Evaluation of Mutation Testing in a Nuclear Industry Case Study

For software quality assurance, many safety-critical industries appeal to the use of dynamic testing and structural coverage criteria. However, there are reasons to doubt the adequacy of such practices. Mutation testing has been suggested as an alternative or complementary approach but its cost has traditionally hindered its adoption by industry, and there are limited studies applying it to real safety-critical code. This paper evaluates the effectiveness of state-of-the-art mutation testing on safety-critical code from within the U.K. nuclear industry, in terms of revealing flaws in test suites that already meet the structural coverage criteria recommended by relevant safety standards. It also assesses the practical feasibility of implementing such mutation testing in a real setting. We applied a conventional selective mutation approach to a C codebase supplied by a nuclear industry partner and measured the mutation score achieved by the existing test suite. We repeated the experiment using trivial compiler equivalence (TCE) to assess the benefit that it might provide. Using a conventional approach, it first appeared that the existing test suite only killed 82% of the mutants, but applying TCE revealed that it killed 92%. The difference was due to equivalent or duplicate mutants that TCE eliminated. We then added new tests to kill all the surviving mutants, increasing the test suite size by 18% in the process. In conclusion, mutation testing can potentially improve fault detection compared to structural-coverage-guided testing, and may be affordable in a nuclear industry context. The industry feedback on our results was positive, although further evidence is needed from application of mutation testing to software with known real faults

Scientific Opinion on Flavouring Group Evaluation 63, Revision 3 (FGE.63Rev3): aliphatic secondary alcohols, ketones and related esters evaluated by JECFA (59th and 69th meetings) structurally related to saturated and unsaturated aliphatic secondary alcohols, ketones and esters of secondary alcohols and saturated linear or branched-chain carboxylic acids evaluated by EFSA in FGE.07Rev4

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ILR Research in Progress 2006-07

The production of scholarly research continues to be one of the primary missions of the ILR School. During a typical academic year, ILR faculty members published or had accepted for publication over 25 books, edited volumes, and monographs, 170 articles and chapters in edited volumes, numerous book reviews. In addition, a large number of manuscripts were submitted for publication, presented at professional association meetings, or circulated in working paper form. Our faculty's research continues to find its way into the very best industrial relations, social science and statistics journals.Research_in_Progress_2006_07.pdf: 18 downloads, before Oct. 1, 2020

Emerging prenatal genetic tests : developing a health technology assessment (HTA) framework for informed decision-making

Delphi Process In preparation for the first Delphi exercise, a list of questions was produced from the academic literature, webbased sources and interviews with experts. These questions were structured into broad dimensions and a draft questionnaire piloted. A final list of 73 questions formed the basis of the first Delphi survey. Participants were asked to grade the perceived importance of each question for inclusion in HTA reports on new prenatal genetic tests (4 = Essential; 3 = Desirable, but not essential; 2 = Useful but should not be required; 1 = Of little/ no importance; 0 = I have no basis for judgement). Secondly, they were asked to indicate whether a question should be addressed during test development or whether the question could be addressed later once the technology is ready for implementation. Finally, Panel members were encouraged to identify any other questions which appeared to be missing from the initial list. For copy of questionnaire, see Annex 1: Delphi Round 1 Questionnaire. Respondents were also asked to provide personal details to give some indication of their HTA experience and specialist expertise. Analysis of responses demonstrated that SAFE Delphi panel members represent a highly experienced, multidisciplinary international group of experts with the knowledge required to define which key questions should be addressed in HTA reports on new prenatal genetic tests. Delphi Responses Responses were received from 77/90 (86%) of Panel members. These were analysed with a cut-off of 75% (±3%) applied as an indicator of Panel consensus for all questions. Thus, any question which three out of four respondents rated as essential or desirable was retained, whilst those not achieving this level of agreement were provisionally excluded. In addition, mean scores were also calculated (excluding 0 = I have no basis for judgement) for each question. A mean score >3.25 ± 0.05 was taken as an indication that the Panel had identified a particular question as being of the highest priority to address in HTA

Construction of a novel fungal gus expression plasmid, and its evaluation in Aspergillus nidulans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University

