Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis

Human cytochrome P450 (P450) 2D6 is an important enzyme involved in the metabolism of drugs, many of which are amines or contain other basic nitrogen atoms. Asp301 has generally been considered to be involved in electrostatic docking with the basic substrates, on the basis of previous modeling studies and site-directed mutagenesis. Substitution of Glu216 with a residue other than Asp strongly attenuated the binding of quinidine, bufuralol, and several other P450 2D6 ligands. Catalytic activity with the substrates bufuralol and 4-methoxyphenethylamine was strongly inhibited by neutral or basic mutations at Glu216 (>95%), to the same extent as the substitution of Asn at Asp301. Unlike the Asp301 mutants, the Gln216 mutant (E216Q) retained 40% enzyme efficiency with the substrate spirosulfonamide, devoid of basic nitrogen, suggesting that the substitutions at Glu216 affect binding of amine substrates more than other catalytic steps. Attempts to induce catalytic specificity toward new substrates by substitutions at Asp301 and Glu216 were unsuccessful. Collectively, the results provide evidence for electrostatic interaction of amine substrates with Glu216, and we propose that both of these acidic residues plus at least another residue(s) is (are) involved in binding the repertoire of P450 2D6 ligands.     

Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic Acid

Amylose-conjugated linoleic acid (CLA) complexes were produced by water/dimethyl sulfoxide (DMSO) and KOH/HCl complexation methods. The formation of amylose V form was confirmed by X-ray diffraction (XRD), and complexes formed at 30, 60, and 90 degrees C exhibit melting temperatures exceeding 88 degrees C. Atomic force microscopy (AFM) images showed distinct difference in complex organization, with complexes formed in water/DMSO showing spherical shape with typical diameter of 150 nm. Complexes formed by KOH/HCl showed elongated structure with typical width of 43-160 nm. Water/DMSO complexes exhibit superior protection to CLA against oxidation. All complexes showed high retention of CLA in simulated stomach conditions, and the digestion of complexes by amylases results in high hydrolysis and CLA release by pancreatin and alpha-amylase. Only moderate release was detected following hydrolysis by amyloglucosidase and beta-amylase. It is therefore suggested that amylose-CLA complexes can serve as molecular nanocapsules for protection and delivery of CLA.     

Subclavian artery stenting: Immediate and mid term clinical follow-up results

BACKGROUND: Intravascular stents are increasingly being used to treat subclavian artery obstructive disease. This study aimed to assess the immediate and mid-term clinical outcome of subclavian artery stenting. METHODS AND RESULTS: Total occlusion of the subclavian artery was seen in 7 (28%) out of the 25 consecutive patients treated for subclavican artery stenosis. Mean lesion length was 14 +/- 4.3 mm. The mean preprocedure diameter stenosis was reduced from 83.2 +/- 14.9% to 9.6 +/- 5.4% postprocedure. Procedural success was achieved in all patients. Clinical follow-up was obtained in all patients. The initial success was maintained at follow-up (mean = 12 +/- 4 months) in 24 (96%) patients. Recurrence of symptoms occurred in 1 (4%) patient who had an angiographically documented restenosis four months after the procedure. It was successfully redilated. CONCLUSION: Stenting for subclavian artery obstructive disease is safe, technically feasible and has favorable clinical outcomes. It may be considered as the therapy of choice for subclavian artery obstructive disease.     

Non-hydraulic signals from maize roots in drying soil: inhibition of leaf elongation but not stomatal conductance

Conditions of soil drying and plant growth that lead to non-hydraulic inhibition of leaf elongation and stomatal conductance in maize (Zea mays L.) were investigated using plants grown with their root systems divided between two containers. The soil in one container was allowed to dry while the other container was kept well-watered. Soil drying resulted in a maximum 35% inhibition of leaf elongation rate which occurred during the light hours, with no measurable decline in leaf water potential (_w). Leaf area was 15% less than in control plants after 18 d of soil drying. The inhibition of elongation was observed only when the soil _w declined to below that of the leaves and, thus, the drying soil no longer contributed to transpiration. However, midday root _w in the dry container (-0.29 MPa) remained much higher than that of the surrounding soil (-1.0 MPa) after 15 d of drying, indicating that the roots in drying soil were rehydrated in the dark.To prove that the inhibition of leaf elongation was not caused by undetectable changes in leaf water status as a result of loss of half the watergathering capacity, one-half of the root system of control plants was excised. This treatment had no effect on leaf elongation or stomatal conductance. The inhibition of leaf elongation was also not explained by reductions in nutrient supply.Soil drying had no effect on stomatal conductance despite variations in the rate or extent of soild drying, light, humidity or nutrition. The results indicate that non-hydraulic inhibition of leaf elongation may act to conserve water as the soil dries before the occurrence of shoot water deficits.Symbol _w water potential Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 10881     

Intra-Aortic Balloon Pump Counterpulsation during Primary Percutaneous Coronary Intervention for ST-Elevation Myocardial Infarction and Cardiogenic Shock: Insights from the British Columbia Cardiac Registry

Background

Cardiogenic shock complicating ST-elevation myocardial infarction (STEMI) is associated with significant morbidity and mortality. In the primary percutaneous coronary intervention (PPCI) era, randomized trials have not shown a survival benefit with intra-aortic balloon pump (IABP) therapy. This differs to observational data which show a detrimental effect, potentially reflecting bias and confounding. Without robust and valid risk adjustment, findings from non-randomized studies may remain biased.

Methods

We compared long-term mortality following IABP therapy in patients with cardiogenic shock undergoing PPCI during 2008–2013 from the British Columbia Cardiac Registry. We addressed measured and unmeasured confounding using propensity score and instrumental variable methods.

Results

A total of 12,105 patients with STEMI were treated with PPCI during the study period. Of these, 700 patients (5.8%) had cardiogenic shock. Of the patients with cardiogenic shock, 255 patients (36%) received IABP therapy. Multivariable analyses identified IABP therapy to be associated with increased mortality up to 3 years (HR = 1.67, 95% CI:1.20–2.67, p<0.001). This association was lost in propensity-matched analyses (HR = 1.23, 95% CI: 0.84–1.80, p = 0.288). When addressing measured and unmeasured confounders, instrumental variable analyses demonstrated that IABP therapy was not associated with mortality at 3 years (Δ = 16.7%, 95% CI: -12.7%, 46.1%, p = 0.281). Subgroup analyses demonstrated IABP was associated with increased mortality in non-diabetics; patients not undergoing multivessel intervention; patients without renal disease and patients not having received prior thrombolysis.

Conclusions

In this observational analysis of patients with STEMI and cardiogenic shock, when adjusting for confounding, IABP therapy had a neutral effect with no association with long-term mortality. These findings differ to previously reported observational studies, but are in keeping with randomized trial data.     

Computational Tension Mapping of Adherent Cells Based on Actin Imaging

Forces transiting through the cytoskeleton are known to play a role in adherent cell activity. Up to now few approaches haves been able to determine theses intracellular forces. We thus developed a computational mechanical model based on a reconstruction of the cytoskeleton of an adherent cell from fluorescence staining of the actin network and focal adhesions (FA). Our custom made algorithm converted the 2D image of an actin network into a map of contractile interactions inside a 2D node grid, each node representing a group of pixels. We assumed that actin filaments observed under fluorescence microscopy, appear brighter when thicker, we thus presumed that nodes corresponding to pixels with higher actin density were linked by stiffer interactions. This enabled us to create a system of heterogeneous interactions which represent the spatial organization of the contractile actin network. The contractility of this interaction system was then adapted to match the level of force the cell truly exerted on focal adhesions; forces on focal adhesions were estimated from their vinculin expressed size. This enabled the model to compute consistent mechanical forces transiting throughout the cell. After computation, we applied a graphical approach on the original actin image, which enabled us to calculate tension forces throughout the cell, or in a particular region or even in single stress fibers. It also enabled us to study different scenarios which may indicate the mechanical role of other cytoskeletal components such as microtubules. For instance, our results stated that the ratio between intra and extra cellular compression is inversely proportional to intracellular tension.     

Complex Interactions Between Polyamines and Calpain-Mediated Proteolysis in Rat Brain

Polyamine synthesis is induced by various extracellular signals, and it is widely held that this biochemical response participates in cell growth and differentiation. Certain of the triggers for synthesis in brain tissues also increase the breakdown of high-molecular-weight structural proteins, apparently by activating calcium-dependent proteases (calpains). The present experiments tested the possibility that calpain activity is modulated by polyamines. Spermine, spermidine, and putrescine all increased calcium-dependent proteolysis of [14C]casein by soluble fractions of rat brain. The order of potency was spermine greater than spermidine greater than putrescine, with apparent affinities of 30, 300, and 6,000 microM, respectively. Each of the three polyamines at physiological concentrations also potentiated the calcium-dependent breakdown of two endogenous high-molecular-weight structural proteins known to be substrates of calpain, in both supernatant and membrane fractions. The thiol protease inhibitor leupeptin, a known calpain inhibitor, also inhibited calcium-dependent proteolysis in the presence and absence of polyamines. The polyamines did not increase the activity of purified calpain I or calpain II determined with either [14C]casein or purified spectrin as the substrate, nor did they interfere with the inhibitory effects of calpastatin, an endogenous inhibitor of calpain. However, polyamines potentiated the stimulation of endogenous but not purified calpain activity produced by an endogenous calpain activator. These results suggest a role for polyamines in protein degradation as well as protein synthesis.     

Model uncertainty analysis using data analytics for life-cycle assessment (LCA) applications

Purpose

Objective uncertainty quantification (UQ) of a product life-cycle assessment (LCA) is a critical step for decision-making. Environmental impacts can be measured directly or by using models. Underlying mathematical functions describe a model that approximate the environmental impacts during various LCA stages. In this study, three possible uncertainty sources of a mathematical model, i.e., input variability, model parameter (differentiate from input in this study), and model-form uncertainties, were investigated. A simple and easy to implement method is proposed to quantify each source.

Methods

Various data analytics methods were used to conduct a thorough model uncertainty analysis; (1) Interval analysis was used for input uncertainty quantification. A direct sampling using Monte Carlo (MC) simulation was used for interval analysis, and results were compared to that of indirect nonlinear optimization as an alternative approach. A machine learning surrogate model was developed to perform direct MC sampling as well as indirect nonlinear optimization. (2) A Bayesian inference was adopted to quantify parameter uncertainty. (3) A recently introduced model correction method based on orthogonal polynomial basis functions was used to evaluate the model-form uncertainty. The methods are applied to a pavement LCA to propagate uncertainties throughout an energy and global warming potential (GWP) estimation model; a case of a pavement section in Chicago metropolitan area was used.

Results and discussion

Results indicate that each uncertainty source contributes to the overall energy and GWP output of the LCA. Input uncertainty was shown to have significant impact on overall GWP output; for the example case study, GWP interval was around 50%. Parameter uncertainty results showed that an assumption of ±?10% uniform variation in the model parameter priors resulted in 28% variation in the GWP output. Model-form uncertainty had the lowest impact (less than 10% variation in the GWP). This is because the original energy model is relatively accurate in estimating the energy. However, sensitivity of the model-form uncertainty showed that even up to 180% variation in the results can be achieved due to lower original model accuracies.

Conclusions

Investigating each uncertainty source of the model indicated the importance of the accurate characterization, propagation, and quantification of uncertainty. The outcome of this study proposed independent and relatively easy to implement methods that provide robust grounds for objective model uncertainty analysis for LCA applications. Assumptions on inputs, parameter distributions, and model form need to be justified. Input uncertainty plays a key role in overall pavement LCA output. The proposed model correction method as well as interval analysis were relatively easy to implement. Research is still needed to develop a more generic and simplified MCMC simulation procedure that is fast to implement.