Producing medium-chain-length polyhydroxyalkanoate from diverse feedstocks by deregulating unsaturated fatty acid biosynthesis in Escherichia coli.

The fatty acid metabolism in Escherichia coli has served as a basic metabolic chassis for medium-chain-length polyhydroxyalkanoate (mcl-PHA) production. In this study, the phaG and phaC1 genes from Pseudomonas entomophila L48 were first cloned as pGRN08. E. coli BL21P (E. coli BL21(DE3) ΔptsG) containing pGRN08 was able to produce 23 ± 3 and 7 ± 0 mg/L homopolymer poly(3-hydroxydecanoate)(P(3HD)) from glucose and xylose, respectively. Next, a gene, PSEEN0908 (encoding a putative 3-hydroxyacyl-CoA ligase), from P. entomophila L48 was found to increase the performance of mcl-PHA production. The induction of the fatty acid biosynthesis repressor (FabR), a transcription regulator that represses UFA biosynthesis, in E. coli substantially increased the mcl-PHA production by an order of magnitude from both unrelated and related carbon source conversion. A mcl-PHA concentration of 179 ± 1 mg/L and a content of 5.79 ± 0.16 % were obtained, where 31 mol% was 3-hydroxyoctanoate (3HO) and 69 mol% was 3HD.

The transcription activator AtxA from Bacillus anthracis was employed for developing a tight-control, high-level, modulable and stationary-phase-specific transcription activity in Escherichia coli.

The strong transcriptional activity of the virulent gene pagA in Bacillus anthracis has been proven to be anthrax toxin activator (AtxA)-regulated. However, the obscure pagA transcription mechanism hinders practical applications of this strong promoter. In this study, a 509-bp DNA fragment [termed 509sequence, (-508)-(+1) relative to the P2 transcription start site] was cloned upstream of rbs-GFPuv as pTOL02B to elucidate the AtxA-regulated transcription. The 509sequence was dissected into the -10 sequence, -35 sequence, ATrich tract, SLI/SLII and upstream site. In conjunction with the heterologous co-expression of AtxA (under the control of the T7 promoter), the -10 sequence (TATACT) was sufficient for the AtxA-regulated transcription. Integration of pTOL02F + pTOLAtxA as pTOL03F showed that the AtxA-regulated transcription exhibited a strong specific fluorescence intensity/common analytical chemistry term (OD600) of 40 597 ± 446 and an induction/repression ratio of 122. An improved induction/repression ratio of 276 was achieved by cultivating Escherichia coli/pTOL03F in M9 minimal medium. The newly developed promoter system termed PAtxA consists of AtxA, the -10 sequence and Escherichia RNA polymerase. These three elements synergistically and cooperatively formed a previously undiscovered transcription system, which exhibited a tight-control, high-level, modulable and stationary-phase-specific transcription. The PAtxA was used for phaCAB expression for the stationary-phase polyhydroxybutyrate production, and the results showed that a PHB yield, content and titer of 0.20 ± 0.27 g/g-glucose, 68 ± 11% and 1.5 ± 0.4 g/l can be obtained. The positive inducible PAtxA, in contrast to negative inducible, should be a useful tool to diversify the gene information flow in synthetic biology. Graphical Abstract.

Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production.

An antibiotic- and inducer-free culture condition was proposed for polyhydroxybutyrate (PHB) production in recombinant Escherichia coli. First, antibiotic-free vectors were constructed by installing the plasmid maintenance system, alp7, hok/sok, and the hok/sok and alp7 combination into the pUC19 vector. The plasmid stability test showed that pVEC02, the pUC19 vector containing the hok/sok system, was the most effective in achieving antibiotic-free cultivation in the E. coli B strain but not in the K strain. Second, the putative phaCAB operon derived from Caldimonas manganoxidans was inserted into pVEC02 to yield pPHB01 for PHB production in E. coli BL21 (DE3). The putative phaCAB operon was first shown function properly for PHB production and thus, inducer-free conditions were achieved. However, the maintenance of pPHB01 in E. coli requires antibiotics supplementation. Finally, an efficient E. coli ρ factor-independent terminator, thrLABC (ECK120033737), was inserted between the phaCAB operon and the hok/sok system to avoid possible transcriptional carry-over. The newly constructed plasmid pPHB01-1 facilitates an antibiotic- and inducer-free culture condition and induces the production of PHB with a concentration of 3.0 on0.2 g/L, yield of 0.26 /L0.07 g/g-glucose, and content of 44 /g3%. The PHB production using E. coli BL21 (DE3)/pPHB01-1 has been shown to last 84 and 96 h in the liquid and solid cultures.