A GUS expression plasmid, pFunGus, was constructed containing a multi-cloning site for the insertion of gene regulatory elements, to be used in fungal reporter gene studies. A derivative of pFunGus (pFG-gpd) was constructed by the insertion of the gpdA promoter (glyceradehyde-3-phosphatc dehydrogenase) into the multi-cloning site of pFunGus for the assessment of the plasmid's transformation and expression properties in Aspergillus niduans. The correct construction of pFunGus and pFG-gpd was verified by analytical restriction digests and by its property of GUS expression in A. nidulans. The plasmid was integrated into the A. nidulans genome via cotransformation with the phleomycin resistance plasmid, pAN8-l. Transformation frequencies of between 3 and 250 transformants per µg of pAN8-l DNA were obtained. Initial screening for cotransformation yielded no pFG-gpd transformants. Attempts to improve cotransformation frequencies by optimisation of cotransformation conditions were unsuccessful. However, large scale screenings of transformants lead to cotransformants being isolated at a very low cotransformation frequency. Approximately 0.45% of pAN8-l transformants possessed the GUS phenotype. The eight pFG-gpd transformants obtained were analysed by Southern hybridisation. Six out of the eight transformants had a single copy integration. Of the remaining two transformants, one had three copies integrated at separate locations, one of which was disrupted, and the other had four copies integrated as tandem repeats, one of which was disrupted. All the transforming DNA appeared to be integrated ectopically. The physiology of the transformants was assessed by dry weight increase, colony extension and total protein content. These showed that the transformants biology was not significantly compromised by the transforming DNA. Finally, high levels of GUS expression were observed in all pFG-gpd transformants and the GUS expression per copy of the GUS expression cassette integrated into the genome was constant. These results showed that the transformed gene copy number determined the levels of gene activity rather than the position of integration in the genome. Overall these results demonstrate the potential application of the versatile GUS expression plasmid, pFunGus for reporter gene studies in filamentous fungi

Programmed cell death and genetic stability in conifer embryogenesis

Somatic embryogenesis, the generation of embryos from somatic cells, is a valuable tool for studying embryology. In addition, somatic embryos can be used for large-scale vegetative propagation, an application of great interest for forestry. A critical event during early embryo differentiation in conifers is the apical basal polarization, which proceeds through the establishment of two embryonic parts: the proliferating embryonal mass and the terminally differentiated suspensor. The development of both parts is strictly coordinated and imbalance causes embryonic defects. The suspensor cells are eliminated by programmed cell death (PCD). In animals, caspase family proteases are the main executioners of PCD. In this work we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity during early embryo differentiation in Norway spruce (Picea abies L. Karst.). We found that VEIDase is the principal caspase-like activity. This activity is localized specifically in suspensor cells, and its inhibition prevents normal embryo development by blocking the suspensor differentiation. The in vitro VEIDase activity was shown to be highly sensitive to pH, ionic strength, temperature and zinc concentration. In vivo studies with Zinquin, a zinc-specific fluorescent probe, revealed a high accumulation of intracellular free zinc in the embryonal masses and an abrupt decrease in the suspensor. Increased zinc concentration in the culture medium suppresses terminal differentiation and PCD of the suspensor. In accordance, exposure of early embryos to TPEN, a zinc-specific chelator, induces ectopic cell death affecting embryonal masses. This establishes zinc as an important factor affecting cell fate specification during plant embryogenesis. Before somatic embryos can be accepted for clonal propagation it is important to show that the regenerated plants have similar growth to that of seedlings and are genetically uniform. The genetic integrity during zygotic and somatic embryogenesis in Norway spruce and Scots pine (Pinus sylvestris L.) was investigated by comparing the stability of variable nuclear microsatellite loci. The stability varied significantly among families in both species during somatic embryogenesis. Scots pine families showing low genetic stability during establishment of embryogenic cultures had a higher embryogenic potential than those that were genetically more stable. In contrast, embryo development was suppressed in genetically unstable families. The stability of microsatellites was in general higher in zygotic embryos than in somatic embryos. No deviation in growth was observed in somatic embryo plants of Norway spruce carrying mutated microsatellites

Patient and Disease-Specific Induced Pluripotent Stem Cells for Discovery of Personalized Cardiovascular Drugs and Therapeutics.

