Registry-derived stage (RD-Stage) for capturing cancer stage at diagnosis for endometrial cancer.

BACKGROUND: Capture of cancer stage at diagnosis is important yet poorly reported by health services to population-based cancer registries. In this paper we describe current completeness of stage information for endometrial cancer available in Australian cancer registries; and develop and validate a set of rules to enable cancer registry medical coders to calculate stage using data available to them (registry-derived stage or 'RD-Stage'). METHODOLOGY: Rules for deriving RD-stage (Endometrial carcinoma) were developed using the American Joint Commission on Cancer (AJCC) TNM (tumour, nodes, metastasis) Staging System (8th Edition). An expert working group comprising cancer specialists responsible for delivering cancer care, epidemiologists and medical coders reviewed and endorsed the rules. Baseline completeness of data fields required to calculate RD-Stage, and calculation of the proportion of cases for whom an RD stage could be assigned, was assessed across each Australian jurisdiction. RD-Stage (Endometrial cancer) was calculated by Victorian Cancer Registry (VCR) medical coders and compared with clinical stage recorded by the patient's treating clinician and captured in the National Gynae-Oncology Registry (NGOR). RESULTS: The necessary data completeness level for calculating RD-Stage (Endometrial carcinoma) across various Australian jurisdictions varied from 0 to 89%. Three jurisdictions captured degree of spread of cancer, rendering RD-Stage unable to be calculated. RD-Stage (Endometrial carcinoma) could not be derived for 64/485 (13%) cases and was not captured for 44/485 (9%) cases in NGOR. At stage category level (I, II, III, IV), there was concordance between RD-Stage and NGOR captured stage in 393/410 (96%) of cases (95.8%, Kendall's coefficient = 0.95). CONCLUSION: A lack of consistency in data captured by, and data sources reporting to, population-based cancer registries meant that it was not possible to provide national endometrial carcinoma stage data at diagnosis. In a sample of Victorian cases, where surgical pathology was available, there was very good concordance between RD-Stage (Endometrial carcinoma) and clinician-recorded stage data available from NGOR. RD-Stage offers promise in capturing endometrial cancer stage at diagnosis for population epidemiological purposes when it is not provided by health services, but requires more extensive validation.

2,4-Dinitrophenol: 'diet' drug death following major trauma.

There has been a resurgence in the illicit use of 2,4-dinitrophenol by people wishing to achieve rapid weight loss. Despite its availability, the drug is banned for human consumption as it is toxic and can have fatal consequences. We present the case of a 23-year-old man who regularly consumed 2,4-dinitrophenol to generate fat loss without apparent ill effect. He was involved in a high-speed road traffic collision and sustained limb-threatening injuries. The combination of emergency surgery, trauma and 2,4-dinitrophenol consumption culminated in deterioration under anaesthesia, with subsequent death from multiorgan failure in the intensive care unit 48 h later. Previous cases have reported death from 2,4-dinitrophenol toxicity alone. We believe this is the first reported case of 2,4-dinitrophenol toxicity triggered by the additional physiological stress of polytrauma and emergency surgery.

Spectral characterization of flash and high flux x-ray radiographic sources with a magnetic Compton spectrometer.

In this work, we present a new analysis method applied to revitalize permanent magnet Compton spectrometers used to measure photon energy spectra in the MeV range. The inversion of the measured electron distribution to determine the original photon distribution is achieved via a method of consistent coupled radiation transport and magnetic field mapping of the input photon spectra to the measured electron distribution. The method of linear least squares was used to perform the unfolding of the electron distribution to the initial photon spectra, without any assumptions made regarding the electron distribution. We present an application of this method to data from a nominal 19.4 MeV flash radiographic source (the first axis of the Dual Axis Radiographic Hydro-Test Facility) capable of generating 500 R @ 1 m in ∼60 ns and a medical therapy source (a Scanditronix M22, Microtron) capable of variable energies with nominal endpoints of 6, 10, 15, and 20 MeV and an output of ∼1000-2000 R/min @ 1 m. The results provide agreement between the modeled and unfolded experimentally measured photon spectra as quantified by statistical tests, from 1.5 to 20 MeV. Experimental results are presented as well as a discussion of the novel MCNP6-based simulations and methods for reconstruction of the spectra.

