Volatile Constituents of Some Selected Plant Species Traditionally Used as Tea in the Sharri Mountains (Kosovo).

The study evaluates the chemical composition of the volatile constituents of ten plant species traditionally used as herbal tea in the Sharri Mountain regions (Kosovo and North Macedonia). Volatile constituents responsible for the flavour and fragrance of selected species (Crataegus monogyna, Cydonia oblonga, Malus sylvestris, Matricaria chamomilla, Morus alba, Morus nigra, Rosa canina, Sambucus nigra, Tilia cordata, and Vaccinium myrtillus) were separated and then identified using GC-MS, whereas GC-FID is employed for the quantitative analysis. Experimental data revealed different patterns of volatile constituents depending on plant species. Monoterpenes, sesquiterpenes, diterpenes, and norisoprenoids were the most abundant volatile constituents. Principal component analysis (PCA) was deployed for data analysis and resulted in grouping these ten species in four principal clusters. The combination of various volatile constituents present in specific plant species may play an important role in the specific aroma and taste sensation of these species used as recreational teas.

The potentially harmful excipients in prescribed medications in a Neonatal Intensive Care Unit in Kosovo and available safer alternatives.

BACKGROUND: Medicinal products contain excipients that might be associated with toxicity in neonates. The aim of this study was to investigate the administration of medication containing potentially harmful excipients to neonates hospitalized in Kosovo and to identify the possibility of reducing neonatal exposure to these excipients through product substitution. METHODS: Data on all medication administered to hospitalized neonates from 1st of February to 1st of August 2018 along with patients` demographic data were collected from medical records for each neonate. Excipients were identified from the Summaries of Product Characteristics. Three stage criteria for product substitution were: (1) same active pharmaceutical ingredient (API) and route of administration; (2) 1 plus same dosage form; (3) 1 and 2 plus same strength. RESULTS: In total, 100 excipients were found in 2388 prescriptions comprising 67 medications and 60 API administered to 294 (183 preterm and 111 term) hospitalized neonates. The excipients of interest (EOI) were present in 409 (17.1%) prescriptions and were administered to 131 (71.6%) preterm and 52 (46.8%) term neonates through a relatively small number of products (n=27; 32.8%). In relation to prescription frequency, the most common EOI was polysorbate 80, found in 229 (56%) of EOI-containing prescriptions. Substitution with EOIfree products was possible for 14 (63.6%), 12 (54.5%) and 5 (22.7%) products, according to the first-, second- and third-stage criteria, respectively. CONCLUSIONS: We have provided the first detailed description of neonatal exposure to potentially harmful excipients among neonates admitted to a neonatal intensive care unit in Kosovo. Unnecessary exposure could be reduced by using EOI-free products available in the local medicine market. Collaborative initiative is required to build up the evidence on the use of EOI in neonates and raising awareness among health care professionals on use of products without EOI where possible.

Phytochemical Study of Eight Medicinal Plants of the Lamiaceae Family Traditionally Used as Tea in the Sharri Mountains Region of the Balkans.

In the present study, eight plant species belonging to Lamiaceae family were identified as ingredients for herbal teas in the region of Sharri Mountains: Thymus serpyllum, Rosmarinus officinalis, Melissa officinalis, Origanum vulgare, Mentha longifolia, Ocimum basilicum, Teucrium chamaedrys, and Sideritis scardica, respectively. Chemical composition of essential oils obtained from these species was analyzed using GC-MS and GC-FID with the aim of examining their volatile compound profiles, responsible for their respective flavors and fragrance. Principal Component Analysis (PCA) was performed with the aim of grouping plant species under study on the basis of their chemical composition. Experimental data revealed the typical volatile constituent pattern for the Lamiaceae family. Monoterpenes and sesquiterpenes, responsible for flavor and medicinal use of these plants, were the most abundant groups of the volatile constituents. PCA data analysis resulted in the grouping of these analyzed species in four principal clusters.

Secondary Metabolite Dereplication and Phylogenetic Analysis Identify Various Emerging Mycotoxins and Reveal the High Intra-Species Diversity in Aspergillus flavus.

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.

The Glycogen Synthase Kinase-3 in the Regulation of Ion Channels and Cellular Carriers.

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-3ß) encoded by two distinct genes. Recently, GSK-3ß has been reported to function as a powerful regulator of various transport processes across the cell membrane. This kinase, GSK-3ß, either directly or indirectly, may stimulate or inhibit many different types of transporter proteins, including ion channel and cellular carriers. More specifically, GSK-3ß-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-3ß-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-3ß in many other types of cellular transporters as well as further elucidating the underlying mechanisms of GSK-3ß-mediated cellular transport regulation.

Unravelling the Diversity of the Cyclopiazonic Acid Family of Mycotoxins in Aspergillus flavus by UHPLC Triple-TOF HRMS.

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.

Essential Oil Composition of Pinus peuce Griseb. Needles and Twigs from Two National Parks of Kosovo.

The principal aim of this study was to analyze the chemical composition and qualitative and quantitative variability of essential oils obtained from seven naturally grown populations of the Pinus peuce Grisebach, Pinaceae in Kosovo. Plant materials were collected from three populations in the Sharri National Park and from four other populations in the Bjeshkët e Nemuna National Park, in Kosovo. Essential oils were obtained by steam distillation and analyzed by GC-FID (Gas Chromatography-Flame Ionization Detection) and GC-MS (Gas Chromatography-Mass Spectrometry). The results showed that the yield of essential oils (v/w dry weight) varied depending on the origin of population and the plant organs and ranged from 0.7 to 3.3%. In total, 51 compounds were identified. The main compounds were α-pinene (needles: 21.6-34.9%; twigs: 11.0-24%), ß-phellandrene (needles: 4.1-27.7; twigs: 29.0-49.8%), and ß-pinene (needles: 10.0-16.1; twigs: 6.9-20.7%). HCA (Hierarchical Cluster Analysis) and PCA (Principal Component Analyses) were used to assess geographical variations in essential oil composition. Statistical analysis showed that the analyzed populations are grouped in three main clusters which seem to reflect microclimatic conditions on the chemical composition of the essential oils.

Chemical Composition of Juniperus communis L. Cone Essential Oil and Its Variability among Wild Populations in Kosovo.

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 ß-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.

Quality control and stability studies with the monoclonal antibody, trastuzumab: application of 1D- vs. 2D-gel electrophoresis.

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.

The challenge to quantify proteins with charge trains due to isoforms or conformers.

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.

Comparison of two-dimensional gel electrophoresis patterns and MALDI-TOF MS analysis of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab.

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 analysis of therapeutic fusion protein abatacept.

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.

Comparison of aragonitic molluscan shell proteins.

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.