The effect of metabolic stress on genome stability of a synthetic biology chassis Escherichia coli K12 strain.

BACKGROUND: Synthetic organism-based biotechnologies are increasingly being proposed for environmental applications, such as in situ sensing. Typically, the novel function of these organisms is delivered by compiling genetic fragments in the genome of a chassis organism. To behave predictably, these chassis are designed with reduced genomes that minimize biological complexity. However, in these proposed applications it is expected that even when contained within a device, organisms will be exposed to fluctuating, often stressful, conditions and it is not clear whether their genomes will retain stability. RESULTS: Here we employed a chemostat design which enabled us to maintained two strains of E. coli K12 under sustained starvation stress: first the reduced genome synthetic biology chassis MDS42 and then, the control parent strain MG1655. We estimated mutation rates and utilised them as indicators of an increase in genome instability. We show that within 24 h the spontaneous mutation rate had increased similarly in both strains, destabilizing the genomes. High rates were maintained for the duration of the experiment. Growth rates of a cohort of randomly sampled mutants from both strains were utilized as a proxy for emerging phenotypic, and by association genetic variation. Mutant growth rates were consistently less than rates in non-mutants, an indicator of reduced fitness and the presence of mildly deleterious mutations in both the strains. In addition, the effect of these mutations on the populations as a whole varied by strain. CONCLUSIONS: Overall, this study shows that genome reductions in the MDS42 did not stabilize the chassis under metabolic stress. Over time, this could compromise the effectiveness of synthetic organisms built on chassis in environmental applications.

Dual antiplatelet response during PCI: VerifyNow P2Y12 predicts myocardial necrosis and thromboxane B2 generation confirms wide variation in aspirin response.

INTRODUCTION: There remains concern that the antiplatelet effects of aspirin and clopidogrel vary between patients and poor responders may be at increased risk of adverse events. However, the optimal method of measuring aspirin and/or clopidogrel response remains unresolved. We compared three methods of measuring clopidogrel response recommended by a recent consensus statement for the European Society of Cardiology, and investigated a novel approach to measuring aspirin response in patients established on both aspirin and clopidogrel. In addition, we investigated whether any of these assays predict peri-procedural myocardial necrosis following percutaneous coronary intervention (PCI). METHODS: A cross-section of 323 patients attending for PCI was tested for clopidogrel response using VerifyNow P2Y12, VASP Platelet Reactivity Index (VASP-PRI) and whole blood impedance aggregometry (WBPA). Aspirin response was assessed by measuring the residual ability of platelets to generate thromboxane, calculated as the difference between thromboxane B2 levels in serum and plasma, [TxB2]S-P. Peri-procedural myocardial necrosis was determined by a change in troponin I >0.2 µmol/l. RESULTS: Patients demonstrated wide variation in response to both aspirin and clopidogrel. Correlation between VerifyNow P2Y12 and VASP-PRI was good (r=0.702, p<0.001). Correlation was moderate between WBPA and VerifyNow P2Y12 (r=0.639, p<0.001) and weak for WBPA and VASP-PRI (r=0.353, p<0.001). Only VerifyNow P2Y12 predicted peri-procedural myocardial necrosis. CONCLUSIONS: The three methods of measuring response to clopidogrel identify different patients as poor responders. Poor response to clopidogrel assessed by VerifyNow P2Y12 predicts myocardial necrosis. Measurement of [TxB2]S-P demonstrates a wide variation in aspirin response in patients taking dual antiplatelet therapy.

Variation in thromboxane B2 concentrations in serum and plasma in patients taking regular aspirin before and after clopidogrel therapy.

Dual antiplatelet therapy with aspirin and a P2Y12 antagonist is widely prescribed for the prevention of thrombotic events in patients with an acute coronary syndrome or undergoing percutaneous coronary intervention (PCI). It is recognised that there is inter-individual variation in the antiplatelet effects of both drugs. Recent data also suggest that P2Y12 antagonists can affect the response to aspirin. A direct indicator of the effect of aspirin on platelets is their ability to generate thromboxane, which if measured as the difference between the level of thromboxane B2 in serum and plasma ([TxB2]S-P) avoids the confounding effect of endogenous TxB2 production from other cells. We therefore analysed [TxB2]S-P as a measure of aspirin response in a group of 123 patients undergoing elective PCI before and after the introduction of clopidogrel. In a subgroup of 40 patients taking aspirin alone, we compared [TxB2]S-P and VerifyNow Aspirin for the assessment of aspirin response. There was a wide variation in plasma and serum TxB2 concentrations both before and after clopidogrel therapy but only 3.5% of patients had residual serum concentration of TxB2 > 10 ng/ml. There was a strong correlation between the pre and post clopidogrel levels of TxB2 (r ≥ 0.78; p = 0.001) and no significant difference in [TxB2]S-P. There was no correlation between the magnitude of response to clopidogrel response and the generation of thromboxane B2. Correlation between [TxB2]S-P and VerifyNow Aspirin was poor. We conclude that the use of a P2Y12 antagonist does not influence the effect of aspirin on the ability of platelets to generate thromboxane. Therefore, measurement of TxB2 levels in serum, after subtracting the contribution from plasma, provides a measure of the response to aspirin in patients taking dual antiplatelet therapy.

Autologous blood transfusion for cardiopulmonary bypass: effects of storage conditions on platelet function.

OBJECTIVES: Cardiopulmonary bypass impairs formation of large stable platelet aggregates (macroaggregation), although formation of small aggregates (microaggregation) is preserved. A factor in the uncertain benefits of intraoperative autologous blood transfusion may be the effects of storage on platelet function. The effects of citrate preservative and heparinization before storage on platelet function was therefore assessed. METHODS: Twenty-seven patients undergoing elective coronary artery bypass grafting were randomly allocated to have 450 to 1,000 mL of blood taken into CPDA anticoagulant bags either before (n = 14) or after heparinization (n = 13). Samples from the patients and stored blood were anticoagulated with rhirudin, 200 U/mL. The macroaggregatory response to submaximal collagen was measured by impedance aggregometry and microaggregation by single platelet counting. RESULTS: During macroaggregation, before cardiopulmonary bypass, the ex vivo median (interquartile range) response was 16.3 (12.4-18.7) Omega. This decreased 10 minutes after heparin to 8.9 (3.3-11.0) Omega (p < 0.0001). In the blood bags (in vitro), the initial response for nonheparinized blood was 4.8 (0.1-7.5) Omega (p < 0.002 v ex vivo) and at end-cardiopulmonary bypass was 2.4 (1.6-8.2) Omega. During microaggregation, in vivo heparinization decreased microaggregation both ex vivo and in vitro in CPDA blood; the in vitro response of nonheparinized blood at end-cardiopulmonary bypass was greater than that seen after in vivo heparinization (p < 0.007). No difference in bleeding or transfusion requirements was seen. CONCLUSIONS: Collecting blood into CPDA anticoagulant caused a marked deterioration in platelet function. This was worse after in vivo heparinization and included depression of microaggregation.