Cystatin C-Based Renal Function in Predicting the Long-Term Outcomes of Chronic Total Occlusion After Percutaneous Coronary Intervention.

Renal function estimated by various biomarkers predicting for adverse cardiovascular events has not been well-identified in received percutaneous coronary intervention (PCI) for chronic total occlusion (CTO), the advanced stages of atherosclerosis. We aim to determine whether the serum cystatin C-based-estimated glomerular filtration rate (eGFR) can have an improved predictive value in patients with CTO lesions undergoing PCI as compared with multiple creatinine-based estimates of kidney function. Six hundred and seventy-one patients received CTO PCI were retrospectively included in the study. The eGFR was calculated by modification of diet in renal disease equation for Chinese (cMDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations at baseline, respectively. Then, the cohort was categorized into three groups according to standard KDIGO kidney stages based on eGFR. The primary endpoint was all-cause mortality, and the secondary endpoint was cardiac death. Strikingly, cystatin C-based eGFR showed a better performance with the greater area being under the receiver operating characteristic (ROC) curve (0.73 for all-cause mortality and 0.73 for cardiac death, separately) and a better assessment for survival free from adverse event across renal levels among four eGFR equations. Compared with eGFR calculated by other formulas, serum cystatin C-based eGFR showed the highest prognostic value for both all-cause mortality (adjusted HR 3.6, 95% CI 1.6-8.1, P = 0.002) and cardiac death (adjusted HR 2.9, 95% CI 1.0-8.1, P = 0.028). Moreover, cystatin C-based eGFR significantly improved the risk reclassification of event with a high value of net reclassification improvement and integrated discrimination improvement. This study may prove that cystatin C-based eGFR is a better predictor of both all-cause mortality and cardiac death than other equations in populations with CTO undergoing PCI.

miR-590-3p Is a Novel MicroRNA in Myocarditis by Targeting Nuclear Factor Kappa-B in vivo.

OBJECTIVE: Nuclear factor kappa-B (NF-954;B)-induced inflammation leads to myocarditis and heart dysfunction. How microRNAs (miRNAs) contribute to this process is poorly defined. The aim of this study was to investigate whether miRNAs regulate NF-954;B-induced inflammation in experimental autoimmune myocarditis (EAM) in vivo. METHODS AND RESULTS: NF-954;B and its related proinflammatory genes, including interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), were activated in EAM. Profiling of NF-954;B-related miRNAs revealed that miR-590-3p was strikingly reduced in EAM. We found IL-6-induced proinflammatory signaling via miR-590-3p reduction, p50 induction, NF-954;B activation and IL-6/TNF-a expression. Moreover, a luciferase reporter assay demonstrated that miR-590-3p directly interacted with the 3' UTR (untranslated region) of the p50 subunit, and that miR-590-3p overexpression inhibited p50 expression. Finally, miR-590-3p transfection through adeno-associated virus significantly inhibited p50 expression, suppressed NF-954;B activity and blocked IL-6/TNF-a expression in vivo, reducing the lesion area and improving cardiac function in EAM. CONCLUSION: miR-590-3p is a novel NF-954;B-related miRNA that directly targets the p50 subunit. This may provide a novel strategy for the treatment of myocarditis.

Drug-eluting stent, but not bare metal stent, accentuates the systematic inflammatory response in patients.

OBJECTIVE: The systematic pro-inflammatory responses after percutaneous coronary intervention with drug-eluting stents (DES) remain poorly defined. Therefore, we compared the systematic pro-inflammatory state of circulating mononuclear cells (MNCs) between DES and bare metal stent (BMS) implantation. METHODS: Patients with indications for treatment with stents were randomized in a 1:1 ratio to placement of DES or BMS. The primary endpoint was a change of pro-inflammatory state at 12 weeks post-procedure. RESULTS: Thirty-six consecutive patients received DES or BMS. At 12 weeks after stent implantation, the lipid profile and high-sensitivity C-reactive protein (hs-CRP) improved significantly in both groups. The mRNA levels and plasma concentrations of interleukin-6, tumor necrosis factor-α and matrix metalloproteinase-9 were significantly elevated in the DES group, which was not observed in the BMS group. An increase in NF-κB binding activity and a decrease in PPAR-γ expression in MNCs were observed in the DES group, along with increases in IκB phosphorylation and p50 expression. However, similar changes were not observed in the BMS group. CONCLUSIONS: Systematic inflammatory responses were accentuated after the patients were treated percutaneously with DES, despite their improved lipid profile and hs-CRP. These data may provide fundamental information for optimizing therapeutic strategy in the era of DES.

Isoproterenol instigates cardiomyocyte apoptosis and heart failure via AMPK inactivation-mediated endoplasmic reticulum stress.

