Cultivation of E. coli carrying a plasmid-based Measles vaccine construct (4.2 kbp pcDNA3F) employing medium optimisation and pH-temperature induction techniques

<p>Abstract</p> <p>Background</p> <p>Plasmid-based measles vaccines offer great promises over the conventional fertilised egg method such as ease of manufacture and mimic wild-type intracellular antigen expression. The increasing number of clinical trials on plasmid-based measles vaccines has triggered the need to make more in less time.</p> <p>Results</p> <p>In this work, we investigated the process variables necessary to improve the volumetric and specific yields of a model plasmid-based measles vaccine (pcDNA3F) harboured in <it>E. coli </it>DH5<it>α</it>. Results from growth medium optimisation in 500 mL shake flasks by response surface methodology (RSM) generated a maximum volumetric yield of 13.65 mg/L which was 1.75 folds higher than that of the base medium. A controlled fed-batch fermentation employing strategic glycerol feeding and optimised growth conditions resulted in a remarkable pcDNA3F volumetric yield of 110 mg/L and a specific yield of 14 mg/g. In addition, growth pH modification and temperature fluctuation between 35 and 45°C were successfully employed to improve plasmid production.</p> <p>Conclusion</p> <p>Production of a high copy number plasmid DNA containing a foreign gene of interest is often hampered by the low plasmid volumetric yield which results from the over expression of foreign proteins and metabolic repressors. In this work, a simple bioprocess framework was employed and successfully improved the production of pcDNA3F.</p

Association of dietary sodium intake with atherogenesis in experimental diabetes and with cardiovascular disease in patients with Type 1 diabetes

Abstract It is recommended that individuals with diabetes restrict their dietary sodium intake. However, although salt intake is correlated with BP (blood pressure), it also partly determines the activation state of the RAAS (reninangiotensin-aldosterone system), a key mediator of diabetes-associated atherosclerosis. apoE KO (apolipoprotein E knockout) mice were allocated for the induction of diabetes with streptozotocin or citrate buffer (controls) and further randomized to isocaloric diets containing 0.05 %, 0.3 % or 3.1 % sodium with or without the ACEi [ACE (angiotensin-converting enzyme) inhibitor] perindopril. After 6 weeks of study, plaque accumulation was quantified and markers of atherogenesis were assessed using RT-PCR (reverse transcription-PCR) and ELISA. The association of sodium intake and adverse cardiovascular and mortality outcomes were explored in 2648 adults with Type 1 diabetes without prior CVD (cardiovascular disease) from the FinnDiane study. A 0.05 % sodium diet was associated with increased plaque accumulation in diabetic apoE KO mice, associated with activation of the RAAS. By contrast, a diet containing 3.1 % sodium suppressed atherogenesis associated with suppression of the RAAS, with an efficacy comparable with ACE inhibition. In adults with Type 1 diabetes, low sodium intake was also associated with an increased risk of all-cause mortality and new-onset cardiovascular events. However, high sodium intake was also associated with adverse outcomes, leading to a J-shaped relationship overall. Although BP lowering is an important goal for the management of diabetes, off-target actions to activate the RAAS may contribute to an observed lack of protection from cardiovascular complications in patients with Type 1 diabetes with low sodium intake

Oxidative Stress and Inflammation in Cardiovascular Diseases

Cardiovascular diseases (CVD), which include a number of cardiac and vascular conditions, resulted in approximately 17 [...

Circulating Soluble ACE2 Plays an Independent Role to Protect against Vascular Damage in Diabetic Mice