Steering an intense relativistic electron beam in a linear induction accelerator.

The best electron beam transport through a linear induction accelerator (LIA) is achieved when the beam is well centered on the magnetic axis of the focusing solenoids. Since the beam may be injected offset from, or at an angle to, the centerline, dipole magnets are usually provided as a means to steer the beam and center it. Steering may be accomplished by trial and error, but this is very time consuming, especially for accelerators with a low repetition rate for beam position measurements and dipole adjustments. This article presents a steering method requiring a minimal number of measurements and adjustments to position the beam at any desired location, including positioning on the centerline.

Helicobacter pylori physiology predicted from genomic comparison of two strains.

Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outcome of H. pylori-related disease.

Calibration of two compact permanent magnet spectrometers for high current electron linear induction accelerators.

In this work, two compact, permanent magnet, electron spectrometers have been built to measure the electron beam energy at the Dual Axis Radiographic Hydrodynamic Test facility. Using H- and OH- anions, the spectrometers were calibrated at the Special Technologies Laboratory in Santa Barbara, California (USA). The spectrometers were mounted on a custom drift tube that allows the magnet assemblies to be translated, which increases the path length of the electrons traveling through the magnetic field and therefore increases the upper bound of the measurable electron kinetic energy. The measurable range of electron kinetic energies is between 2.8 MeV-4.1 MeV for the first spectrometer and 14.1 MeV-21.1 MeV for the second spectrometer, with an overall measurement uncertainty of 0.32%.

Influence of structural and functional modifications of selected genotoxic carcinogens on metabolism and mutagenicity - a review.

Alterations in molecular structure are responsible for the differential biological response(s) of a chemical inside a biosystem. Structural and functional parameters that govern a chemical's metabolic course and determine its ultimate outcome in terms of mutagenic/carcinogenic potential are extensively reviewed here. A large number of environmentally-significant organic chemicals are addressed under one or more broadly classified groups each representing one or more characteristic structural feature. Numerous examples are cited to illustrate the influence of key structural and functional parameters on the metabolism and DNA adduction properties of different chemicals. It is hoped that, in the event of limited experimental data on a chemical's bioactivity, such knowledge of the likely roles played by key molecular features should provide preliminary information regarding its bioactivation, detoxification and/or mutagenic potential and aid the process of screening and prioritising chemicals for further testing.

Cloning of ALL-1, the locus involved in leukemias with the t(4;11)(q21;q23), t(9;11)(p22;q23), and t(11;19)(q23;p13) chromosome translocations.

Chromosomal region 11q23 participates in a number of reciprocal translocations with specific regions of chromosomes 4, 9, 19, and others. These translocations are associated with acute lymphocytic leukemia and acute myelomonocytic, monocytic, and myelogenous leukemia. From a yeast artificial chromosome containing human DNA derived from 11q23 we cloned a DNA fragment which can be used as a probe to detect rearrangements in leukemic cells from the majority of patients with the t(4;11), t(9;11), and t(11;19) translocations. The breakpoints cluster in a small DNA region of less than 5.8 kilobases.

Dynamics and orientation of glycolipid headgroups by 2H-NMR: gentiobiose.

Deuterium nuclear magnetic resonance has been used to investigate the dynamics and determine the orientation of the headgroup of the glycolipid 1,2-di-O-tetradecyl-3-O-(6-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl )-sn- glycerol (beta-DTDGL), in aqueous multilamellar dispersions. In addition, its anomeric analog, having an alpha glucose-glycerol linkage, was prepared and examined. The lipids were labelled with deuterium at specific positions in the disaccharide moiety. Analysis of the deuterium quadrupolar splittings for the first glucose ring (glycerol-linked) gave segmental order parameters of 0.43 and 0.35 for the beta and alpha isomers, respectively. Both isomers had similar orientations of the sugar ring relative to the bilayer surface, as determined for lipid in the liquid-crystalline phase. 2H-NMR results for the lipid labelled at C-6' are consistent with a single conformation about the C-5'-C-6' bond of the first glucose residue, with a dihedral angle (O-5'-C-5'-C-6'-O-6') of -17 degrees. The results obtained for the second sugar ring suggest that two conformers may be present, which are in slow exchange on the 2H-NMR timescale. Measurements of longitudinal relaxation times, T1z, gave similar values for both sugar moieties in the headgroup, suggesting that the disaccharide does not exhibit the flexibility expected about the 1----6 linkage. Since T1z for 2H in these compounds decreases with increasing temperature and increases with magnetic field strength, the motion(s) dominating relaxation is in the long-correlation-time regime [omega 0 tau c)2 greater than 1). Thus, the gentiobiosyl headgroup undergoes the slowest motion of the glycolipid headgroups studied to date.

