Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization

Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at −20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process

To Assess the Association between Glucose Metabolism and Ectopic Lipid Content in Different Clinical Classifications of PCOS - Fig 3

<p>Bee swarm plot of ectopic lipids in different subgroups: controls (CONT), females with PCOS classified by NIH 1999 criteria (PCOS-NIH) as well as females with PCOS additionally classified by the Rotterdam 2003 criteria (PCOS-ROT): A: Hepatocellular lipids (HCL), B: intramyocellular lipids (IMCL) in soleus muscle, C: intramyocellular lipids (IMCL) in tibialis muscle. Lines indicate first, second (median) and third quartiles.</p

Sensitivity analysis excluding 10 controls with systemic contraceptive agents during the study period.

<p>Sensitivity analysis excluding 10 controls with systemic contraceptive agents during the study period.</p

Summary of missing OGTT data in PCOS and controls.

<p>Summary of missing OGTT data in PCOS and controls.</p

Comparision of glucometabolic parameters.

<p>Comparision of glucometabolic parameters.</p

Correlation map representing the amount of association of clinical and metabolic parameters with hepatic (HCL) and intramyocellular (IMCL) lipid content of females with PCOS.

<p>The magnitude of correlation is indicated by the shape of the ellipses (circle represents no correlation) and color (dark color represents higher correlation; positive = blue, negative = red).</p

Estimator of nonparametric relative effects and 95% simultaneous confidence intervals (PCOS phenotypes vs. subgroups) for HOMA-IR (homeostatic model assessment of insulin resistance); ISI-Comp (Composite index), AUC-I (area under the concentration curve of insulin) as well as Sec-Total (total insulin secretion) for PCOS subgroups and controls, respectively.

<p>A: ovulatory dysfunction + hyperandogenism + polycystic ovaries (n = 28); B: ovulatory dysfunction + hyperandrogenism (n = 7); C: ovulatory dysfunction + polycystic ovary (n = 11); D: hyperandrogenism + polycystic ovary (n = 7); cont: controls (n = 20).</p

Spaghetti plots of plasma glucose, insulin and C-Peptide dynamics during a 2h-oral glucose tolerance test (OGTT) in control subjects, patients with PCOS classified by NIH 1999 criteria (PCOS-NIH) as well as patients with PCOS additionally classified by the Rotterdam 2003 criteria (PCOS-ROT).

<p>Spaghetti plots of plasma glucose, insulin and C-Peptide dynamics during a 2h-oral glucose tolerance test (OGTT) in control subjects, patients with PCOS classified by NIH 1999 criteria (PCOS-NIH) as well as patients with PCOS additionally classified by the Rotterdam 2003 criteria (PCOS-ROT).</p

Basic characteristics of the study sample.

<p>Basic characteristics of the study sample.</p

A structured open dataset of government interventions in response to COVID-19

International audienceIn response to the COVID-19 pandemic, governments have implemented a wide range of non-pharmaceutical interventions (NPIs). Monitoring and documenting government strategies during the COVID-19 crisis is crucial to understand the progression of the epidemic. Following a content analysis strategy of existing public information sources, we developed a specific hierarchical coding scheme for NPIs. We generated a comprehensive structured dataset of government interventions and their respective timelines of implementation. To improve transparency and motivate collaborative validation process, information sources are shared via an open library. We also provide codes that enable users to visualise the dataset. Standardization and structure of the dataset facilitate inter-country comparison and the assessment of the impacts of different NPI categories on the epidemic parameters, population health indicators, the economy, and human rights, among others. This dataset provides an in-depth insight of the government strategies and can be a valuable tool for developing relevant preparedness plans for pandemic. We intend to further develop and update this dataset until the end of December 2020