Engineered biosynthesis of β‐alkyl tryptophan analogs

Noncanonical amino acids (ncAAs) with dual stereocenters at the α and β positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive β‐branched ncAAs, their availability is limited due to the inefficiency of the multistep methods used to prepare them. Herein we report a stereoselective biocatalytic synthesis of β‐branched tryptophan analogues using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB^(7E6). PfTrpB^(7E6) is the first biocatalyst to synthesize bulky β‐branched tryptophan analogues in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of PfTrpB^(7E6) was explored through X‐ray crystallography and UV/Vis spectroscopy, which revealed that a combination of active‐site and remote mutations increase the abundance and persistence of a key reactive intermediate. PfTrpB^(7E6) provides an operationally simple and environmentally benign platform for the preparation of β‐branched tryptophan building blocks

Fractional Flow Reserve\u2013Guided Deferred Versus Complete Revascularization in Patients With Diabetes Mellitus

To assess the safety and efficacy of deferred versus complete revascularization using a fractional flow reserve (FFR)\u2013guided strategy in patients with diabetes mellitus (DM), we analyzed all DM patients who underwent FFR-guided revascularization from January 1, 2010, to December 12, 2013. Patients were divided into 2 groups: those with 651 remaining FFR-negative (>0.80) medically treated lesions [FFR( 12)MT] and those with only FFR-positive lesions ( 640.80) who underwent complete revascularization [FFR(+)CR] and were followed until July 1, 2015. The primary end point was the incidence of major adverse cardiovascular events (MACE), a composite of death, myocardial infarction (MI), target lesion (FFR assessed) revascularization, and rehospitalization for acute coronary syndrome. A total of 294 patients, 205 (69.7%) versus 89 (30.3%) in FFR( 12)MT and FFR(+)CR, respectively, were analyzed. At a mean follow-up of 32.6 \ub1 18.1\ua0months, FFR( 12)MT was associated with higher MACE rate 44.0% versus 26.6% (log-rank p\ua0=\ua00.02, Cox regression\u2013adjusted hazard ratio [HR] 2.01, 95% confidence interval [CI] 1.21 to 3.33, p\ua0<0.01), and\ua0driven by both safety and efficacy end points: death/MI (HR 2.02, 95% CI 1.06 to 3.86, p\ua0= 0.03), rehospitalization for acute coronary syndrome (HR 2.06, 95% CI 1.03 to 4.10, p\ua0= 0.04), and target lesion revascularization (HR 3.38, 95% CI 1.19 to 9.64, p\ua0= 0.02). Previous MI was a strong effect modifier within the FFR( 12)MT group (HR 1.98, 95% CI 1.26 to 3.13, p <0.01), whereas this was not the case in the FFR(+)CR group (HR 0.66, 95% CI 0.27 to 1.62, p\ua0= 0.37). Significant interaction for MACE was present between FFR groups and previous MI (p\ua0= 0.03). In conclusion, in patients with DM, particularly those\ua0with previous MI, deferred revascularization is associated with poor medium-term outcomes. Combining FFR with imaging techniques may be required to guide our treatment strategy in these patients with high-risk, fast-progressing atherosclerosis

Is There Evidence to Show that Fetal Alcohol Syndrome can be Prevented?

Fetal Alcohol Syndrome (FAS) is currently the major cause of mental retardation in the Western world. Since FAS is not a natural phenomenon and is created by mixing alcohol and pregnancy, the solution to decreasing the incidence of all alcohol-related birth defects is therefore entirely preventable. To date, little is known about the effectiveness of prevention programs in reducing the incidence of FAS. Therefore, it is the intention of this article to review the effectiveness of prevention programs in lowering the incidence of FAS. The present review revealed that prevention programs, to date, have been successful in raising awareness of FAS levels across the groups examined. However, this awareness has not been translated into behavioral changes in 'high risk' drinkers as consumption levels in this group have decreased only marginally, indicating prevention programs have had minimal or no impact in lowering the incidence of FAS. Urgent steps must now be taken to fully test prevention programs, and find new strategies involving both sexes, to reduce and ultimately eliminate the incidence of FAS