A prolonged or excessive adrenergic activation leads to myocyte loss and heart dysfunction; however, how it contributes to heart failure remains poorly defined. Here we show that isoproterenol (ISO) induced aberrant endoplasmic reticulum (ER) stress and apoptotic cell death, which was inhibited by activating the AMP-activated protein kinase (AMPK) in vitro and in vivo. Persistent ISO stimulation suppressed the AMPK phosphorylation and function, resulting in enhanced ER stress and the subsequent cell apoptosis in cardiomyocytes in vitro and in vivo. AMPK activation decreased the aberrant ER stress, apoptosis, and brain natriuretic peptide (BNP) release in ISO-treated cardiomyocytes, which was blocked by AMPK inhibitor Compound C. Importantly, increased ER stress and apoptosis were observed in ISO-treated cardiomyocytes isolated from AMPKα2(-/-) mice. Inhibition of ER stress attenuated the apoptosis but failed to reverse AMPK inhibition in ISO-treated cardiomyocytes. Moreover, metformin administration activated AMPK and reduced both ER stress and apoptosis in ISO-induced rat heart failure in vivo. We conclude that ISO, via AMPK inactivation, causes aberrant ER stress, cardiomyocyte injury, BNP release, apoptosis, and hence heart failure in vivo, all of which are inhibited by AMPK activation.

Improvement of cardiac function and reversal of gap junction remodeling by Neuregulin-1ß in volume-overloaded rats with heart failure.

OBJECTIVE: We performed experiments using Neuregulin-1ß (NRG-1ß) treatment to determine a mechanism for the protective role derived from its beneficial effects by remodeling gap junctions (GJs) during heart failure (HF). METHODS: Rat models of HF were established by aortocaval fistula. Forty-eight rats were divided randomly into the HF (HF, n = 16), NRG-1ß treatment (NRG, n = 16), and sham operation (S, n = 16) group. The rats in the NRG group were administered NRG-1ß (10 µg/kg per day) for 7 days via the tail vein, whereas the other groups were injected with the same doses of saline. Twelve weeks after operation, Connexin 43 (Cx43) expression in single myocytes obtained from the left ventricle was determined by immunocytochemistry. Total protein was extracted from frozen left ventricular tissues for immunoblotting assay, and the ultrastructure of myocytes was observed by transmission electron microscopy. RESULTS: Compared with the HF group, the cardiac function of rats in the NRG group was markedly improved, irregular distribution and deceased Cx43 expression were relieved. The ultrastructure of myocytes was seriously damaged in HF rats, and NRG-1ß reduced these pathological damages. CONCLUSIONS: Short-term NRG-1ß treatment can rescue pump failure in experimental models of volume overload-induced HF, which is related to the recovery of GJs structure and the improvement of Cx43 expression.

Mesenchymal stem cells neither fully acquire the electrophysiological properties of mature cardiomyocytes nor promote ventricular arrhythmias in infarcted rats.

Electrophysiological properties of implanted mesenchymal stem cells (MSCs) in infarcted hearts remain unclear, and their proarrhythmic effect is still controversial. The intent of this study was to investigate electrophysiological properties and proarrhythmic effects of MSCs in infarcted hearts. Rats were randomly divided into a myocardial infarction (MI) group, a MI-DMEM group (received DMEM medium injection) and MI-MSCs group (received MSCs injection). Survival analysis showed that the majority of engrafted MSCs died at day 9 after transplantation. Engrafted MSCs expressed cardiac markers (MYH, cTnI, Cx43), cardiac ion channel genes (Kv1.4, Kv4.2 and Kir2.1) and potassium currents (I (to), I (K1) and I (KDR)), but did not express Nav1.5, Cav1.2, Na(+) current and Ca(2+) current during their survival. When induced by Ca(2+), implanted MSCs exhibited no contraction ability after being isolated from the heart. Following 8-week electrocardiography monitoring, the cumulative occurrence of ventricular arrhythmias (VAs) was not different among the three groups. However, the prolonged QRS duration in infarcted rats without VAs was significantly decreased in the MI-MSCs group compared with the other two groups. The inducibility of VAs in the MI-MSCs group was much lower than that in the MI and MI-DMEM groups (41.20 vs. 86.67 % and 92.86 %; P < 0.0125). The ventricular effective refractory period in MI-MSCs group was prolonged in comparison with that in the MI and MI-DMEM groups (56.0 ± 8.8 vs. 47.7 ± 8.8 ms and 45.7 ± 6.2 ms; P < 0.01). These results demonstrate that MSCs do not acquire the electrophysiological properties of mature cardiomyocytes during the survival period in the infarcted hearts. However, they can alleviate the electrical vulnerability and do not promote ventricular arrhythmias.