Circulating levels of soluble ACE2 are increased by diabetes. Although this increase is associated with the presence and severity of cardiovascular disease, the specific role of soluble ACE2 in atherogenesis is unclear. Previous studies suggested that, like circulating ACE, soluble ACE2 plays a limited role in vascular homeostasis. To challenge this hypothesis, we aimed to selectively increase circulating ACE2 and measure its effects on angiotensin II dependent atherogenesis. Firstly, in Ace2/ApoE DKO mice, restoration of circulating ACE2 with recombinant murine soluble (rmACE219-613; 1 mg/kg/alternate day IP) reduced plaque accumulation in the aortic arch, suggesting that the phenotype may be driven as much by loss of soluble ACE2 as the reduction in local ACE2. Secondly, in diabetic ApoE KO mice, where activation of the renin angiotensin system drives accelerated atherosclerosis, rmACE219-613 also reduced plaque accumulation in the aorta after 6 weeks. Thirdly, to ensure consistent long-term delivery of soluble ACE2, an intramuscular injection was used to deliver a DNA minicircle encoding ACE219-613. This strategy efficiently increased circulating soluble ACE2 and reduced atherogenesis and albuminuria in diabetic ApoE KO mice followed for 10 weeks. We propose that soluble ACE2 has independent vasculoprotective effects. Future strategies that increase soluble ACE2 may reduce accelerated atherosclerosis in diabetes and other states in which the renin angiotensin system is upregulated

ACE2 deficiency shifts energy metabolism towards glucose utilization

Abstract BACKGROUND: This study aimed at investigating the effects of genetic angiotensin-converting enzyme (ACE) 2 deficiency on glucose homeostasis in the pancreas and skeletal muscle and their reversibility following ACE inhibition. PROCEDURES: ACE2-knockout and C57bl6J mice were placed on a standard diet (SD) or a high-fat diet (HFD) for 12weeks. An additional group of ACE2-knockout mice was fed a SD and treated with the ACE inhibitor, perindopril (2mgkg(-1)day(-1)). Glucose and insulin tolerance tests, indirect calorimetry measurements and EchoMRI were performed. Non-esterfied 'free' fatty acid oxidation rate in skeletal muscle was calculated by measuring the palmitate oxidation rate. \u3b2-cell mass was determined by immunostaining. Insulin, collectrin, glucose transporter protein, and peroxisome proliferator-activated receptor-\u3b3 expression were analysed by RT-PCR. Markers of mithocondrial biogenesis/content were also evaluated. MAIN FINDINGS: ACE2-knockout mice showed a \u3b2-cell defect associated with low insulin and collectrin levels and reduced compensatory hypertrophy in response to a HFD, which were not reversed by perindopril. On the other hand, ACE2 deficiency shifted energy metabolism towards glucose utilization, as it increased the respiratory exchange ratio, reduced palmitate oxidation and PCG-1\u3b1 expression in the skeletal muscle, where it up-regulated glucose transport proteins. Treatment of ACE2-knockout mice with perindopril reversed the skeletal muscle changes, suggesting that these were dependent on Angiotensin II (Ang II). PRINCIPAL CONCLUSIONS: ACE2-knockout mice display a \u3b2-cell defect, which does not seem to be dependent on Ang II but may reflect the collectrin-like action of ACE2. This defect seemed to be compensated by the fact that ACE2-knockout mice shifted their energy consumption towards glucose utilisation via Ang II

End-organ weights, physiological and RAS parameters, and urine biochemistry.

<p>Data expressed as mean±SEM. AU, arbitrary units;</p><p>*P<0.05</p><p>**P<0.01</p><p>***P<0.001 disease effect (Control vehicle vs. STNx Vehicle).</p><p>#P<0.05 treatment effect (Vehicle vs. DIZE)</p><p>End-organ weights, physiological and RAS parameters, and urine biochemistry.</p

Increased urinary ACE2 activity is associated with decreased cortical ACE2 and impaired renal function.

<p>Urinary ACE2 activity (A) of Control (vehicle, n = 10; DIZE, n = 8) and STNx (vehicle, n = 10; DIZE n = 10) rats. Data expressed as mean±SEM. **P<0.01 disease effect (Control vehicle vs. STNx Vehicle). Increased urinary ACE2 activity was associated with increased urinary protein (B) and reduced creatinine clearance (CrCl; C), while cortical ACE2 activity decreased with impaired renal function (D). Increased urinary ACE2 activity was associated with reduced cortical ACE2 activity (E) but not medullary ACE2 activity (F). Only non-treated groups were used for correlation analysis (n = 20). Open squares represent Control rats; closed squares represent STNx rats.</p