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Glycogen synthase kinase-3 (GSK-3) is a highly evolutionarily conserved and ubiquitously expressed serine/threonine kinase, an enzyme protein profoundly specific for glycogen synthase (GS). GSK-3 is involved in various cellular functions and physiological processes, including cell proliferation, differentiation, motility, and survival as well as glycogen metabolism, protein synthesis, and apoptosis. There are two isoforms of human GSK-3 (named GSK-3α and GSK-3β) encoded by two distinct genes. Recently, GSK-3β has been reported to function as a powerful regulator of various transport processes across the cell membrane. This kinase, GSK-3β, either directly or indirectly, may stimulate or inhibit many different types of transporter proteins, including ion channel and cellular carriers. More specifically, GSK-3β-sensitive cellular transport regulation involves various calcium, chloride, sodium, and potassium ion channels, as well as a number of Na+-coupled cellular carriers including excitatory amino acid transporters EAAT2, 3 and 4, high-affinity Na+ coupled glucose carriers SGLT1, creatine transporter 1 CreaT1, and the type II sodium/phosphate cotransporter NaPi-IIa. The GSK-3β-dependent cellular transport regulations are a part of the kinase functions in numerous physiological and pathophysiological processes. Clearly, additional studies are required to examine the role of GSK-3β in many other types of cellular transporters as well as further elucidating the underlying mechanisms of GSK-3β-mediated cellular transport regulation.

Aspergillus flavus is one of the most important mycotoxigenic species from the genus Aspergillus, due to its ability to synthesize the potent hepatocarcinogen, aflatoxin B1. Moreover, this fungus is capable of producing several other toxic metabolites from the class of indole-tetramates, non-ribosomal peptides, and indole-diterpenoids. Populations of A. flavus are characterized by considerable diversity in terms of morphological, functional and genetic features. Although for many years A. flavus was considered an asexual fungus, researchers have shown evidence that at best these fungi can exhibit a predominantly asexual existence. We now know that A. flavus contains functional genes for mating, uncovering sexuality as potential contributor for its diversification. Based on our results, we reconfirm that A. flavus is a predominant producer of B-type aflatoxins. Moreover, this fungus can decisively produce AFM1 and AFM2. We did not observe any clear relationship between mating-type genes and particular class of metabolites, probably other parameters such as sexual/asexual ratio should be investigated. A dynamic secondary metabolism was found also in strains intended to be used as biocontrol agents. In addition we succeeded to provide mass spectrometry fragmentation spectra for the most important classes of A. flavus metabolites, which will serve as identification cards for future studies. Both, metabolic and phylogenetic analysis proved a high intra-species diversity for A. flavus. These findings contribute to our understanding about the diversity of Aspergillus section Flavi species, raising the necessity for polyphasic approaches (morphological, metabolic, genetic, etc.) when dealing with this type of complex group of species.

Ripe cones of Juniperus communis L. (Cupressaceae) were collected from five wild populations in Kosovo, with the aim of investigating the chemical composition and natural variation of essential oils between and within wild populations. Ripe cones were collected, air dried, crushed, and the essential oils obtained by hydrodistillation. The essential-oil constituents were identified by GC-FID and GC/MS analyses. The yield of essential oil differed depending on the population origins and ranged from 0.4 to 3.8% (v/w, based on the dry weight). In total, 42 compounds were identified in the essential oils of all populations. The principal components of the cone-essential oils were α-pinene, followed by β-myrcene, sabinene, and D-limonene. Taking into consideration the yield and chemical composition, the essential oil originating from various collection sites in Kosovo fulfilled the minimum requirements for J. communis essential oils of the European Pharmacopoeia. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) were used to determine the influence of the geographical variations on the essential-oil composition. These statistical analyses suggested that the clustering of populations was not related to their geographic location, but rather appeared to be linked to local selective forces acting on the chemotype diversity.

Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.

Cyclopiazonic acid (α-cyclopiazonic acid, α-CPA) is an indole-hydrindane-tetramic acid neurotoxin produced by various fungal species, including the notorious food and feed contaminant Aspergillus flavus. Despite its discovery in A. flavus cultures approximately 40 years ago, its contribution to the A. flavus mycotoxin burden is consistently minimized by our focus on the more potent carcinogenic aflatoxins also produced by this fungus. Here, we report the screening and identification of several CPA-type alkaloids not previously found in A. flavus cultures. Our identifications of these CPA-type alkaloids are based on a dereplication strategy involving accurate mass high resolution mass spectrometry data and a careful study of the α-CPA fragmentation pattern. In total, 22 CPA-type alkaloids were identified in extracts from the A. flavus strains examined. Of these metabolites, 13 have been previously reported in other fungi, though this is the first report of their existence in A. flavus. Two of our metabolite discoveries, 11,12-dehydro α-CPA and 3-hydroxy-2-oxo CPA, have never been reported for any organism. The conspicuous presence of CPA and its numerous derivatives in A. flavus cultures raises concerns about the long-term and cumulative toxicological effects of these fungal secondary metabolites and their contributions to the entire A. flavus mycotoxin problem., Valdet Uka was financially supported by Project Basileus V (Erasmus Mundus Action 2) funding from the European Commission.