Density-Fluctuations in the Tortur Tokamak

Quantitative measurements of the electron density fluctuation spectra, S(omega, k), are presented as a result of collective scattering measurements with 2 mm waves at various radial positions in the equatorial plane of the TORTUR Tokamak plasma. The spectra are determined in a wide frequency range up to 100 MHz and in a k-range between 400 and 4400 m-1 corresponding to scattering angles between 8-degrees and 90-degrees. It was found by performing experiments for various directions of k, that the fluctuations are isotropic in the poloidal plane, i.e. k0 almost-equal-to k(r). The total fluctuation level rises from 2% in the plasma centre to more than 10% near the plasma periphery. The theoretically expected frequency ranges for various models, like density gradient-driven modes, trapped electron modes and the high-frequency high m-number magnetic tearing modes are much smaller than the experimental values. This can be explained by a frequency shift due to toroidal rotation. Fluctuations at frequencies above 10 MHz in the ion cyclotron frequency range, for which the set-up was especially built, turned out to be absent. The decay coefficients of the k-spectra are found to be - 3.6 +/- 0.6 which is larger than the Kolmogorov value (8/3). Predictions for the radial dependence of by the filamentation hypothesis of Taylor are found to be consistent with experimental values for r/a less-than-or-equal-to 0.5. Values for the electron thermal diffusivity calculated by the heuristic expression chi(e) = gamma/2, with gamma and k(perpendicular-to) derived from the scattering data, were found to be in agreement with values derived from power balance and perturbation measurements

Chronic phosphocreatine depletion by the creatine analogue beta-guanidinopropionate is associated with increased mortality and loss of ATP in rats after myocardial infarction.

BACKGROUND: The failing myocardium is characterized by reductions of phosphocreatine (PCr) and free creatine content and by decreases of energy reserve via creatine kinase (CK), ie, CK reaction velocity (Flux(CK)). It has remained unclear whether these changes contribute directly to contractile dysfunction. In the present study, myocardial PCr stores in a heart failure model were further depleted by feeding of the PCr analogue beta-guanidinopropionate (GP). Functional and metabolic consequences were studied. METHODS AND RESULTS: Rats were subjected to sham operation or left coronary artery ligation (MI). Surviving rats were assigned to 4 groups and fed with 0% (n=7, Sham; n=5, MI) or 1% (n=7 Sham+GP, n=8 MI+GP) GP. Two additional groups were fed GP for 2 or 4 weeks before MI. After 8 weeks, hearts were isolated and perfused, and left ventricular pressure-volume curves were obtained. High-energy phosphate metabolism was determined with (31)P NMR spectroscopy. After GP feeding or MI, left ventricular pressure-volume curves were depressed by 33% and 32%, respectively, but GP feeding in MI hearts did not further impair mechanical function. Both MI and GP feeding reduced PCr content and Flux(CK), but here, effects were additive. In MI+GP rats, PCr levels and Flux(CK) were reduced by 87% and 94%, respectively. Although ATP levels were maintained in the GP and MI groups, ATP content was reduced by 18% in MI+GP hearts. Furthermore, 24-hour mortality in GP-prefed rats was 100%. CONCLUSIONS: Rats with an 87% predepletion of myocardial PCr content cannot survive an acute MI. Chronically infarcted hearts subjected to additional PCr depletion cannot maintain ATP homeostasis

Preservation of cardiac function and energy reserve by the angiotensin-converting enzyme inhibitor quinapril during postmyocardial infarction remodeling in the rat.

