Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform.

Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors.

Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses.

Diversification and selection mechanisms for the production of protein repertoires: lessons from the immune system.

The physiological mechanism for producing antigen-specific antibodies is based on a two-phase neo-Darwinian process: the first phase consists of diversity generation (formation of the repertoire), and the second phase is antigen-mediated selection. In this article, we consider how the natural immunoglobulin gene-diversification processes can be exploited both in vivo and in vitro in order to allow the generation of novel antibody (and heterologous protein) repertoires.

Allelic inclusion of T cell receptor alpha genes poses an autoimmune hazard due to low-level expression of autospecific receptors.

Organ-specific autoimmune disease can be caused by alphabeta T cells that have escaped self-tolerance induction. Here we show that one of the causes of escape from self-tolerance is the coexpression of two different T cell receptors by the same cell, which can occur in up to 30% of all T cells in normal mice and can lead to low-level surface expression of an autospecific TCR. We found that double receptor-expressing T cells can escape tolerance even to ubiquitously expressed antigens but can nevertheless induce autoimmune diabetes when the relevant protein is expressed in pancreatic tissue. Such diabetogenic T cells are absent, however, among T cells expressing the autospecific TCR as the sole receptor.

Changes in function of antigen-specific lymphocytes correlating with progression towards diabetes in a transgenic model.

Mice that express influenza hemagglutinin under control of the rat insulin promoter (INS-HA) as well as a class II major histocompatibility complex (MHC)-restricted HA-specific transgenic TCR (TCR-HA), develop early insulitis with huge infiltrates, but progress late and irregularly to diabetes. Initially, in these mice, INS-HA modulates the reactivity of antigen-specific lymphocytes, such that outside the pancreas they do not cause lethal shock like their naive counterparts in single transgenic TCR-HA mice, when stimulated with high doses of antigen. Inside the pancreas, the antigen-specific cells do not initially attack the islet cells, and produce some IFN-gamma as well as IL-10 and IL-4. Spontaneous progression to diabetes, which can be accelerated by cyclophosphamide injection, is accompanied by a 10-fold increase in IFN-gamma and a 3-fold decrease in IL-10 and IL-4 production by the locally residing antigen-specific T cells. Also, total islets from non-diabetic mice contain more TNF-alpha, compared with diabetic mice. This scenario is consistent with the view that beta cell destruction depends upon the increased production of certain pro-inflammatory cytokines by infiltrating T cells. Our inability to detect Fas expression on beta cells, but not on lymphoid cells, in diabetic and non-diabetic mice, puts some constraints on the role of Fas in beta cell destruction.

Interleukin 10 secretion and impaired effector function of major histocompatibility complex class II-restricted T cells anergized in vivo.

Continuous antigenic stimulation in vivo can result in the generation of so-called "anergic" CD4(+) or CD8(+) T cells that fail to proliferate upon antigenic stimulation and fail to develop cytolytic effector functions. Here we show that class II major histocompatibility complex-restricted T cells specific for influenza hemagglutinin (HA) that become anergic in mice expressing HA under control of the immunoglobulin kappa promoter exhibit an impaired effector function in causing diabetes in vivo, as compared to their naive counterparts, when transferred into immunodeficient recipients expressing HA under the control of the insulin promoter. Furthermore, HA-specific T cells anergized in vivo contain higher levels of interleukin (IL)-4 messenger RNA (mRNA) than naive and recently activated T cells with the same specificity and more than a 100-fold higher levels of IL-10 mRNA. The higher expression of the IL-10 gene is also evident at the protein level. These findings raise the interesting possibility that T cells rendered anergic in vivo have in fact become regulatory T cells that may influence neighboring immune responses through the release of IL-10.

Conditions that induce tolerance in mature CD4+ T cells.

