Preimplantation genetic diagnostic protocols for α- and β-thalassaemias using multiplex fluorescent PCR

Haemoglobinopathies including α- and β-thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for β-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for β-thalassaemia have been introduced using restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contamination and allele dropout (ADO) remain an important concern for all of these strategies. In the present study two PGD protocols for detecting β-thalassaemia mutations (codon 41-42 and IVSI-110) and one for α-thalassaemia (SEA mutation) have been designed and tested. These methods contain failsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contamination and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplification efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarity between the DNA sequences of β-globin and δ-globin was also found to be an important issue that necessitated careful design of primers for the β-globin gene. Copyright © 2001 John Wiley & Sons, Ltd.     

First application of preimplantation genetic diagnosis to neurofibromatosis type 2 (NF2)

Neurofibromatosis type 2 (NF2) is a dominantly inherited cancer predisposition syndrome that is caused bymutations in the NF2 gene. We report here the first clinical preimplantation genetic diagnosis (PGD) forNF2. A protocol was developed to simultaneously amplify the mutation and a single nucleotide polymorphism (SNP) located within the gene. The mutation and polymorphism were analysed by simultaneous fluorescent single-strand conformation polymorphism (SSCP) on an automated DNA sequencer. The mutation, carried by the male partner, was a single base pair substitution affecting a splice site in intron 4 of the gene. The female partner was infertile due to polycystic ovary syndrome and would require IVF to conceive. The couple was found to be informative at a linked intragenic SNP situated in the 5′ untranslated region of the gene. The SNP was included in the assay to reduce the risk of misdiagnosis due to allele dropout (ADO). The couple underwent three cycles of treatment during which a total of 43 blastomeres were biopsied from 31 embryos. Amplification at both loci was obtained in 35 cells (81%). A total of five embryos were transferred, two in the first cycle, two in the second and one in the third. No pregnancy ensued. The results of the diagnoses indicated that, in this couple, the inheritance of the mutation may be non-Mendelian. Out of a total of 32 embryos tested only four were found not to carry the mutation. The reasons for this apparent skew remain unknown. Copyright © 2002 John Wiley & Sons, Ltd.     

Turning rhetoric into practice: Integrating tqm into plant operations

Often a management theory fails in practice not because the theory is without merit, but because it was improperly applied. The integration of TQM into plant operations can be accomplished through a four-step circular process: (1) analysis and identification, (2) design, (3) implementation, and (4) evaluation and adjustment. The author outlines these steps in detail and discusses some practical parameters that should be considered before embarking on a TQM program, including the current management philosophy, plant size, and time management.     

Uptake rates of thorium progeny in a semiarid environment

The release rates and transformation processes that influence the mobility, biological uptake, and transfer of radionuclides are essential to the assessment of the health effects in the food chain and ecosystem. This study examined concentrations of 222Th in both soil and vegetation at a closed military training site, Kirtland Air Force Base (KAFB), New Mexico. Brazilian sludge was intentionally introduced into the topsoil in the early 1960s to simulate nuclear weapon accidents. Soil (60) and vegetation (120) samples were collected from 1996 to 2000 and analyzed for radionuclides and progeny. High-resolution gamma-ray spectroscopy was used to determine radionuclide activities. The results indicate that the thorium progeny were the predominant contaminant in soil and vegetation. Concentration ratios (CRs) were calculated based on actinium levels.     

Discrimination of flicker frequency rates in the reptile tuatara (Sphenodon )

By investigating the mechanisms that underlie the perception of environmental cues, we may begin to understand how the sensory system governs behavioral responses. This is the first empirical study to examine learning and visual sensitivity in a reptile species, the tuatara (Sphenodon punctatus). We established a non-intrusive psychophysical method by employing an instrumental paradigm in order to examine discrimination learning and the ability to distinguish different flicker frequencies in the tuatara. Seventeen tuatara were trained under an operant conditioning task to respond to various discriminative stimuli flickering between 2.65 and 65.09 Hz. Tuatara were able to learn the operant task and discriminate between a constant light and flicker frequency rates between 2.65 and 45.61 Hz, but not at 65.09 Hz. We demonstrated a reliable psychophysical method where these reptiles could learn a basic operant task and discriminate visual stimuli in the form of flicker frequency rates. The tuatara’s ability to perceive flickering light is comparable to that of avian, mammalian, and other reptilian species. This method is thus suitable for more comprehensive examinations of vision and additional sensory abilities in other reptiles. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Compliance with Canada’s Fisheries Act: A Field Audit of Habitat Compensation Projects

