Simultaneous RP‐HPLC‐DAD Separation,and Determination of Flavonoids and Phenolic Acids in Plantago L. Species

A rapid reversed‐phase (RP) high‐performance liquid chromatography method was developed and applied for simultaneous separation, and determination of flavonoids and phenolic acids in eight Plantago L. taxa (P. altissima L., P. argentea Chaix , P. coronopus L., P. holosteum Scop . ssp. depauperata Pilger , P. holosteum ssp. holosteum, P. holosteum ssp. scopulorum (Degen) Horvati? , P. lagopus L., and P. maritima L.) growing in Croatia. Chromatographic separation was carried out on Zorbax Eclipse XDB‐C18 using gradient elution with a H₂O (pH 2.5, adjusted with CF₃COOH) and MeCN mixture at 30°. The contents of analyzed phenolic compounds (% of the dry weight of the leaves, dw) varied among examined species: rutin (max. 0.024%, P. argentea), hyperoside (max. 0.020%, P. lagopus), quercitrin (max. 0.013%, P. holosteum ssp. holosteum), quercetin (max. 0.028%, P. holosteum ssp. scopulorum), chlorogenic acid (max. 0.115%, P. lagopus), and caffeic acid (max. 0.046%, P. coronopus). Isoquercitrin was detected only in P. argentea (0.020%), while isochlorogenic acid content was below limit of quantification in all investigated species. Multivariate analyses (UPGMA and PCA) showed significant differences in contents of investigated polyphenolic compounds between different Plantago taxa. Accordingly, investigated substances might be employed as chemotaxonomic markers in the study of the complex genus Plantago.     

Variability of the Needle Essential Oils of Juniperus deltoides R.P.Adams from Different Populations in Serbia and Croatia

The essential‐oil compositions of one Croatian and three Serbian populations of Juniperus deltoides R.P.Adams have been determined by GC/MS analysis. In total, 147 compounds were identified, representing 97.3–98.3% of the oil composition. The oils were dominated by monoterpenes, which are characteristic components for the species of the section Juniperus. Two monoterpenes, α‐pinene and limonene, were the dominant constituents, with a summed‐up average content of 49.45%. Statistical methods were used to determine the diversity of the terpene classes and the common terpenes between the newly described J. deltoides populations from Serbia and Croatia. Only reports on several specimens from this species have been reported so far, and there are no studies that treat population diversity. Cluster analysis of the oil contents of 21 terpenes showed high correlation with the geographical distribution of the populations, separating the Croatian from the Serbian populations. The comparison of the essential‐oil compositions obtained in the present study with literature data, showed the separation of J. deltoides and J. oxycedrus ssp. oxycedrus from the western Mediterranean region.     

Interactions of Platinum and Ruthenium Coordination Complexes with Pancreatic Phospholipase A2 and Phospholipids Investigated by MALDI TOF Mass Spectrometry

Phospholipase A₂ is involved in propagation of inflammatory processes and carcinogenesis through its role in phospholipid metabolism, and release of arachidonic acid and lysophospholipids. Recent findings on correlation between elevated PLA₂ activity and metastatic cancer render this enzyme an attractive target for cancer therapy. On the other hand, due to a broad range of oxidation states under physiological conditions and a high affinity for protein binding, platinum and ruthenium coordination complexes are promising candidates for PLA₂ inhibitors. In this article, we discuss the interactions of Pt and Ru coordination complexes with PLA₂ and phospholipids, as well as the application of MALDI‐TOF mass spectrometry for screening PLA₂ inhibitors. Owing to the ability of this technique to simultaneously detect and monitor changes in substrate and product concentrations, the inhibitor mechanisms of both Pt and Ru complexes with various ligands were determined.     

Multi-objective optimal control of dynamic bioprocesses using ACADO Toolkit

The optimal design and operation of dynamic bioprocesses gives in practice often rise to optimisation problems with multiple and conflicting objectives. As a result typically not a single optimal solution but a set of Pareto optimal solutions exist. From this set of Pareto optimal solutions, one has to be chosen by the decision maker. Hence, efficient approaches are required for a fast and accurate generation of the Pareto set such that the decision maker can easily and systematically evaluate optimal alternatives. In the current paper the multi-objective optimisation of several dynamic bioprocess examples is performed using the freely available ACADO Multi-Objective Toolkit (http://www.acadotoolkit.org). This toolkit integrates efficient multiple objective scalarisation strategies (e.g., Normal Boundary Intersection and (Enhanced) Normalised Normal Constraint) with fast deterministic approaches for dynamic optimisation (e.g., single and multiple shooting). It has been found that the toolkit is able to efficiently and accurately produce the Pareto sets for all bioprocess examples. The resulting Pareto sets are added as supplementary material to this paper.     

