Grounding knowledge and normative valuation in agent-based action and scientific commitment

Philosophical investigation in synthetic biology has focused on the knowledge-seeking questions pursued, the kind of engineering techniques used, and on the ethical impact of the products produced. However, little work has been done to investigate the processes by which these epistemological, metaphysical, and ethical forms of inquiry arise in the course of synthetic biology research. An attempt at this work relying on a particular area of synthetic biology will be the aim of this chapter. I focus on the reengineering of metabolic pathways through the manipulation and construction of small DNA-based devices and systems synthetic biology. Rather than focusing on the engineered products or ethical principles that result, I will investigate the processes by which these arise. As such, the attention will be directed to the activities of practitioners, their manipulation of tools, and the use they make of techniques to construct new metabolic devices. Using a science-in-practice approach, I investigate problems at the intersection of science, philosophy of science, and sociology of science. I consider how practitioners within this area of synthetic biology reconfigure biological understanding and ethical categories through active modelling and manipulation of known functional parts, biological pathways for use in the design of microbial machines to solve problems in medicine, technology, and the environment. We might describe this kind of problem-solving as relying on what Helen Longino referred to as “social cognition” or the type of scientific work done within what Hasok Chang calls “systems of practice”. My aim in this chapter will be to investigate the relationship that holds between systems of practice within metabolic engineering research and social cognition. I will attempt to show how knowledge and normative valuation are generated from this particular network of practitioners. In doing so, I suggest that the social nature of scientific inquiry is ineliminable to both knowledge acquisition and ethical evaluations

Remineralization of demineralized dentin using a dual analog system.

ObjectiveImproved methods are needed to remineralize dentin caries in order to promote conservation of dentin tissue and minimize the surgical interventions that are currently required for clinical treatment. Here, we test the hypothesis that bulk substrates can be effectively mineralized via a dual analog system proposed by others, using a tripolyphosphate (TPP) "templating analog" and a poly(acrylic acid) (PAA) or poly(aspartic acid) (pAsp) "sequestration analog," the latter of which generates the polymer-induced liquid-precursor (PILP) mineralization process studied in our laboratory.Material & methodsDemineralized human dentin slices were remineralized with and without pre-treatment with TPP, using either PAA or pAsp as the PILP process-directing agent. A control experiment with no polymer present was used for comparison.ResultsNo mineralization was observed in any of the PAA groups. In both the pAsp and no polymer groups, TPP inhibited mineralization on the surfaces of the specimens but promoted mineralization within the interiors. Pre-treatment with TPP enhanced overall mineralization of the pAsp group. However, when analysed via TEM, regions with little mineral were still present.ConclusionPoly(acrylic acid) was unable to remineralize demineralized dentin slices under the conditions employed, even when pre-treated with TPP. However, pre-treatment with TPP enhanced overall mineralization of specimens that were PILP-remineralized using pAsp

Entecavir versus lamivudine for patients with HBeAg-negative chronic hepatitis B

BACKGROUND: Entecavir is a potent and selective antiviral agent that has demonstrated efficacy in phase 2 studies in patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B. METHODS: In this phase 3, double-blind trial, we randomly assigned 648 patients with HBeAg-negative chronic hepatitis B who had not previously been treated with a nucleoside analogue to receive 0.5 mg of entecavir or 100 mg of lamivudine once daily for a minimum of 52 weeks. The primary efficacy end point was histologic improvement (a decrease by at least two points in the Knodell necroinflammatory score, without worsening of fibrosis). RESULTS: Histologic improvement after 48 weeks of treatment occurred in 208 of 296 patients in the entecavir group who had adequate baseline liver-biopsy specimens that could be evaluated (70 percent), as compared with 174 of 287 such patients in the lamivudine group (61 percent, P=0.01). More patients in the entecavir group than in the lamivudine group had undetectable serum hepatitis B virus (HBV) DNA levels according to a polymerase-chain- reaction assay (90 percent vs. 72 percent, P<0.001) and normalization of alanine aminotransferase levels (78 percent vs. 71 percent, P = 0.045). The mean reduction in serum HBV DNA levels from baseline to week 48 was greater with entecavir than with lamivudine (5.0 vs. 4.5 log [on a base-10 scale] copies per milliliter, P<0.001). There was no evidence of resistance to entecavir. Safety and adverse-event profiles were similar in the two groups. CONCLUSIONS: Among patients with HBeAg-negative chronic hepatitis B who had not previously been treated with a nucleoside analogue, the rates of histologic improvement, virologic response, and normalization of alanine aminotransferase levels were significantly higher at 48 weeks with entecavir than with lamivudine. The safety profile of the two agents was similar, and there was no evidence of viral resistance to entecavir. Copyright © 2006 Massachusetts Medical Society.published_or_final_versio

