A précis of philosophy of computing and information technology

The authors recently finished a comprehensive chapter on “Philosophy of Computing and Information Technology” for the forthcoming (fall 2009) Philosophy of Technology and Engineering Sciences (Ed.: A. Meijers), Volume IX in the Elsevier series Handbook of the Philosophy of Science (Eds.: D. Gabbay, P. Thagard and J. Woods). The purpose of the chapter is to review and discuss the main developments, concepts, topics, and contributors in the intersection between philosophy and computing, as well as provide some suggestions on how to structure the many subcategories within what is loosely referred to as philosophy of computing. In this short synopsis, we will give an outline of the kinds of issues raised in this chapter

Engineering and optimization of Bacillus megaterium for the biosynthesis and biotransformation of terpenoid derived compounds

Over the past decades, Bacillus megaterium has been employed for the industrial production of recombinant proteins and active pharmaceutical compounds. However, the limited repertoire of promoter sequences has largely impaired the potential of B. megaterium for the heterologous protein expression and the rational strain design towards a more efficient biosynthesis of these compounds. In the present work, 19 innovative promoter elements of diverse promoter classes were identified based on differential gene expression analyses and characterized via β-galactosidase screening. Their activities ranged from 15% to 145% compared to the reference promoter. Selected promoters were successfully applied to establish the currently most efficient B. megaterium based whole-cell systems for the cholesterol oxidase mediated conversion of pregnenolone to progesterone and the 11β- hydroxysteroid dehydrogenase mediated conversion of cortisol to cortisone. Multigram scaled steroid yields were achieved. Moreover, the novel promoters were used to establish the complex biosynthesis of C30 carotenoids. The involved enzymes in B. megaterium had previously been identified within this work along with the underlying biosynthetic route towards the production of the pharmaceutically relevant C30 carotenoid diaponeurosporene. In summary, the versatile range of applications demonstrated the promising potential of the novel promoters for a future use in diverse biotechnological processes.Bacillus megaterium wird seit Jahren für die industrielle Produktion von rekombinanten Proteinen und pharmazeutisch aktiven Substanzen eingesetzt. Aufgrund der beschränkten Auswahl an Promotorsequenzen wurde das Potenzial von B. megaterium für die heterologe Proteinexpression und das rationale Stammdesign allerdings weitestgehend vernachlässigt. Im Rahmen der vorliegenden Arbeit wurden 19 innovative Promotoren unterschiedlicher Promotorklassen auf Grundlage von differentiellen Genexpressionsanalysen im B. megaterium Stamm MS941 identifiziert und mittels β- Galaktosidase-Screening charakterisiert. Das Spektrum der Promotoraktivitäten lag im Vergleich zum Referenzpromotor zwischen 15% und 145%. Durch die Anwendung ausgewählter Promotoren konnten die zurzeit effizientesten B. megaterium basierten Ganzzellsysteme für den Cholesteroloxidase vermittelten Umsatz von Pregnenolon zu Progesteron sowie für die 11β- Hydroxysteroid Dehydrogenase katalysierte Produktion von Cortison etabliert werden. Die Steroidausbeuten lagen hierbei im Multigramm Bereich. Des Weiteren konnten die neuen Promotoren erfolgreich für die komplexere Biosynthese von C30-Carotinoiden angewendet werden. Die beteiligten Enzyme wurden zuvor in B. megaterium identifiziert und der Syntheseweg wurde bis zum pharmazeutisch interessanten C30-Carotinoid Diaponeurosporen aufgeklärt. Das breite Anwendungsspektrum der neuen Promotoren zeigt deren enormes Potenzial für eine zukünftige biotechnologische Nutzung