Establishment of antigen-specific tolerance among mature T cells has been a long debated, yet poorly understood issue. In this study we have used transgenic mice bearing a class II--restricted TCR specific for the hemmagglutinin of the influenza virus in order to test the behavior of CD4+ T cells upon exposure to antigen in different forms and doses. We first studied the fate of T cells expressing the transgenic TCR (6.5) in double transgenic mice where HA was expressed as a self antigen by hemapoietic cells. In these mice, we found some mature T cells in periphery that had escaped thymic deletion and that showed signs of activation but which were anergic. Mature CD4+6.5+ cells that were transferred into antigen-containing recipients went through an initial phase of expansion after which most cells were deleted and those remaining became unresponsive, as previously described for CD8+ cells. Inducing tolerance in CD4+6.5+ cells in situ in single transgenic mice proved a difficult task: classical protocols using single doses of soluble or deaggregated antigen as well as feeding antigen all failed to induce antigen-specific unresponsiveness. It was only after decreasing cell numbers by CD4 antibody treatment and by repeatedly reintroducing antigen thereafter that unresponsiveness of 6.5+ cells was achieved and maintained. In no case could we observe the appearance of antigen-specific T cells with a Th2 cytokine profile among the remaining cells and therefore conclude that deletion and anergy represent the major mechanisms of tolerance in our studies.

A pain management documentation tool.

PURPOSE/OBJECTIVES: To describe the development, implementation, and evaluation of a tool to standardize assessment, monitoring, management, and documentation of acute and chronic pain in hospitalized patients. DATA SOURCES: A multidisciplinary hospital committee, nursing staff, a pain consultant, published standards, and hospital standards. DATA SYNTHESIS: A comprehensive pain flow sheet and pain protocol was developed to assess, monitor, document, and evaluate clients at risk for or with actual pain. The tool was designed to assess and document acute and chronic pain in patients with cancer. CONCLUSIONS: Based on an initial review of quality assurance indicators, the tool has demonstrated increased consistency in documentation of pain management activities. IMPLICATIONS FOR NURSING PRACTICE: The use of a pain assessment and management flow sheet with a pain protocol provides an organized, consistent approach that maximizes quality, cost-effective care.

Milrinone, dobutamine, and nitroprusside: comparative effects on hemodynamics and myocardial energetics in patients with severe congestive heart failure.

To assess their comparative effects on hemodynamics and myocardial energetics, we administered nitroprusside (1.5 +/- 0.6 microgram/kg-min), dobutamine (10 +/- 3 micrograms/kg-min), and milrinone (67 +/- 13 micrograms/kg-min) sequentially to 10 patients with severe (NYHA class III or IV) congestive heart failure. Each agent led to a significant (p = .001) increase in cardiac index (1.9 +/- 0.5 to 2.7 +/- 0.6 liters/min/m2; 1.7 +/- 0.4 to 2.6 +/- 0.6 liters/min/m2; and 1.8 +/- 0.5 to 2.7 +/- 0.5 liters/min/m2, for nitroprusside, dobutamine, and milrinone, respectively). Dobutamine did not produce a significant change in the pulmonary capillary wedge pressure (27 +/- 5 to 24 +/- 6 mm Hg, NS) nor in mean arterial pressure (83 +/- 9 to 86 +/- 10 mm Hg, NS), but caused a significant rise in heart rate (85 +/- 16 to 99 +/- 17 beats/min, p = .001) and in myocardial oxygen consumption (8.7 +/- 2.1 to 11.1 +/- 3.8 ml O2/min, p = .03). In contrast, nitroprusside and milrinone each caused a significant (p = .001) fall in the pulmonary capillary wedge pressure (27 +/- 6 to 19 +/- 7 mm Hg and 26 +/- 6 to 19 +/- 9 mm Hg, respectively), without significantly increasing either the heart rate (87 +/- 18 to 85 +/- 17 beats/min and 86 +/- 17 to 89 +/- 17 beats/min, respectively) or myocardial oxygen consumption (8.8 +/- 2.3 to 7.6 +/- 2.1 ml O2/min and 8.8 +/- 2.1 to 8.4 +/- 2.1 ml O2/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of long-term therapy with milrinone and the effects of milrinone withdrawal.