A Systematic Review of Side Effects of Nucleoside and Nucleotide Drugs Used for Treatment of Chronic Hepatitis B

Although nucleosides and nucleotides have a good safety record for the treatment of hepatitis B, there have been no systematic reviews on this topic. We searched Medline to include studies of the oral antiviral agents for hepatitis B and adverse events, with at least 48 weeks of follow-up from the initiation of treatment with the drug. Important toxicities include nephrotoxicity, myopathy, and resistance. It is often difficult to ascertain whether an adverse effect is from the study drug or the natural progression of the disease. Further safety data are needed for the newer agents and for all agents with regard to patients with decompensated liver disease, renal dysfunction, the elderly, children, and pregnant women

Informational entropy : a failure tolerance and reliability surrogate for water distribution networks

Evolutionary algorithms are used widely in optimization studies on water distribution networks. The optimization algorithms use simulation models that analyse the networks under various operating conditions. The solution process typically involves cost minimization along with reliability constraints that ensure reasonably satisfactory performance under abnormal operating conditions also. Flow entropy has been employed previously as a surrogate reliability measure. While a body of work exists for a single operating condition under steady state conditions, the effectiveness of flow entropy for systems with multiple operating conditions has received very little attention. This paper describes a multi-objective genetic algorithm that maximizes the flow entropy under multiple operating conditions for any given network. The new methodology proposed is consistent with the maximum entropy formalism that requires active consideration of all the relevant information. Furthermore, an alternative but equivalent flow entropy model that emphasizes the relative uniformity of the nodal demands is described. The flow entropy of water distribution networks under multiple operating conditions is discussed with reference to the joint entropy of multiple probability spaces, which provides the theoretical foundation for the optimization methodology proposed. Besides the rationale, results are included that show that the most robust or failure-tolerant solutions are achieved by maximizing the sum of the entropies

ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair

Background: The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum-drug resistance. Resistance was not associated with increased cellular glutathione or decreased accumulation of platinum, rather the resistant cell lines have a cell cycle alteration allowing them to rapidly proliferate post drug treatment. Results: A decrease in ERCC1 protein expression and an increase in RAD51B foci activity was observed in association with the platinum induced cell cycle arrest but these changes did not correlate with resistance or altered DNA repair capacity. The H69 cells and resistant cell lines have a p53 mutation and consequently decrease expression of p21 in response to platinum drug treatment, promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. Conclusion: Decreased ERCC1 protein and increased RAD51B foci may in part be mediating the maintenance of the cell cycle arrest in the sensitive cells. Resistance in the H69CIS200 and H69OX400 cells may therefore involve the regulation of ERCC1 and RAD51B independent of their roles in DNA repair. The novel mechanism of platinum resistance in the H69CIS200 and H69OX400 cells demonstrates the multifactorial nature of platinum resistance which can occur independently of alterations in DNA repair capacity and changes in ERCC1

The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

Redox proteomics of the inflammatory secretome identifies a common set of redoxins and other glutathionylated proteins released in inflammation, influenza virus infection and oxidative stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions

An empirical approach towards the efficient and optimal production of influenza-neutralizing ovine polyclonal antibodies demonstrates that the novel adjuvant CoVaccine HT(TM) is functionally superior to Freund's adjuvant

Passive immunotherapies utilising polyclonal antibodies could have a valuable role in preventing and treating infectious diseases such as influenza, particularly in pandemic situations but also in immunocompromised populations such as the elderly, the chronically immunosuppressed, pregnant women, infants and those with chronic diseases. The aim of this study was to optimise current methods used to generate ovine polyclonal antibodies. Polyclonal antibodies to baculovirus-expressed recombinant influenza haemagglutinin from A/Puerto Rico/8/1934 H1N1 (PR8) were elicited in sheep using various immunisation regimens designed to investigate the priming immunisation route, adjuvant formulation, sheep age, and antigen dose, and to empirically ascertain which combination maximised antibody output. The novel adjuvant CoVaccine HT™ was compared to Freund’s adjuvant which is currently the adjuvant of choice for commercial production of ovine polyclonal Fab therapies. CoVaccine HT™ induced significantly higher titres of functional ovine anti-haemagglutinin IgG than Freund’s adjuvant but with fewer side effects, including reduced site reactions. Polyclonal hyperimmune sheep sera effectively neutralised influenza virus in vitro and, when given before or after influenza virus challenge, prevented the death of infected mice. Neither the age of the sheep nor the route of antigen administration appeared to influence antibody titre. Moreover, reducing the administrated dose of haemagglutinin antigen minimally affected antibody titre. Together, these results suggest a cost effective way of producing high and sustained yields of functional ovine polyclonal antibodies specifically for the prevention and treatment of globally significant diseases.Natalie E. Stevens, Cara K. Fraser, Mohammed Alsharifi, Michael P. Brown, Kerrilyn R. Diener, John D. Haybal