homologous chromatids pair at centromeres and telomeres, (2) all paired telomeres join end-to-end with other paired telomeres in all mononuclear and some syncytial metakaryotic cells, and (3) telomere junctions may open and close during the syncytial phase of development. Twenty-three telomeric joining figures could be accounted by 23 rings of one chromatid pair each, a single pangenomic ring of 23 joined chromatid pairs, or any of many possible sets of oligo-chromatid pair rings. As telomeric end-joining may affect peri-telomeric gene expression, a programmed sequence of telomeric end-joining associations in metakaryotic stem cells could guide developmental arboration and errors in, or interruptions of, this program could contribute to carcinogenesis.National Institute of Environmental Health SciencesUnited Therapeutics, Inc

Bacterial Mutagenicity of Urban Organic Aerosol Sources in Comparison to Atmospheric Samples

The bacterial mutagenicity of a comprehensive set of urban particulate air pollution source samples is examined using the Salmonella typhimurium forward mutation assay. Each of the combustion source samples examined, including the exhaust from catalyst-equipped autos, noncatalyst autos, heavy-duty diesel trucks, plus natural gas, distillate oil, and wood combustion sources, is mutagenic in this assay, with a response per microgram of organic carbon in these samples generally greater than that of cigarette smoke aerosol. The noncombustion source samples tested generally are not mutagenic at the levels examined. The specific mutagenicity (mutant fraction per microgram of organic carbon) of ambient aerosol samples collected in southern California is compared to a weighted average of the specific mutagenicity of the primary source samples assembled in proportion to their emission rates in the Los Angeles area. In most cases where a comparison can be made, the specific mutagenicity of the source composites and the ambient samples are of similar magnitude, with the exception that the -PMS mutagenicity of the aerosol at Long Beach, CA, during the first half of the calendar year 1982 and at Azusa, CA, during the April-June 1982 period is much higher than can be explained by direct emissions from the sources studied here

The study of calcified atherosclerotic arteries: an alternative to evaluate the composition of a problematic tissue reveals new insight including metakaryotic cells

Background Calcifications of atherosclerotic plaques represent a controversial issue as they either lead to the stabilization or rupture of the lesion. However, the cellular key players involved in the progression of the calcified plaques have not yet been described. The primary reason for this lacuna is that decalcification procedures impair protein and nucleic acids contained in the calcified tissue. The aim of our study was to preserve the cellular content of heavily calcified plaques with a new rapid fixation in order to simplify the study of calcifications. Methods Here we applied a fixation method for fresh calcified tissue using the Carnoy’s solution followed by an enzymatic tissue digestion with type II collagenase. Immunohistochemistry was performed to verify the preservation of nuclear and cytoplasmic antigens. DNA content and RNA preservation was evaluated respectively with Feulgen staining and RT-PCR. A checklist of steps for successful image analysis was provided. To present the basic features of the F-DNA analysis we used descriptive statistics, skewness and kurtosis. Differences in DNA content were analysed with Kruskal-Wallis and Dunn’s post tests. The value of P < 0.05 was considered significant. Results Twenty-four vascular adult tissues, sorted as calcified (14) or uncalcified (10), were processed and 17 fetal tissues were used as controls (9 soft and 8 hard). Cells composing the calcified carotid plaques were positive to Desmin, Vimentin, Osteocalcin or Ki-67; the cellular population included smooth muscle cells, osteoblasts and osteoclasts-like cells and metakaryotic cells. The DNA content of each cell type found in the calcified carotid artery was successfully quantified in 7 selected samples. Notably the protocol revealed that DNA content in osteoblasts in fetal control tissues exhibits about half (3.0 ng) of the normal nuclear DNA content (6.0 ng). Conclusion Together with standard histology, this technique could give additional information on the cellular content of calcified plaques and help clarify the calcification process during atherosclerosis.United Therapeutics Corporatio

Seasonal and Spatial Variation of the Bacterial Mutagenicity of Fine Organic Aerosol in Southern California

The bacterial mutagenicity of a set of 1993 urban particulate air pollution samples is examined using the Salmonella typhimurium TM677 forward mutation assay. Ambient fine particulate samples were collected for 24 hr every sixth day throughout 1993 at four urban sites, including Long Beach, central Los Angeles, Azusa, and Rubidoux, California, and at an upwind background site on San Nicolas Island. Long Beach and central Los Angeles are congested urban areas where air quality is dominated by fresh emissions from air pollution sources; Azusa and Rubidoux are located farther downwind and receive transported air pollutants plus increased quantities of the products of atmospheric chemical reactions. Fine aerosol samples from Long Beach and Los Angeles show a pronounced seasonal variation in bacterial mutagenicity per cubic meter of ambient air, with maximum in the winter and a minimum in the summer. The downwind smog receptor site at Rubidoux shows peak mutagenicity (with postmitochondrial supernatant but no peak without postmitochondrial supernatant) during the September-October periods when direct transport from upwind sources can be expected. At most sites the mutagenicity per microgram of organic carbon from the aerosol is not obviously higher during the summer photochemical smog period than during the colder months. Significant spatial variation in bacterial mutagenicity is observed: mutagenicity per cubic meter of ambient air, on average, is more than an order of magnitude lower at San Nicolas Island than within the urban area. The highest mutagenicity values per microgram of organics supplied to the assay are found at the most congested urban sites at central Los Angeles and Long Beach. The highest annual average values of mutagenicity per cubic meter of air sampled occur at central Los Angeles. These findings stress the importance of proximity to sources of direct emissions of bacterial mutagens and imply that if important mutagen-forming atmospheric reactions occur, they likely occur in the winter and spring seasons as well as the photochemically more active summer and early fall periods

Non-Invasive Prenatal Detection of Trisomy 21 Using Tandem Single Nucleotide Polymorphisms

BACKGROUND: Screening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA. METHODS AND FINDINGS: Highly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects. CONCLUSIONS: In summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma