Saponins from European Licorice Roots (<i>Glycyrrhiza glabra</i>)

European licorice roots (<i>Glycyrrhiza glabra</i>), used in the food and beverage industry due to their distinctive sweet and typical licorice flavor, were fractionated, with the triterpenoid saponins isolated and their chemical structures determined by means of ESIMS, ESIMS/MS, HRESIMS, and 1D/2D NMR experiments. Next to the quantitatively predominant saponin glycyrrhizin (<b>11</b>) and some previously known saponins, the structures of 10 monodesmosidic saponins were assigned unequivocally for the first time, namely, 30-hydroxyglycyrrhizin (<b>1</b>), glycyrrhizin-20-methanoate (<b>2</b>), 24-hydroxyglucoglycyrrhizin (<b>3</b>), rhaoglycyrrhizin (<b>4</b>), 11-deoxorhaoglycyrrhizin (<b>5</b>), rhaoglucoglycyrrhizin (<b>6</b>), rhaogalactoglycyrrhizin (<b>7</b>), 11-deoxo-20α-glycyrrhizin (<b>8</b>), 20α-galacturonoylglycyrrhizin (<b>9</b>), and 20α-rhaoglycyrrhizin (<b>10</b>)

A Simple, Non-Invasive Score to Predict Paroxysmal Atrial Fibrillation

<div><p>Paroxysmal atrial fibrillation (pAF) is a major risk factor for stroke but remains often unobserved. To predict the presence of pAF, we developed model scores based on echocardiographic and other clinical parameters from routine cardiac assessment. The scores can be easily implemented to clinical practice and might improve the early detection of pAF. In total, 47 echocardiographic and other clinical parameters were collected from 1000 patients with sinus rhythm (SR; n = 728), pAF (n = 161) and cAF (n = 111). We developed logistic models for classifying between pAF and SR that were reduced to the most predictive parameters. To facilitate clinical implementation, linear scores were derived. To study the pathophysiological progression to cAF, we analogously developed models for cAF prediction. For classification between pAF and SR, amongst 12 selected model parameters, the most predictive variables were tissue Doppler imaging velocity during atrial contraction (TDI, A’), left atrial diameter, age and aortic root diameter. Models for classifying between pAF and SR or between cAF and SR showed areas under the ROC curves of 0.80 or 0.93, which resembles classifiers with high discriminative power. The novel risk scores were suitable to predict the presence of pAF based on variables readily available from routine cardiac assessment. Modelling helped to quantitatively characterize the pathophysiologic transition from SR via pAF to cAF. Applying the scores may improve the early detection of pAF and might be used as decision aid for initiating preventive interventions to reduce AF-associated complications.</p></div

Classification performance of linear model scores.

<p>Sensitivities (red) and specificities (grey) are indicated for different values of the pAF/SR classification scores with 12 or 4 parameters. Threshold score values for classification with 80% sensitivity are indicated (L₁₂ = 58.35 for the model with 12 variables and L₄ = 63.32 for the model with 4 variables, error bars: standard deviations, shaded areas: 95% confidence intervals).</p

Odds ratios for variables of the model for classification between pAF and SR.

<p>(A) Odds ratios for the reduced logistic model with 12 variables ordered according to their magnitude. Odds ratios reflect the effects of binary variable changes, indicated by a ‘+’, or continuous variable changes by the indicated unit intervals, on the risk for the presence of pAF (error bars: 95% confidence intervals, shaded bars: echocardiographic variables). The most predictive variables can be recognized by small confidence intervals. (B) Odds ratios for the simplified logistic model that was reduced to the most predictive 4 variables as in panel A.</p

Reliable classification is possible between pAF and SR, and between cAF and SR.

<p>ROC curves are plotted for logistic models reduced to the most predictive variables for classification between pAF, cAF and SR groups after 100-fold cross-validation (areas: 95% confidence intervals). AUCs indicate reliable classification between pAF and SR (AUC = 0.80), and between cAF and SR (AUC = 0.93).</p

Comparison of demographic and clinical parameters between groups.

<p>Comparison of demographic and clinical parameters between groups.</p

Model coefficients and odds ratios for reduced logistic models.