Ambient intelligence and problems with interferring desires from behaviour

In this paper the authors argue that many of the ethical problems raised by Ambient Intelligence stems from presupposing a behaviourist conception of the relation between human desires and behaviour. Insofar as Ambient Intelligence systems take overt, natural behaviour as input, they are likely to suffer from many of the same problems that have fuelled the widespread criticism of behaviourist explanations of human behaviour. If these limitations of the technology are not sufficiently recognized, the technology is likely to be insufficiently successful in supporting the needs and desires of human users. We will focus on four distinct challenges that result from this behaviourist presupposition, all of which ought to be taken into consideration at the design stage: reciprocal adaptation, bias towards isolated use, culture-specific behaviour, and inability to manually configure the system. By considering these issues, our purpose is to raise awareness of the ethical problems that can arise because of intelligent user interfaces that rely on natural, overt behaviour

Full incorporation of the noncanonical amino acid hydroxylysine as a surrogate for lysine in green fluorescent protein

The canonical set of amino acids leads to an exceptionally wide range of protein functionality, nevertheless, this set still exhibits limitations. The incorporation of noncanonical amino acids into proteins can enlarge its functional scope. Although proofreading will counteract the charging of tRNAs with other amino acids than the canonical ones, the translation machinery may still accept noncanonical amino acids as surrogates and incorporate them at the canonically prescribed locations within the protein sequence. Here, we use a cell-free expression system to demonstrate the full replacement of L-lysine by L-hydroxylysine at all lysine sites of recombinantly produced GFP. In vivo, as a main component of collagen, post-translational L-hydroxylysine generation enables the formation of cross-links. Our work represents a first step towards in vitro production of (modified) collagens, more generally of proteins that can easily be crosslinke

A CYPome-wide study reveals new potential players in the pathogenesis of Parkinson's disease

Genetic and environmental factors lead to the manifestation of Parkinson's disease (PD) but related mechanisms are only rudimentarily understood. Cytochromes P450 (P450s) are involved in the biotransformation of toxic compounds and in many physiological processes and thus predestinated to be involved in PD. However, so far only SNPs (single nucleotide polymorphisms) in CYP2D6 and CYP2E1 have been associated with the susceptibility of PD. Our aim was to evaluate the role of all 57 human P450s and their redox partners for the etiology and pathophysiology of PD and to identify novel potential players which may lead to the identification of new biomarkers and to a causative treatment of PD. The PPMI (Parkinson's Progression Markers Initiative) database was used to extract the gene sequences of all 57 P450s and their three redox partners to analyze the association of SNPs with the occurrence of PD. Applying statistical analyses of the data, corresponding odds ratios (OR) and confidence intervals (CI) were calculated. We identified SNPs significantly over-represented in patients with a genetic predisposition for PD (GPD patients) or in idiopathic PD (IPD patients) compared to HC (healthy controls). Xenobiotic-metabolizing P450s show a significant accumulation of SNPs in PD patients compared with HC supporting the role of toxic compounds in the pathogenesis of PD. Moreover, SNPs with high OR values (>5) in P450s catalyzing the degradation of cholesterol (CYP46A1, CY7B1, CYP39A1) indicate a prominent role of cholesterol metabolism in the brain for PD risk. Finally, P450s participating in the metabolism of eicosanoids show a strong over-representation of SNPs in PD patients underlining the effect of inflammation on the pathogenesis of PD. Also, the redox partners of P450 show SNPs with OR > 5 in PD patients. Taken together, we demonstrate that SNPs in 26 out of 57 P450s are at least 5-fold over-represented in PD patients suggesting these P450s as new potential players in the pathogenesis of PD. For the first time exceptionally high OR values (up to 12.9) were found. This will lead to deeper insight into the origin and development of PD and may be applied to develop novel strategies for a causative treatment of this disease.The work was supported by a research grant from the “Dr. Rolf M. Schwiete Stiftung” Mannheim/Germany

Protocol to identify amino acids bound to tRNA by aminoacylation using mass spectrometry

tRNA-bound amino acids often need to be identified, for instance, in cases where different amino acids compete for binding to the same tRNA. Here, we present a mass-spectrometry-based protocol to determine the amino acids bound to tRNA by aminoacylation. We detail how to perform the aminoacylation reaction, the preparation of the aminoacyl-tRNA for measurement, and the mass spectrometric analysis. We use arginine acylation as an example; however, this protocol can be applied to any other amino acid

