Jožef Stefan Institute

About: Jožef Stefan Institute is a facility organization based out in Ljubljana, Slovenia. It is known for research contribution in the topics: Liquid crystal & Dielectric. The organization has 3828 authors who have published 12614 publications receiving 291025 citations.

The Preparation of Catalytically Active Human Cathepsin B from Its Precursor Expressed in Escherichia coli in the Form of Inclusion Bodies

Abstract: A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/l E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three-amino-acid extension at its N-terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens

Abstract: The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. Here we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility (P. aureginosa only). We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. We conclude that, while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. We finally report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bioontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

$D \pi$ scattering and $D$ meson resonances from lattice QCD

Abstract: A first exploratory lattice QCD simulation is presented, aimed at extracting the masses and widths of the broad scalar ${D}_{0}^{*}(2400)$ and the axial ${D}_{1}(2430)$ charm-light resonances. For that purpose $D\ensuremath{\pi}$ and ${D}^{*}\ensuremath{\pi}$ scattering are simulated, and the resonance parameters are extracted using a Breit-Wigner fit of the resulting phase shifts. We use a single two-flavor dynamical ensemble with ${m}_{\ensuremath{\pi}}\ensuremath{\approx}266\text{ }\text{ }\mathrm{MeV}$, $a\ensuremath{\simeq}0.124\text{ }\text{ }\mathrm{fm}$ and a rather small volume $V={16}^{3}\ifmmode\times\else\texttimes\fi{}32$. The resulting ${D}_{0}^{*}(2400)$ mass is $351\ifmmode\pm\else\textpm\fi{}21\text{ }\text{ }\mathrm{MeV}$ above the spin average $\frac{1}{4}({m}_{D}+3{m}_{{D}^{*}})$, in agreement with the experimental value of $347\ifmmode\pm\else\textpm\fi{}29\text{ }\text{ }\mathrm{MeV}$ above. The resulting ${D}_{0}^{*}\ensuremath{\rightarrow}D\ensuremath{\pi}$ coupling, ${g}^{\mathrm{lat}}=2.55\ifmmode\pm\else\textpm\fi{}0.21\text{ }\text{ }\mathrm{GeV}$, is close to the experimental value ${g}^{\mathrm{exp} }\ensuremath{\le}1.92\ifmmode\pm\else\textpm\fi{}0.14\text{ }\text{ }\mathrm{GeV}$, where $g$ parametrizes the width $\ensuremath{\Gamma}\ensuremath{\equiv}{g}^{2}{p}^{*}/s$. The resonance parameters for the broad ${D}_{1}(2430)$ are also found close to the experimental values; these are obtained by appealing to the heavy quark limit, where the neighboring resonance ${D}_{1}(2420)$ is narrow. The calculated $I=1/2$ scattering lengths are ${a}_{0}=0.81\ifmmode\pm\else\textpm\fi{}0.14\text{ }\text{ }\mathrm{fm}$ for $D\ensuremath{\pi}$ and ${a}_{0}=0.81\ifmmode\pm\else\textpm\fi{}0.17\text{ }\text{ }\mathrm{fm}$ for ${D}^{*}\ensuremath{\pi}$ scattering. The simulation of the scattering in these channels incorporates quark-antiquark as well as multihadron interpolators, and the distillation method is used for contractions. In addition, the ground and several excited charm-light and charmonium states with various ${J}^{P}$ are calculated using standard quark-antiquark interpolators.

Trace-level determination of pharmaceutical residues by LC-MS/MS in natural and treated waters. A pilot-survey study

Abstract: A pilot-survey study was performed by collecting samples (influent and effluent wastewaters, rivers and tap waters) from different locations in Europe (Spain, Belgium, Germany and Slovenia). A solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry method was applied for the determination of pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid). Method detection limits and method quantification limits were at the parts-per-trillion level (7.5-75 ng/L). The recovery rates of the SPE from deionized water and effluent wastewater samples spiked at 100- and 1,000-ng/L levels ranged from 87 to 95%. Identification criteria in compliance with the EU regulation for confirmatory methods of organic residues were applied. A detailed study of signal suppression evaluation for analysis of pharmaceutical residues in effluent wastewaters is presented.