Furthermore, in terms of the moral duty to provide care, it seems that most HCWs are more concerned about the availability of protective measures than about whether they had been informed of a particular risk beforehand. It seems important that further research be carried out to improve understanding of the opinions and perceptions of HCWs and the basis of their definitions, as this information could prove very helpful in defining a duty to treat that can be effectively put into practice. It is also important that a well-organized ongoing educational program that is needs-based and easily accessible be provided to HCWs at both the graduate and postgraduate levels. In particular, this program must be continuously updated regarding AIDS and other diseases that may cause the HCWs to behave discriminatively towards patients, even though these diseases are below the SLR. Such continuing medical education is the best answer to the justification "When I chose the profession/when I graduated, this disease did not exist!" for refusing treatment. Emphasizing the social role of HCWs, and educating them about the professional obligations derived from the social contract betweeen the profession and the wider social order, would further reduce that kind of reasoning. In addition, stricter standards for the duty to provide care should established by determining the criteria for a SLR and identifying the situations and conditions that would exceed this SLR. Each of these measures could serve to remind HCWs that they have a moral responsibility, as individual HCWs, to be aware of professional obligations and to act as responsible members of the profession. Moreover, the working environment of HCWs should be provided with preventative measures that can be applied both generally and specifically and should emphasize their use. For a circumstance in which a preventative measure has been developed for a disease but is not available for treating a particular case, it would not be easy to justify the claim that there is an undeniable duty to provide care at that moment. Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system BACKGROUND: Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control. RESULTS: By applying on-line pO(2 )monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles. By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels. CONCLUSION: The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems. The methylotrophic yeast Pichia pastoris is a favored yeast species as a host for heterologous protein production (for reviews see [1] [2] [3] [4] [5] [6] ). P. pastoris has the potential for high expression levels, efficient secretion of target proteins, posttranslational modifications, and is easily grown to high cell densities on mineral salt medium in bioreactors. It has been demonstrated that P. pastoris is an efficient production system also for very large and complex proteins, such as collagens, which besides the recombinant gene(s) needed for the collagen polypeptide chain(s) needs the parallel expression of two different genes coding for collagen prolyl 4-hydroxylase (C-P4H), an enzyme required for the thermal stability of collagens [7] [8] [9] .