Determination of the order of gene function in the yeast nuclear division pathway using cs and ts mutants.

Cold-sensitive (cs) and heat-sensitive (ts) conditional-lethal mutations that affect specifically the cell division cycle of budding yeast (Saccharomyces cerevisiae) were used to determine the order of gene function. Reciprocal temperature-shift experiments using cs-ts double mutants revealed a detailed order of function among genes whose execution points and mutant phenotypes are very similar. The data suggest that the nuclear branch of the overall cell-cycle pathway itself contains at least one branch.

Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies.

We isolated 18 independent recessive cold-sensitive cell-division-cycle (cdc) mutants of Saccharomyces cerevisiae, in nine complementation groups. Terminal phenotypes exhibited include medial nuclear division, cytokinesis, and a previously undescribed terminal phenotype consisting of cells with a single small bud and an undivided nucleus. Four of the cold-sensitive mutants proved to be alleles of CDC11, while the remaining mutants defined at least six new cell-division-cycle genes: CDC44, CDC45, CDC48, CDC49, CDC50 and CDC51.--Spontaneous revertants from cold-sensitivity of four of the medial nuclear division cs cdc mutants were screened for simultaneous acquisition of a temperature-sensitive phenotype. The temperature-sensitive revertants of four different cs cdc mutants carried single new mutations, called Sup/Ts to denote their dual phenotype: suppression of the cold-sensitivity and concomitant conditional lethality at 37 degrees. Many of the Sup/Ts mutations exhibited a cell-division-cycle terminal phenotype at the high temperature, and they defined two new cdc genes (CDC46 and CDC47). Two cold-sensitive medial nuclear division cdc mutants representing two different cdc genes were suppressed by different Sup/Ts alleles of another gene which also bears a medial nuclear division function (CDC46). In addition, the cold-sensitive medial nuclear division cdc mutant csH80 was suppressed by a Sup/Ts mutation yielding an unbudded terminal phenotype with an undivided nucleus at the high temperature. This mutation was an allele of CDC32. These results suggest a pattern of interaction among cdc gene products and indicate that cdc gene proteins might act in the cell cycle as complex specific functional assemblies.

Characterization of the surface enhanced raman scattering (SERS) of bacteria.

The surface enhanced Raman scattering (SERS) of a number of species and strains of bacteria obtained on novel gold nanoparticle (approximately 80 nm) covered SiO(2) substrates excited at 785 nm is reported. Raman cross-section enhancements of >10(4) per bacterium are found for both Gram-positive and Gram-negative bacteria on these SERS active substrates. The SERS spectra of bacteria are spectrally less congested and exhibit greater species differentiation than their corresponding non-SERS (bulk) Raman spectra at this excitation wavelength. Fluorescence observed in the bulk Raman emission of Bacillus species is not apparent in the corresponding SERS spectra. Despite the field enhancement effects arising from the nanostructured metal surface, this fluorescence component appears "quenched" due to an energy transfer process which does not diminish the Raman emission. The surface enhancement effect allows the observation of Raman spectra of single bacterial cells excited at low incident powers and short data acquisition times. SERS spectra of B. anthracis Sterne illustrate this single cell level capability. Comparison with previous SERS studies reveals how the SERS vibrational signatures are strongly dependent on the morphology and nature of the SERS active substrates. The potential of SERS for detection and identification of bacterial pathogens with species and strain specificity on these gold particle covered glassy substrates is demonstrated by these results.

An investigation into exposure of pigs to lead from contaminated zinc oxide in 2007-2008.

