Metabolic engineering of Escherichia coli for the production of isoflavonoid-4'-O-methoxides and their biological activities.

Isoflavonoid representatives such as genistein and daidzein are highly potent anticancer, antibacterial, and antioxidant agents. It have been demonstrated that methylation of flavonoids enhanced the transporting ability, which lead to facilitated absorption and greatly increased bioavailability. In this paper, genetically engineered Escherichia coli was reconstructed by harboring E. coli K12-derived metK encoding S-adenosine-l-methionine (SAM) synthase (accession number: K02129) for enhancement of SAM as a precursor and Streptomyces avermitilis originated SaOMT2 (O-methyltransferase, accession number: NP_823558) for methylation of daidzein and genistein as preferred substrates. The formation of desired products via biotransformation including 4'-O-methyl-genistein and 4'-O-methyl-daidzein was confirmed individually by using chromatographical methods such as high-performance liquid chromatography, liquid chromatography/time-of-flight/mass spectrometry (LC-TOF-MS), and nuclear magnetic resonance (NMR), and NMR (1 H and 13 C). Furthermore, substrates concentration, incubation time, and media parameters were optimized using flask culture. Finally, the most fit conditions were applied for fed-batch fermentation with scale-up to 3 L (working volume) to obtain the maximum yield of the products including 164.25 µM (46.81 mg/L) and 382.50 µM (102.88 mg/L) for 4'-O-methyl genistein and 4'-O-methyl daidzein, respectively. In particular, potent inhibitory activities of those isoflavonoid methoxides against the growth of cancer line (B16F10, AGS, and HepG2) and human umbilical vein endothelial cells were investigated and demonstrated. Taken together, this research work described the production of isoflavonoid-4'-O-methoxides by E. coli engineering, improvement of production, characterization of produced compounds, and preliminary in vitro biological activities of the flavonoids being manufactured.

The prognostic value of regadenoson myocardial perfusion imaging.

BACKGROUND: Regadenoson (REGA), a selective adenosine A2A receptor agonist, is the most widely used stress agent for SPECT myocardial perfusion imaging (MPI) in the United States. The diagnostic accuracy of REGA MPI is comparable to Adenosine MPI, but its prognostic value is not well defined. METHODS: We categorized 1,400 patients (700 consecutive normal and 700 consecutive abnormal REGA-MPIs) into 4 groups based on the perfusion defect size using automated quantitative analysis: Group 1: normal perfusion; Group 2: <10% of left ventricle; Group 3: 10%-20%; Group 4: >20%. The primary outcome was a composite of cardiac death, myocardial infarction (MI), and late coronary revascularization (CR >90 days after MPI). RESULTS: Of the 1,400 patients (42% male, 37% diabetes, 21% heart failure, 26% end-stage renal disease), the primary outcome occurred in 23% (17% cardiac death, 4% MI, 6% late CR) during 46 ± 18 months of follow-up and 8% had early CR (within 90 days of MPI). Early CR occurred in 0.4%, 9%, 17%, and 17% and the primary outcome in 10%, 27%, 31%, and 43% in Groups 1-4, respectively (P < .001 for both). In an adjusted Cox proportional model, the hazard ratio for the primary outcome was 2.68 (1.77-4.06), 3.32 (2.28-4.83), and 4.05 (2.78-5.91) for Groups 2-4 compared to Group 1. CONCLUSION: REGA MPI provides powerful prognostic information that has important implications in patient management and can guide clinical practice.

Modification of emodin and aloe-emodin by glycosylation in engineered Escherihia coli.