Human induced pluripotent stem cells (iPSCs) have emerged as an effective platform for regenerative therapy, disease modeling, and drug discovery. iPSCs allow for the production of limitless supply of patient-specific somatic cells that enable advancement in cardiovascular precision medicine. Over the past decade, researchers have developed protocols to differentiate iPSCs to multiple cardiovascular lineages, as well as to enhance the maturity and functionality of these cells. Despite significant advances, drug therapy and discovery for cardiovascular disease have lagged behind other fields such as oncology. We speculate that this paucity of drug discovery is due to a previous lack of efficient, reproducible, and translational model systems. Notably, existing drug discovery and testing platforms rely on animal studies and clinical trials, but investigations in animal models have inherent limitations due to interspecies differences. Moreover, clinical trials are inherently flawed by assuming that all individuals with a disease will respond identically to a therapy, ignoring the genetic and epigenomic variations that define our individuality. With ever-improving differentiation and phenotyping methods, patient-specific iPSC-derived cardiovascular cells allow unprecedented opportunities to discover new drug targets and screen compounds for cardiovascular disease. Imbued with the genetic information of an individual, iPSCs will vastly improve our ability to test drugs efficiently, as well as tailor and titrate drug therapy for each patient

Permitted daily exposure for diisopropyl ether as a residual solvent in pharmaceuticals

Solvents can be used in the manufacture of medicinal products provided their residual levels in the final product comply with the acceptable limits based on safety data. At worldwide level, these limits are set by the "Guideline Q3C (R6) on impurities: guideline for residual solvents" issued by the ICH. Diisopropyl ether (DIPE) is a widely used solvent but the possibility of using it in the pharmaceutical manufacture is uncertain because the ICH Q3C guideline includes it in the group of solvents for which "no adequate toxicological data on which to base a Permitted Daily Exposure (PDE) was found". We performed a risk assessment of DIPE based on available toxicological data, after carefully assessing their reliability using the Klimisch score approach. We found sufficiently reliable studies investigating subchronic, developmental, neurological toxicity and carcinogenicity in rats and genotoxicity in vitro. Recent studies also investigated a wide array of toxic effects of gasoline/DIPE mixtures as compared to gasoline alone, thus allowing identifying the effects of DIPE itself. These data allowed a comprehensive toxicological evaluation of DIPE. The main target organs of DIPE toxicity were liver and kidney. DIPE was not teratogen and had no genotoxic effects, either in vitro or in vivo. However, it appeared to increase the number of malignant tumors in rats. Therefore, DIPE could be considered as a non-genotoxic animal carcinogen and a PDE of 0.98 mg/day was calculated based on the lowest No Observed Effect Level (NOEL) value of 356 mg/m3 (corresponding to 49 mg/kg/day) for maternal toxicity in developmental rat toxicity study. In a worst-case scenario, using an exceedingly high daily dose of 10 g/day, allowed DIPE concentration in pharmaceutical substances would be 98 ppm, which is in the range of concentration limits for ICH Q3C guideline class 2 solvents. This result might be considered for regulatory decisions

Scientific Opinion on Flavouring Group Evaluation 7, Revision 5 (FGE.07Rev5) : saturated and unsaturated aliphatic secondary alcohols, ketones and esters of secondary alcohols and saturated linear or branched‐chain carboxylic acids from chemical group 5

Acknowledgements: The Panel wishes to thank the members of the Working Group on Flavourings: Ulla Beckman Sundh, Leon Brimer, Karl-Heinz Engel, Paul Fowler, Rainer Gürtler, Trine Husøy, Wim Mennes and Gerard Mulder for the preparatory work on this scientific opinion and the Working Group on Genotoxicity: Mona-Lise Binderup, Claudia Bolognesi, Riccardo Crebelli, Rainer Gürtler, Francesca Marcon, Daniel Marzin and Pasquale Mosesso for the preparatory work on this scientific opinion and the hearing experts: Vibe Beltoft and Karin Nørby, and EFSA staff: Maria Anastassiadou, Maria Carfi and Annamaria Rossi for the support provided to this scientific opinion.Publisher PD