SNPs in cytochrome P450 genes decide on the fate of individuals with genetic predisposition to Parkinson’s disease

Parkinson’s disease (PD) is one of the most frequent neurological diseases affecting millions of people worldwide. While the majority of PD cases are of unknown origin (idiopathic), about 5%–10% are familial and linked to mutations in different known genes. However, there are also people with a genetic predisposition to PD who do not develop the disease. To elucidate factors leading to the manifestation of PD we compared the occurrence of single nucleotide polymorphisms (SNPs) in various cytochrome P450 (P450) genes in people with a genetic predisposition and suffering from PD (GPD) to that of people, who are genetically predisposed, but show no symptoms of the disease (GUN). We used the PPMI (Parkinson’s Progression Markers Initiative) database and the gene sequences of all 57 P450s as well as their three redox partners. Corresponding odds ratios (OR) and confidence intervals (CI) were calculated to assess the incidence of the various SNPs in the two groups of individuals and consequently their relation to PD. We identified for the first time SNPs that are significantly (up to 10fold!) over- or under-represented in GPD patients compared to GUN. SNPs with OR > 5 were found in 10 P450s being involved in eicosanoid, vitamin A and D metabolism as well as cholesterol degradation pointing to an important role of endogenous factors for the manifestation of PD clinical symptoms. Moreover, 12 P450s belonging to all P450 substrate classes as well as POR have SNPs that are significantly under-represented (OR < 0.2) in GPD compared to GUN, indicating a protective role of those SNPs and the corresponding P450s regarding disease advancement. To the best of our knowledge our data for the first time demonstrate an association between known PD predisposition genes and SNPs in other genes, shown here for different P450 genes and for their redox partner POR, which promote the manifestation of the disease in familial PD. Our results thus shed light onto the pathogenesis of PD, especially the switch from GUN to GPD and might further help to advance novel strategies for preventing the development or progression of the disease

25 Anos de Política Nacional de Saúde na República da Guiné-Bissau: Memórias do seu Planeamento Estratégico em Saúde

A República da Guiné-Bissau (RGB), um Estado frágil marcado por um contexto de instabilidade política e institucional, tem feito desde 1993 o exercício do Planeamento Estratégico para o Sector da Saúde (PES). Os documentos estratégicos sectoriais – Planos Nacionais de Desenvolvimento Sanitário (PNDS) – demonstram capacidade de resistir às adversidades governativas. Esta revisão longitudinal das políticas pretende fazer memória deste exercício com duplo propósito: analisar os processos de PES e destes guardar memória, como trilhos de um passado que permitem entender e contextualizar caminhos percorridos. Constata-se que o PES ocorreu sobretudo respondendo às exigências dos financiadores, nem sempre sendo implementado. Com a implementação do PNDS III (2018-2022), ambiciona-se reverter esta situação com uma estratégia integradora dos diferentes atores em saúde.The Republic of Guinea-Bissau (RGB), a fragile state marked by a context of political and institutional instability, has since 1993 carried out the Strategic Planning for the Health Sector (SPHS). Sectoral strategic documents – National Plans of Sanitary Development (NPSD) – demonstrate a capacity for resistance to governmental adversities. This longitudinal policy review has a double purpose, namely to analyse the SPHS processes and to guard these memories to allow an understanding of the trajectories in question. This paper concludes that the SPHS mainly responded to the requirements of funding institutions, and it has not always been implemented. The NPSD III (2018-2022) aims to reverse this situation by implementing a strategy which aims to integrate the different health actors

Regio- and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X-ray crystallography and computational modeling

The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 to 25-hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio- and stereoselective manner to 16β-hydroxytestosterone. To reveal the structural determinants governing the regio- and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a non-productive site away from the heme-iron. To examine if the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics simulations were performed with testosterone and vitamin D3 present in the active site. These molecular dynamics simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon hydrogen bond was taken into account. Overall, the first-time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site-directed mutagenesis towards improved enzymatic properties of this enzyme.</p