Glycosyltransferase from Bacillus licheniformis DSM13 (YjiC) was used for enzymatic modification of emodin and aloe-emodin in vitro and in vivo. In order to increase the availability of UDP-glucose, three genes involved in the production of precursors of NDP-sugar in Escherichia coli BL21 (DE3) viz. D-glucose phosphate isomerase (pgi), D-glucose-6-phosphate dehydrogenase (zwf), and UDP-sugar hydrolase (ushA) were deleted and glucose-1-phosphate urididyltransferase (galU) gene was over expressed. To improve the yield of the products; substrate, time and media parameters were optimized, and the production was scaled up using a 3 L fermentor. The maximum yield of glycosylated products of emodin (emodin-O-ß-D-glucoside) and aloe-emodin (aloe-emodin-O-ß-D-glucoside) were approximately 144 µM (38 mg/L) and 168 µM (45 mg/L) respectively, representing almost 72 % and 84 % bioconversion of emodin and aloe-emodin when 200 µM of emodin and aloe-emodin were supplemented in the culture. Additionally, the emodin and aloe emodin major glycosylated products exhibited the highest stability at pH 8.0 and the stability of products was up to 70 °C and 60 °C respectively. Furthermore, the biological activities of emodin and its major glucoside (P1) were compared and their anti-cancer activities were assayed in several cancer cell lines. The results demonstrate that YjiC has the capacity to catalyze the glycosylation of these aromatic compounds and that glycosylation of anthraquinones enhances their aqueous solubility while retaining their biological activities.

Enzymatic glycosylation of the topical antibiotic mupirocin.

Mupirocin is a commercially available antibiotic that acts on bacterial isoleucyl-tRNA synthetase, thereby inhibiting protein synthesis and preventing bacterial infection. An in vitro glycosylation approach was applied to synthesize glycoside derivatives of mupirocin using different NDP-sugars and glycosyltransferase from Bacillus licheniformis. Ultra pressure liquid chromatography-photo diode array analyses of the reaction mixtures revealed the generation of product peak(s). The results were further supported by high-resolution quadruple time of flight electrospray ionization mass spectrometry analyses. The product purified from the reaction mixture with UDP-D-glucose was subjected to NMR analysis, and the structure was determined to be mupirocin 6-O-ß-D-glucoside. Other glycoside analogs of mupirocin were determined based on high-resolution mass analyses. Antibacterial activity assays against Staphylococcus aureus demonstrated complete loss of antibacterial activity after glucosylation of mupirocin at the 6-hydroxyl position.

Regadenoson: a focused update.

Since its approval by the Food and Drug Administration in 2008, regadenoson has become the most commonly used vasodilator in the United States. Previous reviews have summarized the pre-clinical and clinical data on the use of regadenoson for myocardial perfusion imaging (MPI). Since then, data have emerged on the safety of this agent in special groups of patients such as those with chronic kidney disease, airway disease (asthma and chronic obstructive pulmonary disease), and liver disease. There has also been recent interest in the use of regadenoson in hybrid protocols with exercise as a way to improve patient tolerance and image quality. Finally, although regadenoson was approved for clinical use based on the agreement rate of regadenoson MPI and adenosine MPI with regards to perfusion abnormalities, data are now available on the prognostic data derived from regadenoson MPI. We will briefly summarize these recent reports here in a focused update on the use of regadenoson for MPI.

Brachial artery low-flow-mediated constriction is increased early after coronary intervention and reduces during recovery after acute coronary syndrome: characterization of a recently described index of vascular function.

AIMS: The endothelium plays a role in regulating vascular tone. Acute and dynamic changes in low-flow-mediated constriction (L-FMC) and how it changes with regard to traditional flow-mediated dilatation (FMD) have not been described. We aimed to investigate the changes in brachial artery L-FMC following percutaneous coronary intervention (PCI) and during recovery from non-ST-segment elevation myocardial infarction (NSTEMI). METHODS AND RESULTS: FMD was performed in accordance with a previously described technique in patients before and after PCI and in the recovery phase of NSTEMI, but in addition, L-FMC data were acquired from the last 30 s of cuff inflation. About 135 scans were performed in 96 participants (10 healthy volunteers and 86 patients). Measurement of brachial L-FMC was reproducible over hours. L-FMC was greater among patients with unstable vs. stable coronary atherosclerosis (-1.33 ±1.09% vs. -0.03 ± 1.26%, P < 0.01). Following PCI, FMD reduced (4.43 ± 2.93% vs. 1.66 ± 2.16%, P < 0.01) and L-FMC increased (-0.33 ± 0.76% vs. -1.63 ± 1.15%, P = 0.02). Furthermore, during convalescence from NSTEMI, L-FMC reduced (-1.37 ± 1.19% vs. 0.01 ± 0.82%, P = 0.02) in parallel with improvements in FMD (2.54 ± 2.19% vs. 5.15 ± 3.07%, P < 0.01). CONCLUSION: Brachial L-FMC can be measured reliably. Differences were observed between patients with stable and unstable coronary disease. L-FMC was acutely increased following PCI associated with reduced FMD and, in the recovery from NSTEMI, L-FMC reduced associated with increased FMD. These novel findings characterize acute and subacute variations in brachial L-FMC. The pathophysiological and clinical implications of these observations require further study.