PURPOSE: Angiotensin-converting enzyme (ACE) inhibitors show beneficial long-term hemodynamic effects in chronically infarcted hearts. The purpose of this study was to test whether prevention of the deterioration of mechanical function by ACE inhibitors is related to beneficial effects on high-energy phosphate metabolism that is deranged in heart failure. METHODS: Twelve-week old rats were randomly assigned to ligation of the left coronary artery [mycardial infarction (MI)] or sham operation (Sham) and to the ACE inhibitor quinapril (+Q) (6 mg/kg/day per gavage) or placebo treatment. Eight weeks later, cardiac function was measured in the isolated heart by a left ventricular balloon (pressure-volume curves), and energy metabolism of residual intact myocardium was analyzed in terms of total and isoenzyme creatine kinase activity (spectrophotometry), steady-state levels [adenosine triptosphate (ATP), phosphocreatine], and turnover rates (creatine kinase reaction velocity) of high-energy phosphates [31P nuclear magnetic resonance (NMR)] and total creatine content [high-performance liquid chromatography (HPLC)]. RESULTS: Quinapril prevented post-MI hypertrophy and partially prevented left ventricular contractile dysfunction [maximum left ventricular developed pressure 166+/-6, 83+/-16 (p &lt; 0.05 MI vs. Sham), 139+/-13 mm Hg (p &lt; 0.05 quinapril treated vs. untreated) in Sham, MI and MI+Q hearts]. Residual intact failing myocardium showed a 17% decrease of MM-CK and a 16% decrease of mito-CK activity. Total creatine was reduced by 23%, phosphocreatine by 26% and CK reaction velocity by 30%. Parallel to improved function, treatment with quinapril largely prevented the impairment of energy metabolism occuring post-MI. CONCLUSIONS: quinapril treatment results in an improvement of high-energy phosphate metabolism, of energy reserve via the creatine kinase reaction, and of contractile performance post-MI

Density-Fluctuations in the Tortur Tokamak

Quantitative measurements of the electron density fluctuation spectra, S(omega, k), are presented as a result of collective scattering measurements with 2 mm waves at various radial positions in the equatorial plane of the TORTUR Tokamak plasma. The spectra are determined in a wide frequency range up to 100 MHz and in a k-range between 400 and 4400 m-1 corresponding to scattering angles between 8-degrees and 90-degrees. It was found by performing experiments for various directions of k, that the fluctuations are isotropic in the poloidal plane, i.e. k0 almost-equal-to k(r). The total fluctuation level rises from 2% in the plasma centre to more than 10% near the plasma periphery. The theoretically expected frequency ranges for various models, like density gradient-driven modes, trapped electron modes and the high-frequency high m-number magnetic tearing modes are much smaller than the experimental values. This can be explained by a frequency shift due to toroidal rotation. Fluctuations at frequencies above 10 MHz in the ion cyclotron frequency range, for which the set-up was especially built, turned out to be absent. The decay coefficients of the k-spectra are found to be - 3.6 +/- 0.6 which is larger than the Kolmogorov value (8/3). Predictions for the radial dependence of by the filamentation hypothesis of Taylor are found to be consistent with experimental values for r/a less-than-or-equal-to 0.5. Values for the electron thermal diffusivity calculated by the heuristic expression chi(e) = gamma/2, with gamma and k(perpendicular-to) derived from the scattering data, were found to be in agreement with values derived from power balance and perturbation measurements

Acute changes of myocardial creatine kinase gene expression under beta-adrenergic stimulation.

Creatine kinase (CK) plays a crucial role in myocardial energy metabolism. Alterations in CK gene expression are found in hypertrophied and failing heart, but the mechanisms behind these changes are unclear. This study tests the hypothesis that increased adrenergic stimulation, which is observed in heart failure, induces changes of myocardial CK-activity, -isoenzyme distribution and -gene expression that are characteristic of the failing and hypertrophied heart. Isolated rat hearts were perfused (constant pressure of 80 mmHg) with red cell suspensions. Following a 20-min warm-up period, perfusion for 3 h with 10(-8) M (iso 3 h) or without (control 3 h) isoproterenol was started or experiments were immediately terminated (control 0 h). Left ventricular tissue was analyzed for total CK-activity, CK-isoenzyme distribution and, by use of quantitative RT-PCR, for B-CK, M-CK, mito-CK and GAPDH- (as internal standard) mRNA. After beta-adrenergic stimulation (iso 3 h) but not after control perfusion (control 3 h) a roughly threefold increase in B-CK mRNA levels and a decrease in M-CK mRNA levels by 18% was found. There were no significant differences among the three groups in total CK-activity and in distribution of CK-MM, CK-BB, CK-MB and mito-CK. Thus, beta-adrenergic stimulation induces a switch in CK gene expression from M-CK to B-CK, which is characteristic for the hypertrophied and failing heart. This may be interpreted as an adaptive mechanism making energy transduction via CK more efficient at times of increased metabolic demand