Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring

Advances in synthetic genomics are now well underway in yeasts due to the low cost of synthetic DNA. These new capabilities also bring greater need for quantitating the presence, loss and rearrangement of loci within synthetic yeast genomes. Methods for achieving this will ideally; i) be robust to industrial settings, ii) adhere to a global standard and iii) be sufficiently rapid to enable at-line monitoring during cell growth. The methylotrophic yeast Pichia pastoris (P. pastoris) is increasingly used for industrial production of biotherapeutic proteins so we sought to answer the following questions for this particular yeast species. Is time-consuming DNA purification necessary to obtain accurate end-point polymerase chain reaction (e-pPCR) and quantitative PCR (qPCR) data? Can the novel linear regression of efficiency qPCR method (LRE qPCR), which has properties desirable in a synthetic biology standard, match the accuracy of conventional qPCR? Does cell cultivation scale influence PCR performance? To answer these questions we performed e-pPCR and qPCR in the presence and absence of cellular material disrupted by a mild 30s sonication procedure. The e-pPCR limit of detection (LOD) for a genomic target locus was 50 pg (4.91 × 103 copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300 pg gDNA (2.95 × 104 copies). LRE qPCR matched the accuracy of a conventional standard curve qPCR method. The presence of material from bioreactor cultivation of up to OD600 = 80 did not significantly compromise the accuracy of LRE qPCR. We conclude that a simple and rapid cell disruption step is sufficient to render P. pastoris samples of up to OD600 = 80 amenable to analysis using LRE qPCR which we propose as a synthetic biology standard

Application of medical and analytical methods in Lyme borreliosis monitoring

Lyme borreliosis (LB) is one of the most common tick-borne diseases in the northern hemisphere. It is a chronic inflammatory disease caused by the spirochaete Borrelia burgdorferi. In its early stages, pathological skin lesions, namely erythema chronicum migrans, appear. The lesions, usually localised at the site of the bite, may become visible from a few weeks up to 3 months after the infection. Predominant clinical symptoms of the disease also involve joint malfunctions and neurological or cardiac disorders. Lyme disease, in all its stages, may be successfully treated with antibiotics. The best results, however, are obtained in its early stages. In order to diagnose the disease, numerous medical or laboratory techniques have been developed. They are applied to confirm the presence of intact spirochaetes or spirochaete components such as DNA or proteins in tick vectors, reservoir hosts or patients. The methods used for the determination of LB biomarkers have also been reviewed. These biomarkers are formed during the lipid peroxidation process. The formation of peroxidation products generated by human organisms is directly associated with oxidative stress. Apart from aldehydes (malondialdehyde and 4-hydroxy-2-nonenal), many other unsaturated components such as isoprostenes and neuroprostane are obtained. The fast determination of these compounds in encephalic fluid, urine or plasma, especially in early stages of the disease, enables its treatment. Various analytical techniques which allow the determination of the aforementioned biomarkers have been reported. These include spectrophotometry as well as liquid and gas chromatography. The analytical procedure also requires the application of a derivatization step by the use of selected reagents

Methods for measuring shrinkage

This paper presents findings from research amongst European grocery retailers into their methods for measuring shrinkage. The findings indicate that: there is no dominant method for valuing or stating shrinkage; shrinkage in the supply chain is frequently overlooked; data is essential in pinpointing where and when loss occurs and that many retailers collect data at the stock-keeping unit (SKU) level and do so every 6 months. These findings reveal that it is difficult to benchmark between retailers due to inconsistencies between measurement methods and that there are opportunities for many of the retailers surveyed to improve their shrinkage measurement by adopting known good practice

Understanding the relationships between time and cost to improve supply chain performance

In today's global competitive market, the total cost of sourcing, manufacturing and delivery of products is a major driver of competitive advantage. However, the true cost of operations in many organisations is often unclear or misleading because supply chain processes transcend both functional and company boundaries and the limitations associated with traditional cost reporting systems. Some cost-based approaches have been developed to address these issues but they tend to focus on the traditional accounting functional view of an organisation and not extend to the business processes involved. Alternative approaches to improving supply chain performance, such as time-based methods, seek to improve efficiency by identifying and eliminating areas of non-value added activity in supply chain processes. Although using time as a measure can be an effective approach to increasing value in the supply chain there is limited understanding of the relationship between time and cost across supply chains. The paper investigates, through the use of an industrial case study, how analysis of both time and cost can be combined to provide a more accurate view of supply chain performance which can lead to better informed decision making. The subsequent analysis provides an insight into the relationship between time and cost in supply chain processes and demonstrates how product costs accumulate in the supply chain.Supply chain management Costing Time-based process mapping Value adding

Interactions between Environmental Design and Human Factors Specialists ENVIRONMENTAL DESIGN: Panel

Most of the interactions between human factors specialists, such as ergonomists, and environmental specialists such as facility planners and architects  tend to be task specific and do not follow any accepted process. Consequently, the success of such interactions are usually a function of serendipity rather than informed expectation. It is anticipated that by gathering such specialists in an open discussion, relevant issues may be addressed and successful interaction procedures introduced and discussed. Such a forum is desirable for developing an understanding of the differences, educational and operational, between environmental design specialists, and human factors specialists, as well as for exploring the ways their communications can be enhanced. It is anticipated that by sharing their experiences with the attendees, the presenters will identify relevant on-going knowledge transfer activities, and also introduce and discuss practical problem-solving and communication methods that can be used with assurance by the attendees themselves when faced with similar problems in the future. This panel will focus on issues that arrive out of situations where human factors specialists and environmental design specialists are joined together in project development. The specialties represented include architecture, facility planning, environmental psychology, ergonomic research, industrial design and engineering, and equipment and furniture design and manufacturing.

Collaborative Witnessing of Survival During the Holocaust: An Exemplar of Relational Autoethnography

In this article, a researcher and collaborator present stories about the second author’s survival during the Holocaust. They propose that their approach of collaborative witnessing is a form of “relational autoethnography” that allows researchers to focus on and evocatively tell the lives of others in shared storytelling and conversation. The authors address the benefits and complications of collaborative witnessing and how it extends an autoethnographic perspective in its emphasis on writing for and with the other, listening and working together with care and compassion, and bearing witness to others as well as to oneself