Achieving routine sub 30 minute door-to-balloon times in a high volume 24/7 primary angioplasty center with autonomous ambulance diagnosis and immediate catheter laboratory access.

BACKGROUND: In primary angioplasty (primary percutaneous coronary intervention [PPCI]) for acute myocardial infarction, institutional logistical delays can increase door-to-balloon times, resulting in increased mortality. METHODS: We moved from a thrombolysis (TL) service to 24/7 PPCI for direct access and interhospital transfer in April 2004. Using autonomous ambulance diagnosis with open access to the myocardial infarction center catheter laboratory, we compared reperfusion times and clinical outcomes for the final 2 years of TL with the first 3 years of PPCI. RESULTS: Comparison was made between TL (2002-2004, n = 185) and PPCI (2004-2007, n = 704); all times are medians in minutes (interquartile range): for TL, symptom to needle 153 (85-225), call to needle 58 (49-73), first professional contact (FPC) to needle 47 (39-63), door to needle 18 (12-30) (mortality: 7.6% at 30 days, 9.2% at 1 year); for interhospital transfer PPCI (n = 227), symptom to balloon 226 (175-350), call to balloon 135 (117-188), FPC to balloon 121 (102-166), first door-to-balloon 100 (80-142) (mortality: 7.0% at 30 days, 12.3% at 1 year); for direct-access PPCI (n = 477), symptom to balloon 142 (101-238), call to balloon 79 (70-93), FPC to balloon 69 (59-82), door to balloon 20 (16-29) (mortality: 4.6% at 30 days, 8.6% at 1 year). There was no difference between direct-access PPCI and TL times for symptom to needle/balloon. Direct-access PPCI was significantly quicker for the group than in-hospital thrombolysis for door to needle/balloon times due to the lack of any long wait patients (P < .001). CONCLUSIONS: Interhospital transfer remains slow even with rapid institutional door-to-balloon times. With autonomous ambulance diagnosis and open access direct to the catheter laboratory, a median door-to-balloon time of <30 minutes day and night was achieved, and >95% of patients were reperfused within 1 hour.

Improved squalene production via modulation of the methylerythritol 4-phosphate pathway and heterologous expression of genes from Streptomyces peucetius ATCC 27952 in Escherichia coli.

Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.

Squalene-hopene cyclase (Spterp25) from Streptomyces peucetius: sequence analysis, expression and functional characterization.

Squalene-hopene cyclase, which catalyzes the complex cyclization of squalene to the pentacyclic triterpene, hopene, is a key enzyme in the biosynthesis of hopanoids. The deduced amino acid sequence of the Streptomyces peucetius gene (spterp25) had significant similarity to other prokaryotic squalene-hopene cyclases. Like other triterpene cyclases, the S. peucetius squalene-hopene cyclase contains eight so-called QW-motifs with an aspartate-rich domain. The 2,025-bp squalene-hopene cyclase-encoding gene was expressed in Escherichia coli BL21(DE3)pLySs, and the in vitro activity of the recombinant cyclase was demonstrated using purified membrane protein. The cyclization product hopene was identified by gas chromatography/mass spectrometry (GC/MS).

Identification of a cryptic type III polyketide synthase (1,3,6,8-tetrahydroxynaphthalene synthase) from Streptomyces peucetius ATCC 27952.