<p>Model coefficients and odds ratios for reduced logistic models.</p

A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in <i>Pichia pastoris</i>

The heterologous expression of biosynthetic pathways for pharmaceutical or fine chemical production requires suitable expression hosts and vectors. In eukaryotes, the pathway flux is typically balanced by stoichiometric fine-tuning of reaction steps by varying the transcript levels of the genes involved. Regulated (inducible) promoters are desirable to allow a separation of pathway expression from cell growth. Ideally, the promoter sequences used should not be identical to avoid loss by recombination. The methylotrophic yeast Pichia pastoris is a commonly used protein production host, and single genes have been expressed at high levels using the methanol-inducible, strong, and tightly regulated promoter of the <i>alcohol oxidase 1</i> gene (<i>P</i>_<i>AOX1</i>). Here, we have studied the regulation of the P. pastoris methanol utilization (MUT) pathway to identify a useful set of promoters that (i) allow high coexpression and (ii) differ in DNA sequence to increase genetic stability. We noticed a pronounced involvement of the pentose phosphate pathway (PPP) and genes involved in the defense of reactive oxygen species (ROS), providing strong promoters that, in part, even outperform <i>P</i>_<i>AOX1</i> and offer novel regulatory profiles. We have applied these tightly regulated promoters together with novel terminators as useful tools for the expression of a heterologous biosynthetic pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest sets of strong and co-regulated promoters of any microbe, moving it from a protein production host to a general industrial biotechnology host

A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in <i>Pichia pastoris</i>

The heterologous expression of biosynthetic pathways for pharmaceutical or fine chemical production requires suitable expression hosts and vectors. In eukaryotes, the pathway flux is typically balanced by stoichiometric fine-tuning of reaction steps by varying the transcript levels of the genes involved. Regulated (inducible) promoters are desirable to allow a separation of pathway expression from cell growth. Ideally, the promoter sequences used should not be identical to avoid loss by recombination. The methylotrophic yeast Pichia pastoris is a commonly used protein production host, and single genes have been expressed at high levels using the methanol-inducible, strong, and tightly regulated promoter of the <i>alcohol oxidase 1</i> gene (<i>P</i>_<i>AOX1</i>). Here, we have studied the regulation of the P. pastoris methanol utilization (MUT) pathway to identify a useful set of promoters that (i) allow high coexpression and (ii) differ in DNA sequence to increase genetic stability. We noticed a pronounced involvement of the pentose phosphate pathway (PPP) and genes involved in the defense of reactive oxygen species (ROS), providing strong promoters that, in part, even outperform <i>P</i>_<i>AOX1</i> and offer novel regulatory profiles. We have applied these tightly regulated promoters together with novel terminators as useful tools for the expression of a heterologous biosynthetic pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest sets of strong and co-regulated promoters of any microbe, moving it from a protein production host to a general industrial biotechnology host

A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in <i>Pichia pastoris</i>

The heterologous expression of biosynthetic pathways for pharmaceutical or fine chemical production requires suitable expression hosts and vectors. In eukaryotes, the pathway flux is typically balanced by stoichiometric fine-tuning of reaction steps by varying the transcript levels of the genes involved. Regulated (inducible) promoters are desirable to allow a separation of pathway expression from cell growth. Ideally, the promoter sequences used should not be identical to avoid loss by recombination. The methylotrophic yeast Pichia pastoris is a commonly used protein production host, and single genes have been expressed at high levels using the methanol-inducible, strong, and tightly regulated promoter of the <i>alcohol oxidase 1</i> gene (<i>P</i>_<i>AOX1</i>). Here, we have studied the regulation of the P. pastoris methanol utilization (MUT) pathway to identify a useful set of promoters that (i) allow high coexpression and (ii) differ in DNA sequence to increase genetic stability. We noticed a pronounced involvement of the pentose phosphate pathway (PPP) and genes involved in the defense of reactive oxygen species (ROS), providing strong promoters that, in part, even outperform <i>P</i>_<i>AOX1</i> and offer novel regulatory profiles. We have applied these tightly regulated promoters together with novel terminators as useful tools for the expression of a heterologous biosynthetic pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest sets of strong and co-regulated promoters of any microbe, moving it from a protein production host to a general industrial biotechnology host