Identification of a discrete intermediate in the assembly disassembly of physalis mottle tymovirus through mutational analysis

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T = 3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T = 3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T = 3 capsid assembly, in contrast, the deletion of even a single residue from the C terminus (PhN188 Delta 1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75A and D144 are not crucial for the T = 3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188 Delta 1, pR PhH69A and pR PhK143E mutant proteins.

The experience of forging new emergency nursing curriculum development, through a course advisory network

Queensland University of Technology (QUT), School of Nursing (SoN), has offered a postgraduate Graduate Certificate in Emergency Nursing since 2003, for registered nurses practising in an emergency clinical area, who fulfil key entry criteria. Feedback from industry partners and students evidenced support for flexible and extended study pathways in emergency nursing. Therefore, in the context of a growing demand for emergency health services and the need for specialist qualified staff, it was timely to review and redevelop our emergency specialist nursing courses. The QUT postgraduate emergency nursing study area is supported by a course advisory group, whose aim is to provide input and focus development of current and future course planning. All members of the course advisory were invited to form an expert panel to review current emergency course documents. A half day “brainstorm session”, planning and development workshop was held to review the emergency courses to implement changes from 2009. Results from the expert panel planning day include: proposal for a new emergency specialty unit; incorporation of the College of Emergency Nurses (CENA) Standards for Emergency Nursing Specialist in clinical assessment; modification of the present core emergency unit; enhancing the focus of the two other units that emergency students undertake; and opening the emergency study area to the Graduate Diploma in Nursing (Emergency Nursing) and Master of Nursing (Emergency Nursing). The conclusion of the brainstorm session resulted in a clearer conceptualisation, of the study pathway for students. Overall, the expert panel group of enthusiastic emergency educators and clinicians provided viable options for extending the career progression opportunities for emergency nurses. In concluding, the opportunity for collaboration across university and clinical settings has resulted in the design of a course with exciting potential and strong clinical relevance.

Simulated systems optimization through response surface techniques [Optimización de sistemas simulados a través de técnicas de superficie de respuesta]

The purpose of this article is to show the applicability and benefits of the techniques of design of experiments as an optimization tool for discrete simulation models. The simulated systems are computational representations of real-life systems; its characteristics include a constant evolution that follows the occurrence of discrete events along the time. In this study, a production system, designed with the business philosophy JIT (Just in Time) is used, which seeks to achieve excellence in organizations through waste reduction in all the operational aspects. The most typical tool of JIT systems is the KANBAN production control that seeks to synchronize demand with flow of materials, minimize work in process, and define production metrics. Using experimental design techniques for stochastic optimization, the impact of the operational factors on the efficiency of the KANBAN / CONWIP simulation model is analyzed. The results show the effectiveness of the integration of experimental design techniques and discrete simulation models in the calculation of the operational parameters. Furthermore, the reliability of the methodologies found was improved with a new statistical consideration.