BACKGROUND: High levels of lead, up to 3.3 mg/kg fresh weight, were detected in pig liver in Western Australia at the beginning of 2008. This followed the detection of lead at above the maximum level (ML) in a pig liver through the National Residue Survey (NRS). The contamination source was traced back to a zinc oxide feed additive used early post-weaning that contained in excess of 8% lead. METHODS AND RESULTS: Confirmation of the source of lead contamination was obtained by comparing lead isotope ratios for the zinc oxide and the pig livers. The investigation demonstrated the importance of verifying the safety of feed and feed ingredients prior to incorporation in feed. Retrospective analysis of NRS data indicated that the level of lead needed to trigger an investigation for intensively housed pigs should be considerably lower than the ML. As a result, investigations in Australia will now be conducted when levels of lead in pig liver exceed 0.1 mg/kg fresh weight. CONCLUSIONS: Despite the potential for small amounts of non-compliant kidney and liver to enter the human food chain, there was no significant increase in the risk to consumers.

Glycosylation and secretion of human alpha-1-antitrypsin by yeast.

Human alpha-1-antitrypsin (alpha-AT) is a major serum protein with protease inhibitory activity. Three asparagine residues in alpha-AT are glycosylated with the mammalian 'complex' pattern of carbohydrate as the protein is secreted from cells in the liver. To examine the glycosylation and secretion of human alpha-AT by Saccharomyces cerevisiae, the yeast invertase secretion signal codons were substituted for the native secretion signal coding DNA of an alpha-AT cDNA, and the fusion gene was placed on an autonomously replicating yeast--Escherichia coli shuttle vector under control of the yeast triosephosphate isomerase (TPI) promoter. Yeast strains transformed with this plasmid produce human alpha-AT and secrete about one-fifth of it into the culture broth. Approximately 80% of the alpha-AT produced in yeast is not in the culture broth but is inside the cell within the secretory pathway. This internal alpha-AT is heterogeneous, consisting of molecules with core carbohydrate on either two or all three of the asparagine receptors. Human alpha-AT secreted into the culture broth contains, in addition to core carbohydrate, variable numbers of mannose outer chains, typical of secreted yeast proteins such as invertase. All carbohydrate is removed by endoglycosidase H treatment. Examination of alpha-AT secreted from an mnn9 mutant, which blocks addition of variable numbers of outer mannose chains, revealed a homogeneous alpha-AT product which, like alpha-AT isolated from human serum, bears carbohydrate on three asparagine residues per molecule.

A human genome YAC library in a selectable high-copy-number vector.

An experimental yeast artificial chromosome (YAC) library consisting of one genome equivalent of human DNA was prepared in a selectable high-copy-number (hcn) YAC vector. Screening for unique loci was accomplished by PCR of successively smaller DNA pools and by hybridization to high-density microcolony blots. Inserts averaged 200 kb in size, but several YACs with inserts averaging about 650 kb were obtained when polyamines were added prior to yeast transformation. YACs were identified for 17 out of 29 sequence-tagged sites (STS) screened by a PCR-based approach. All YACs in the size range of 100-600 kb that were examined could be obtained at significantly elevated copy numbers following growth of the clones in methotrexate/sulfanilamide/thymidine-supplemented medium. The hcn YACs could also be selected during growth in microtiter dishes, and the resulting clones were used to prepare high-density microcolony DNA blots for hybridization with radiolabeled PCR products. DNA pools for the PCR-based screening of this experimental library are available to investigators interested in applications of hcn YACs.

Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin.

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the Mr-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.

Rapid identification of overlapping YACs in the MEN2 region of human chromosome 10 by hybridization with Alu element-mediated PCR products.