We identified a 1,134-bp putative type III polyketide synthase from the sequence analysis of Streptomyces peucetius ATCC 27952, named Sp-RppA, which is characterized as 1,3,6,8-tetrahydroxynaphthalene synthase and shares 33% identity with SCO1206 from S. coelicolor A3(2) and 32% identity with RppA from S. griseus. The 1,3,6,8-tetrahydroxynaphthalene synthase is known to catalyze the sequential decarboxylative condensation, intramolecular cyclization, and aromatization of an oligoketide derived from five units of malonyl-CoA to give 1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidizes to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). In this study, we report the in vivo expression and in vitro synthesis of flaviolin from purified gene product (Sp-RppA).

Cloning and functional characterization of the germacradienol synthase (spterp13) from Streptomyces peucetius ATCC 27952.

Sequence analysis of the metabolically rich genome of Streptomyces peucetius ATCC 27952 revealed a 2,199 bp sesquiterpene alcohol (germacradienol) synthase-encoding gene from the germacradienol synthase/terpene cyclase gene cluster. The gene was named spterp13, and its putative function is as a germacradienol synthase/terpene cyclase. The amino acid sequence of Spterp13 shows 66% identity with SAV2163 (GeoA) from S. avermitilis MA- 4680 and 65% identity with SCO6073 from S. coelicolor A3(2), which produces germacradienol/geosmin. The fulllength recombinant protein was heterologously expressed as a his-tagged fusion protein in Escherichia coli, purified, and shown to catalyze the Mg2+-dependent conversion of farnesyl diphosphate to the germacradienol, which was verified by gas chromatography/mass spectrometry.

Large-field intravascular ultrasound for annular sizing and predicting paravalvular regurgitation during TAVR: comparisons with multidetector computed tomography and transoesophageal echocardiography.

AIMS: The use of contrast media with multidetector computed tomography (MDCT) may induce acute kidney injury in patients with renal failure undergoing transcatheter aortic valve replacement (TAVR). We investigated the role of large-field intravascular ultrasound (IVUS) vs. MDCT and two-dimensional transoesophageal echocardiography (2D-TEE) for annular sizing and predicting paravalvular regurgitation (PVR) during TAVR. METHODS AND RESULTS: The aortic annulus was measured by large-field IVUS and 2D-TEE, and compared with MDCT in 50 patients undergoing TAVR. The IVUS and MDCT annular areas and diameters were not significantly different (446 ± 87 mm2 and 23.8 ± 84 mm vs. 466 ± 84 mm2 and 24 ± 2.1 mm, respectively; P > 0.05). IVUS and MDCT mean annular diameters were significantly greater than TEE diameter (23.8 ± 2.4 and 24 ± 2.1 vs. 22 ± 0.65 mm, respectively; P < 0.01). PVR ≥ Mild occurred in 13 patients (26%); 5 patients required post-dilation and 2 patients a second valve. Receiver operating characteristic analyses showed that transcatheter heart valve (THV) area - IVUS or MDCT areas equally predicted of ≥ mild PVR (areas under the curve [AUC] 0.79 and 0.81, respectively; P < 0.001), and were greater than THV diameter-TEE diameter (AUC 0.79 and 0.81 vs. 0.56, respectively; P < 0.05). CONCLUSIONS: The aortic annular measurements and predicting PVR by large field IVUS were not significantly different from those of MDCT, but were greater than those of TEE. Large filed IVUS can be reliably used in lieu of MDCT for annular sizing in patients with aortic stenosis and renal failure or suboptimal MDCT images.

Advances in Biochemistry and Microbial Production of Squalene and Its Derivatives.

Squalene is a linear triterpene formed via the MVA or MEP biosynthetic pathway and is widely distributed in bacteria, fungi, algae, plants, and animals. Metabolically, squalene is used not only as a precursor in the synthesis of complex secondary metabolites such as sterols, hormones, and vitamins, but also as a carbon source in aerobic and anaerobic fermentation in microorganisms. Owing to the increasing roles of squalene as an antioxidant, anticancer, and anti-inflammatory agent, the demand for this chemical is highly urgent. As a result, with the exception of traditional methods of the isolation of squalene from animals (shark liver oil) and plants, biotechnological methods using microorganisms as producers have afforded increased yield and productivity, but a reduction in progress. In this paper, we first review the biosynthetic routes of squalene and its typical derivatives, particularly the squalene synthase route. Second, typical biotechnological methods for the enhanced production of squalene using microbial cell factories are summarized and classified. Finally, the outline and discussion of the novel trend in the production of squalene with several updated events to 2015 are presented.