Loss of fish habitat in North America has occurred at an unprecedented rate through the last century. In response, the Canadian Parliament enacted the habitat provisions of the Fisheries Act. Under these provisions, a “harmful alteration, disruption, or destruction to fish habitat” (HADD) cannot occur unless authorised by Fisheries and Oceans Canada (DFO), with legally binding compensatory habitat to offset the HADD. The guiding principle to DFO’s conservation goal is “no net loss of the productive capacity of fish habitats” (NNL). However, performance in achieving NNL has never been evaluated on a national scale. We investigated 52 habitat compensation projects across Canada to determine compliance with physical, biological, and chemical requirements of Section 35(2) Fisheries Act authorisations. Biological requirements had the lowest compliance (58%) and chemical requirements the highest (100%). Compliance with biological requirements differed among habitat categories and was poorest (19% compliance) in riparian habitats. Approximately 86% of authorisations had larger HADD and/or smaller compensation areas than authorised. The largest noncompliance in terms of habitat area occurred in riverine habitat in which HADDs were, on average, 343% larger than initially authorised. In total, 67% of compensation projects resulted in net losses of habitat area, 2% resulted in no net loss, and 31% achieved a net gain in habitat area. Interestingly, probable violations of the Fisheries Act were prevalent at half of the projects. Analyses indicated that the frequency of probable Fisheries Act violations differed among provinces. Habitat compensation to achieve NNL, as currently implemented in Canada, is at best only slowing the rate of habitat loss. In all likelihood, increasing the amount of authorised compensatory habitat in the absence of institutional changes will not reverse this trend. Improvements in monitoring and enforcement are necessary to move towards achieving Canada’s conservation goals.     

A Preliminary Evaluation of the DDT Contamination of Sediments in Lakes Natron and Bogoria (Eastern Rift Valley,Africa)

Dichlorodiphenyltrichloroethane (DDT) is still used in Africa for the indoor control of malaria and it may represent a potential hazard for wildlife. The littoral sediments of two alkaline-saline lakes, Natron (Tanzania) and Bogoria (Kenya), in the Eastern Rift Valley, supporting large populations of lesser flamingos (Phoeniconaias minor), were analysed for DDT residues. Physical–chemical analyses (temperature, conductivity, pH and dissolved oxygen) were also performed on the water of the two lakes and in the tributaries of Lake Natron, to evaluate the influence of the environmental variables on pollutant occurrence. At Lake Natron, around 1 km from the sediment collection sites, tree leaves of Acacia tortilis were also collected. The main metabolite found in all sediment samples was pp’DDE, whilst equal concentrations of pp’DDT and pp’DDE were measured in acacia leaves. The levels of DDTs measured in the sediments were within 5.9–30.9 ng g⁻¹ d.w., reaching the maximum value in a tributary of Lake Natron. On the whole, the contamination of Lake Natron and Lake Bogoria basins seems to be quite moderate. Nevertheless, the pp’DDE/pp’DDT ratio equals 1 in the Acacia tortilis leaves, which makes one suppose that the input of the parent compound was rather recent and could have been from aerial transport or dust from relatively close-by old pesticides storage sites.     

Modelling of discharge and atmospheric dispersion for carbon dioxide releases

This paper discusses the modelling of the discharge and subsequent atmospheric dispersion for carbon dioxide releases using extensions of models in the consequence modelling package Phast. Phast examines the progress of a potential incident from the initial release to the far-field dispersion including the modelling of rainout and subsequent vaporisation. The original Phast discharge and dispersion models allow the released chemical to occur only in the vapour and liquid phases. As part of the current work these models have been extended to also allow for the occurrence of liquid to solid transition or vapour to solid transition. This applies both for the post-expansion state in the discharge model, as well as for the thermodynamic calculations by the dispersion model. Solid property calculations have been added where necessary. The above extensions are generally valid for fluid releases including CO₂. Using the extended dispersion formulation, a sensitivity study has been carried out for mixing of solid CO₂ with air, and it is demonstrated that solid effects may significantly affect the predicted concentrations.     

Tissue heavy metal concentrations of stranded California sea lions (Zalophus californianus) in Southern California

Concentrations of nine heavy metals (As, Cd, Cu, Fe, Hg, Pb, Mn, Mo and Zn) were determined in the hepatic and renal tissues of 80 stranded California sea lions (Zalophus californianus). Significant age-dependant increases were observed in liver and kidney concentrations of cadmium and mercury, and renal zinc concentrations. Hepatic iron concentrations were significantly higher in females than males. Animals with suspected domoic acid associated pathological findings had significantly higher concentrations of liver and kidney cadmium; and significantly higher liver mercury concentrations when compared to animals classified with infectious disease or traumatic mortality. Significantly higher hepatic burdens of molybdenum and zinc were found in animals that died from infectious diseases. This is the largest study of tissue heavy metal concentrations in California sea lions to date. These data demonstrate how passive monitoring of stranded animals can provide insight into environmental impacts on marine mammals.     

Development and preparation of lead-containing paint films and diagnostic test materials

Lead in paint continues to be a threat to children's health in cities across the United States, which means there is an ongoing need for testing and analysis of paint. This ongoing analytical effort and especially development of new methods continue to drive the need for diagnostic testing materials that provide the analytical challenges of real-world paints. To this end, 31 different types of paint test materials were developed and prepared. Preparation of the materials included development of lead-containing paint films yielding an overall relative standard error for one individual test sample being less than 10%. The 31 diagnostic test materials prepared with these paint films included two lead pigments; lead concentrations from nominally 0 to 2.0 mg lead/cm(2) (0 to 5% lead by weight); overlayers of both "lead-free," oil-based and water-based paints; Al, Ba, and Mg as potential chemical interferents; red and black potential color interferents; and substrates of wood, metal, masonry, and plaster. These materials challenge each step in method development and evaluation, including paint sample collection and preparation, lead extraction, and measurement of solubilized lead. When the materials were used to test performance of a new lead-in-paint testing method based on extraction using a rotor/stator method and measurement using turbidimetry, the results agreed to within ±20% of the expected lead values for 30 out of 31 of the diagnostic test materials, thereby demonstrating their levels of quality and utility.