To assess the long-term efficacy of milrinone in patients with severe congestive heart failure, we obtained hemodynamic measurements (systemic arterial and right heart catheterization) in 13 patients at baseline and after intravenous administration of milrinone. After continuous oral milrinone therapy of 8 +/- 4 months duration, repeat hemodynamic study was performed in patients on oral milrinone therapy, after withdrawal of milrinone, and after readministration of milrinone intravenously. Comparison of initial baseline and withdrawal hemodynamic measurements for the group as a whole showed no interval progression of heart failure, as reflected by the pulmonary capillary wedge pressure (27 +/- 8 to 24 +/- 12 mm Hg, NS) or the cardiac index (2.0 +/- 0.4 to 2.1 +/- 0.8 liters/min/m2, NS). Individual comparisons of milrinone-free hemodynamics revealed that five patients had improved hemodynamically, three patients were unchanged, and five patients had deteriorated, four of whom manifested dependence on milrinone with a progressive hemodynamic decline after milrinone withdrawal which required readministration of milrinone on an emergency basis. Continued efficacy of milrinone was observed on readmission after withdrawal: pulmonary capillary wedge pressure fell from 27 +/- 8 to 16 +/- 10 mm Hg (p = .001) initially and from 24 +/- 12 to 13 +/- 11 mm Hg (p = .001) at readministration, while cardiac index rose from 2.0 +/- 0.4 to 2.8 +/- 0.5 liters/min/m2 (p = .001) initially and from 2.1 +/- 0.8 to 2.7 +/- 0.5 liters/min/m2 (p = .005) upon readministration.(ABSTRACT TRUNCATED AT 250 WORDS)

Survival of patients with severe congestive heart failure treated with oral milrinone.

The safety and efficacy of long-term oral milrinone therapy were evaluated over a 2 1/2 year period in 100 patients who had severe congestive heart failure despite conventional therapy. Long-term oral milrinone therapy (27 +/- 8 mg/day initial dose) was well tolerated; drug-related side effects occurred in only 11% of patients and led to drug withdrawal in only 4% of patients. Of 94 patients evaluated after 1 month of therapy, 51% had improved by at least one New York Heart Association functional class. Despite hemodynamic and clinical improvements, life table analysis showed a 39% mortality rate at 6 months and a 63% mortality rate at 1 year of therapy. Characteristics at study entry that predicted death within 6 months included more advanced functional class, impaired renal function, lower right ventricular ejection fraction, presence of nonsustained ventricular tachycardia on 24 hour ambulatory electrocardiography, more impaired baseline hemodynamic function and absence of clinical improvement after 1 month of milrinone therapy. Multivariate analysis selected lower baseline cardiac index and aortic systolic pressure as the most significant variables in predicting death; patients who died of progressive heart failure had less frequent use of antiarrhythmic drugs and greater increases in furosemide and milrinone doses during long-term follow-up than did those who died suddenly. Thus, although milrinone is well tolerated and produces early symptomatic benefits in approximately half of patients with congestive heart failure refractory to conventional therapy, there is no evidence that it improves the high baseline mortality in this disorder.

Effects of milrinone on coronary hemodynamics and myocardial energetics in patients with congestive heart failure.

To examine the effect of milrinone on myocardial energetics in patients with congestive heart failure, we measured systemic, pulmonary, and coronary hemodynamics in 18 patients before and after intravenous administration of milrinone (125 +/- 36 micrograms/kg). There was a 45% increase in cardiac index (2.1 +/- 0.5 to 3.0 +/- 0.6 liters/min/m2; p = .0001), a 39% fall in the pulmonary capillary wedge pressure (28 +/- 8 to 17 +/- 8 mm Hg; p = .0001), and a 42% increase in left ventricular external work (3758 +/- 1419 to 5340 +/- 1598 g-m/min; p = .0001). Both the heart rate-blood pressure product (9624 +/- 2272 to 9380 +/- 2428 mm Hg-beats/min; p = NS) and regional left ventricular myocardial oxygen consumption (7.6 +/- 2.9 to 8.1 +/- 3.1 ml O2/min; p = NS) were unchanged after milrinone, resulting in a 45% increase in calculated left ventricular external efficiency (p = .004). Although myocardial oxygen consumption did not change, regional great cardiac venous blood flow increased significantly (73 +/- 32 to 85 +/- 34 ml/min; p = .02) as a result of a 30% reduction in regional coronary vascular resistance (1.32 +/- 0.99 to 0.93 +/- 0.54 mm Hg-min/ml; p = .004), a decrease comparable to the concurrent 37% and 38% falls seen in systemic and pulmonary vascular resistance, respectively. These changes were associated with an 11% fall in the transcoronary arterial-venous oxygen difference (111 +/- 24 to 99 +/- 21 ml/O2/liter; p = .0001), which is consistent with a primary coronary vasodilator effect of milrinone.(ABSTRACT TRUNCATED AT 250 WORDS)