Methylation of flavonoids: Chemical structures, bioactivities, progress and perspectives for biotechnological production.

Among the natural products, flavonoids have been particularly attractive, highly studied and become one of the most important promising agent to treat cancer, oxidant stress, pathogenic bacteria, inflammations, cardio-vascular dysfunctions, etc. Despite many promising roles of flavonoids, expectations have not been fulfilled when studies were extended to the in vivo condition, particularly in humans. Instability and very low oral bioavailability of dietary flavonoids are the reasons behind this. Researches have demonstrated that the methylation of these flavonoids could increase their promise as pharmaceutical agents leading to novel applications. Methylation of the flavonoids via theirs free hydroxyl groups or C atom dramatically increases their metabolic stability and enhances the membrane transport, leading to facilitated absorption and highly increased oral bioavailability. In this paper, we concentrated on analysis of flavonoid methoxides including O- and C-methoxide derivatives in aspect of structure, bioactivities and description of almost all up-to-date O- and C-methyltransferases' enzymatic characteristics. Furthermore, modern biological approaches for synthesis and production of flavonoid methoxides using metabolic engineering and synthetic biology have been focused and updated up to 2015. This review will give a handful information regarding the methylation of flavonoids, methyltransferases and biotechnological synthesis of the same.

Device closure of periprosthetic paravalvular regurgitation.

Periprosthetic paravalvular regurgitation is an important sequel associated with prosthetic valves whether implanted surgically or via transcatheter approach. They can remain clinically silent or manifest as clinical heart failure, intravascular hemolysis or a combination of both. Periprosthetic defects are becoming increasingly recognized as a source of morbidity and mortality in patients with prosthetic heart valves and in the last few years, the management of this condition has evolved. This review aims to address the current knowledge on the pathophysiology, imaging modalities and management of these defects. It further details the principles, methodology and outcomes of catheter-based device therapy of periprosthetic paravalvular defects.

Activation of Cryptic hop Genes from Streptomyces peucetius ATCC 27952 Involved in Hopanoid Biosynthesis.

Genes encoding enzymes with sequence similarity to hopanoids biosynthetic enzymes of other organisms were cloned from the hopanoid (hop) gene cluster of Streptomyces peucetius ATCC 27952 and transformed into Streptomyces venezuelae YJ028. The cloned fragments contained four genes, all transcribed in one direction. These genes encode polypeptides that resemble polyprenyl diphosphate synthase (hopD), squalene-phytoene synthases (hopAB), and squalenehopene cyclase (hopE). These enzymes are sufficient for the formation of the pentacyclic triterpenoid lipid, hopene. The formation of hopene was verified by gas chromatography/ mass spectrometry.

Genetic evidence for the involvement of glycosyltransferase PdmQ and PdmS in biosynthesis of pradimicin from Actinomadura hibisca.

Pradimicins are potent antifungal antibiotics with effective inhibitory effects against HIV-1. Pradimicin A consists of an unusual dihydrobenzo[α]naphthacenequinone aglycone substituted with a combination of D-alanine and two sugar moieties. Detailed genetic studies revealed most steps in pradimicin A biosynthesis, but the glycosylation mechanism remained inconclusive. The biosynthetic gene cluster of pradimicin A contains two putative glycosyltransferases, pdmQ and pdmS. However, the exact involvement of each gene in biosynthesis and the particular steps required for precise structural modification was unknown. In this study, the exact role of each gene was evaluated by insertional inactivation and complementation studies. Analysis of the metabolite from both of the disruption mutants revealed abolishment of pradimicin A and complementation resulted in the recovery of production. After deletion of pdmQ, pradimicin B was found to accumulate, whereas deletion of pdmS resulted in the accumulation of aglycone of pradimicin. Together, these results suggest that pdmS is responsible for the attachment of thomosamine to form pradimicin B which in turn is glycosylated by pdmQ to form pradimicin A. These results allowed us to deduce the exact order of terminal tailoring by glycosylation and provided insight into the mechanism of pradimicin A biosynthesis.