Mathematical modeling of maize starch liquefaction catalyzed by α-amylases from Bacillus licheniformis: effect of calcium, pH and temperature

The first step of starch hydrolysis, i.e. liquefaction has been studied in this work. Two commercial α-amylases from Bacilllus licheniformis, known as Termamyl and Liquozyme have been used for this purpose. Using starch as the substrate, kinetics of both enzymes has been determined at optimal pH and temperature (pH 7, T = 80 °C) and at 65 °C and pH 5.5. Michaelis–Menten model with uncompetitive product inhibition was used to describe enzyme kinetics. Mathematical models were developed and validated in the repetitive batch and fed-batch reactor. Enzyme inactivation was described by the two-step inactivation model. All experiments were performed with and without calcium ions. The activities of both tested amylases are approximately one hundred times higher at 80 °C than at 65 °C. Lower inactivation rates of enzymes were noticed in the experiments performed at 65 °C without the addition of calcium than in the experiments at 80 °C. Calcium ions in the reaction medium significantly enhance amylase stability at 80 °C and pH 7. At other process conditions (65 °C and pH 5.5) a weaker calcium stabilizing effect was detected.     

Interaction of Insulin-Like Growth Factor-Binding Protein 2 with α2-Macroglobulin in the Circulation

Insulin-like growth factors (IGFs) play active role in mitogenic and metabolic processes. In the peripheral circulation, they are mostly bound to specific IGF-binding proteins (IGFBPs). Proteolysis of IGFBPs releases free, active IGFs. IGFBP-2 is the second most abundant of the six binding proteins and its concentration increases in catabolic states. The possible interaction between IGFBP-2 and other proteins in the circulation was investigated in this study. Our results showed that IGFBP-2 associates with α2-macroglobulin (α2M), a protease inhibitor. Formation of IGFBP-2/α2M complexes most likely contributes to the regulation of IGFBP-2 proteolysis and, thus, the activity of IGFs.     

Focal DNA Copy Number Changes in Neuroblastoma Target MYCN Regulated Genes

Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17∼92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17∼92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17∼92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment.     

Salinity stress response of non-transformed and AtCKX transgenic centaury (Centaurium erythraea Rafn.) shoots and roots grown in vitro

Common centaury (Centaurium erythraea Rafn.) is a plant species that can inhabit saline soils. It is known as a plant with high spontaneous regeneration potential in vitro. In the present work we evaluated shoots and roots salinity tolerance of non-transformed and three AtCKX transgenic centaury lines to graded NaCl concentrations (0, 50, 100, 150, 200 mM) in vitro. Overexpression of AtCKX genes in transgenic centaury plants resulted in an altered cytokinins (CKs) profile leading to a decline of bioactive CK levels and, at the same time, increased contents of storage CK forms, inactive CK forms and/or CK nucleotides. Significant increment of fresh shoot weight was obtained in shoots of non-transformed and AtCKX1 transgenic line only on medium supplemented with 50 mM NaCl. However two analysed AtCKX2 transgenic lines reduced shoot growth at all NaCl concentrations. In general, centaury roots showed higher tolerance to salinity than shoots. Non-transformed and AtCKX1 transgenic lines tolerated up to 100 mM NaCl without change in frequency of regeneration and number of regenerated plants. Roots of two analysed AtCKX2 transgenic lines showed different regeneration potential under salt stress. Regeneration of transgenic AtCKX2-26 shoots even at 200 mM NaCl was recorded. Salinity stress response of centaury shoots and roots was also evaluated at biochemical level. Free proline, malondialdehyde and hydrogen peroxide content as well as antioxidative enzymes activities were investigated in shoots and roots after 1, 2, 4 and 8 weeks. In general, adition of NaCl in culture medium elevated all biochemical parameters in centaury shoots and in roots. Considering that all analysed AtCKX transgenic centaury lines showed altered salt tolerance to graded NaCl concentrations in vitro it can be assumed that CKs might be involved in plant defence to salt stress conditions.