Single proteins separated by 2-DE often show multiple spots spreading along the first dimension. In many cases, such charge trains are explained by isoform differences or by putative post-translational modifications including phosphorylation, glycosylation and others. We now report that individual spots of such charge trains on 2-D gels in fact often represent the same protein, but, apparently due to conformational changes, segregate to different isoelectric points. If MS analysis reveals protein identity, we therefore suggest integrating all individual spots within a charge train for quantification. Especially in quality control of pharmaceutical proteins, the integration of the spot groups of all active contents is preferable in order to obtain reproducible and reasonable quantitative results. However, most commercial software packages for gel analysis integrate the signals spot-wise. We provide an improved quantification tool for proteins with charge train groups. This calculation can be implemented using the MATLAB software and the self-developed "Correct Integration Software System" or the commercial software package Delta2D.

Acidic macromolecules, as a nucleation factor for mollusc shell formation, are a major focus of research. It remains unclear, however, whether acidic macromolecules are present only in calcified shell organic matrices, and which acidic macromolecules are crucial for the nucleation process by binding to chitin as structural components. To clarify these questions, we applied 2D gel electrophoresis and amino acid analysis to soluble shell organic matrices from nacre shell, non-nacre aragonitic shell and non-calcified squid shells. The 2D gel electrophoresis results showed that the acidity of soluble proteins differs even between nacre shells, and some nacre (Haliotis gigantea) showed a basic protein migration pattern. Non-calcified shells also contained some moderately acidic proteins. The results did not support the correlation between the acidity of soluble shell proteins and shell structure.

The principal objective of this study was the evaluation of two-dimensional gel electrophoresis (2-DE) in combination with MALDI-TOF MS, after tryptic digest with regard to suitability for qualitative characterization and identification of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab. Moreover, the impact of post-translational modifications of these glycoproteins on the electrophoresis behavior has been evaluated. 1-D SDS-PAGE, in reducing and non-reducing conditions, and 2-DE were used for the assessment of M(r) and the monitorization of deglycosylation efficiency. In addition, 2-DE was used for the determination of pIs. 2-DE gels revealed characteristic glycoprotein migration behavior, highly complex spot pattern, typical for recombinant monoclonal antibodies. N-linked oligosaccharides were released with PNGase F; enzymatic desialination was studied with sialidase and carboxypeptidase B was used for the study of lysine truncation. Peptide spots resolved in 2-DE gels were in gel tryptically digested, resulting peptides were subjected to MALDI-TOF MS analysis and peptide mass fingerprinting (PMF) has been used for the identity confirmation of both monoclonal antibodies.

2-DE and MALDI-TOF MS are useful techniques for the quality evaluation of medicinal products derived from recombinant DNA technology. The principal objective of this study has been to evaluate the suitability of 2-DE in combination with MALDI-TOF MS for the quality study of the therapeutic recombinant protein, abatacept. 1-DE SDS-PAGE, under reducing and nonreducing conditions, and 2-DE analysis were used for the assessment of M(r) , pI, and enzymatic deglycosylation efficiency of abatacept. 2-DE allowed the assessment of product identity, purity, charge heterogeneity, isoform pattern, and post-translational modifications. Furthermore, optimization of the deglycosylation procedure, charge heterogeneity, and sample preparation for the subsequent MALDI-TOF MS analysis has been addressed. PMF analysis allowed rapid identity confirmation of abatacept.

Polyphenols are known for their antioxidant properties and as modulators of redox signalling pathways, which have a positive effect on human health. Given their superiority in terms of presence in systemic circulation and their often higher bioactivity than parent polyphenols, there is now more interest in the gut microbiota`s pivotal role in the production of low molecular weight metabolites. Thus, we have focused our study on 5-O-caffeoylquinic acid (5-CQA), a dietary polyphenol, subjected to a resting cell biotransformation study using Lactobacillus reuteri, Bacteroides fragilis and Bifidobacterium longum. Our study highlighted the ability of only L. reuteri to bioconverse 5-CQA into several metabolites, including a known natural compound esculetin. This biotransformation capacity was also evaluated in co-culture. This study put in the spotlight, for the first time, an interesting oxidative pathway carried out by gut bacteria. Electrochemical and enzymatic studies led to identify, after LC-MS/MS analysis, the oxidized compounds of 5-CQA and caffeic acid. In addition, esculetin has shown a beneficial effect on the integrity of the epithelial barrier using an original in vitro quadruple intestinal model.; Les polyphénols sont connus pour leurs propriétés antioxydantes et comme modulateurs des voies de signalisation redox, ce qui a un effet positif sur la santé humaine. Compte tenu de leur supériorité en termes de présence dans la circulation systémique et de leur bioactivité souvent plus élevée que celle des polyphénols parents, on s'intéresse aujourd'hui davantage au rôle central du microbiote intestinal dans la production de métabolites de faible poids moléculaire. Ainsi, nous avons concentré notre étude sur l'acide 5-O-caféoylquinique (5-CQA), un polyphénol alimentaire, soumis à une étude de biotransformation en utilisant Lactobacillus reuteri, Bacteroides fragilis et Bifidobacterium longum. Notre étude a mis en évidence la capacité de L. reuteri à bioconvertir le 5-CQA en plusieurs métabolites, et notamment en un composé naturel connu, l'esculétine. Cette capacité de biotransformation a été également évaluée en co-culture. Cette étude a mis en lumière, pour la première fois, une voie oxydative intéressante réalisée par les bactéries intestinales. Des études électrochimiques et enzymatiques ont permis d'identifier, après analyse par LC-MS/MS, les composés oxydés du 5-CQA et de l’acide caféique. De plus, l'esculétine a montré un effet bénéfique sur l'intégrité de la barrière épithéliale par une étude originale réalisée in vitro sur un modèle intestinal constitué de quatre types de cellules.