An overlapping set of 21 yeast artificial chromosomes (YACs) spanning the RET proto-oncogene [Takahashi et al., Oncogene 3 (1988) 571-578] and D10S102 markers on human chromosome 10 was isolated in a series of hybridization-based chromosomal walks in a YAC library. Genetic linkage analyses implicate this chromosomal region as the location of the gene (MEN2A) responsible for multiple endocrine neoplasia type 2A. Four YACs carrying a RET sequence-tagged site (STS) and two YACs carrying a D10S102 STS were used to initiate chromosome walks. These were based on hybridization of Alu element-mediated polymerase chain reaction (Alu-PCR) products from YACs to dot blots of Alu-PCR products from complex pools of YAC clones. The hybridization anchor content of YACs identified in the walks was confirmed by probing blots of Alu-PCR products from individual YACs and by comparing Alu-PCR fingerprints of each YAC. Ten hybridization-based Alu-PCR anchors and three STS anchors were ordered within eleven intervals created by the 21 overlapping YACs. The order of anchors requiring the fewest gaps in the YACs is consistent with the walking results and establishes the STS anchor order as D10S102-D10S94-RET. The overlapping set of YACs represents about 1.55 Mb of the human genome according to restriction mapping of four representative YACs in the contig. These results demonstrate the power of Alu-PCR hybridization for chromosomal walking and provide a rich source of overlapping YACs which can be used to identify candidate MEN2A genes.

Expression of calf prochymosin in Saccharomyces cerevisiae.

A yeast strain which synthesizes activatable calf prochymosin (also known as prorennin) has been constructed by transformation with a vector carrying the methionyl-prochymosin coding sequence attached to efficient yeast transcriptional promoter and terminator sequences. Cloned preprochymosin cDNA was altered by restriction endonuclease cleavage and addition of a synthetic oligonucleotide to yield a DNA sequence encoding methionyl-prochymosin. This methionyl-prochymosin gene was ligated to a yeast chromosomal fragment containing the GAL1 promoter, and the construction was placed in an Escherichia coli-Saccharomyces cerevisiae shuttle vector with or without a transcriptional terminator DNA fragment from the yeast SUC2 gene. In yeast the two constructions result in equal amounts of prochymosin protein and mRNA. The prochymosin from yeast is activatable to chymosin by incubation at low pH and exhibits milk-clotting activity indistinguishable from calf chymosin.

Genetic polymorphism of CYP2D6 and lung cancer risk.

Previous reports of the association of extensive debrisoquine metabolism, controlled by the cytochrome P450 CYP2D6, with increased lung cancer risk have been conflicting. We examined the hypothesis that genetic polymorphism at the CYP2D6 locus identifies individuals at increased risk for lung cancer in a case-control study of 98 incident Caucasian lung cancer patients and 110 age-, race-, and sex-matched controls conducted at the National Naval Medical Center, Bethesda, MD. Using germ line DNA, we identified inactivating mutations at the CYP2D6 locus (CYP2D6*3, CYP2D6*4, CYP2D6*5, and CYP2D6*6A), as well as those mutations that impair but do not abolish enzyme activity (CYP2D6*9 and CYP2D6*10A). Compared to subjects with homozygous inactivating mutations, no association with lung cancer was observed for those with homozygous or heterozygous functional alleles (odds ratios were 0.4 and 0.7, respectively). Furthermore, no excess risk was seen in any histological group or smoking category, and adjustment for smoking and sociodemographic characteristics did not alter the findings. Although the concept that genetic polymorphisms may contribute to differential lung cancer susceptibility is sound, these data do not support the role of CYP2D6 as a marker for elevated lung cancer risk.

Production of proteins by secretion from yeast.

To summarize, a variety of stable vectors and efficient promoters and secretion signals are available in yeast for engineering the secretion of any protein of interest. Since secretion is growth-associated, we have favored the use of constitutive promoters and moderate copy number integrated vectors. This is because (1) heterologous gene expression from very high copy number vectors is frequently deleterious to growth and (2) delaying gene expression until after the most rapid cell growth phase is cumbersome on a large scale. Methods are available for dividing the total process into growth and production/secretion phases, but they appear worthwhile only when expression of the engineered protein compromises growth significantly. Even with these useful tools, it is frequently helpful to enlist the aid of mutant host strains in order to maximize secretion of a desired protein. Mutations in the PMR1 gene have proved effective in a number of different cases. Moreover, it is possible to identify new host strains tailored to specific needs by applying activity screens to mutagenized colonies growing on petri plates. Finally, colony screens such as the ones described here for active secreted enzymes are useful for routine strain construction. For example, they may be applied to identify the most productive strain from a large number of clones following a transformation or genetic cross. In addition, these screens may be used for characterizing the products of random mutagenesis of the gene encoding the secreted enzyme. The resulting structure-function information can be used to identify regions of the enzyme involved in different activities and to build new enzymes with different characteristics.