Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.     

Lipids and Antiplatelet Therapy: Important Considerations and Future Perspectives

Lipids play an essential role in platelet functions. It is known that polyunsaturated fatty acids play a role in increasing platelet reactivity and that the prothrombotic phenotype plays a crucial role in the occurrence of major adverse cardiovascular events. The ongoing increase in cardiovascular diseases’ incidence emphasizes the importance of research linking lipids and platelet function. In particular, the rebound phenomenon that accompanies discontinuation of clopidogrel in patients receiving dual antiplatelet therapy has been associated with changes in the lipid profile. Our many years of research underline the importance of reduced HDL values for the risk of such a rebound effect and the occurrence of thromboembolic events. Lipids are otherwise a heterogeneous group of molecules, and their signaling molecules are not deposited but formed “on-demand” in the cell. On the other hand, exosomes transmit lipid signals between cells, and the profile of such changes can be monitored by lipidomics. Changes in the lipid profile are organ-specific and may indicate new drug action targets.     

Novel strategy for encapsulation and targeted delivery of poorly water-soluble active substances

Carriers for targeted delivery and controlled release of poorly water-soluble active substances (PWSAS) are facing three challenges: (a) the encapsulation issues, (b) limitations of PWSAS water solubility, and (c) burst drug release which can be pharmacologically dangerous and economically inefficient. The present study brings a novel strategy for encapsulation and controlled release of PWSAS—caffeine in concentrations which are higher than its maximal water solubility without the possibility of burst effect. The modification of hydrophilic carrier based on poly(methacylic acid) was done using casein and liposomes. To further increase the maximal caffeine loading inside the carrier nicotinamide was used. The release study of the encapsulated PWSAS was elaborated with respect to morphology of the carriers and interactions that could be established between its structural components. The carriers swelling and the release of caffeine and nicotinamide were also investigated depending on caffeine concentration, the presence of different liposomal formulations and the volume ratio of liposomal formulation, in three media with different pH simulating the path of the carrier through the human gastrointestinal tract. The synthesized carriers are promising candidates for encapsulation of PWSAS in concentrations which are higher than its maximal water solubility and for the targeted delivery of those dosages.     

A Potent Mimetic of the Siglec-8 Ligand 6’-Sulfo-Sialyl Lewisx

Siglecs are members of the immunoglobulin gene family containing sialic acid binding N-terminal domains. Among them, Siglec-8 is expressed on various cell types of the immune system such as eosinophils, mast cells and weakly on basophils. Cross-linking of Siglec-8 with monoclonal antibodies triggers apoptosis in eosinophils and inhibits degranulation of mast cells, making Siglec-8 a promising target for the treatment of eosinophil- and mast cell-associated diseases such as asthma. The tetrasaccharide 6’-sulfo-sialyl Lewis^x has been identified as a specific Siglec-8 ligand in glycan array screening. Here, we describe an extended study enlightening the pharmacophores of 6’-sulfo-sialyl Lewis^x and the successful development of a high-affinity mimetic. Retaining the neuraminic acid core, the introduction of a carbocyclic mimetic of the Gal moiety and a sulfonamide substituent in the 9-position gave a 20-fold improved binding affinity. Finally, the residence time, which usually is the Achilles tendon of carbohydrate/lectin interactions, could be improved.     

The effect of different pollutants exposure on the pigment content of pigmented macrophage aggregates in the spleen of Vardar chub (Squalius vardarensis Karaman, 1928)

Pigmented macrophage aggregates (MAs) are known to change under influence of various factors, such as aging, season, starvation, and/or pollution. In this study, changes in the pigment content of the MAs in the spleen of Vardar chub (Squalius vardarensis, Karaman) (n = 129) collected in spring and autumn, from three rivers with different pollution impact was examined: Zletovska River (metals), Kriva River (metals and municipal wastewater), and Bregalnica River (municipal wastewater). Collected data revealed increased relative volume and number of MAs containing hemosiderin under the influence of metals, significant in autumn (p < .05). In chub exposed to metals combined with municipal wastewater, significant increase of lipochrome accumulation in MAs in autumn, melanin in MAs in fish captured in the spring season, and number of splenic MAs containing combination of melanin and lipochrome was noted. Volumes and number of MAs containing combination of hemosiderin and lipochrome increased in spleen of fish captured in autumn from both Zletovska River and Kriva River, most likely due to the contribution of hemosiderin and lipochrome, respectively. Values measured for the various pigments in splenic MAs in fish captured from Bregalnica River, were overall closer to the values measured for fish captured from Kriva River. Notably, melanin and lipochrome are more likely to be found in fish from waters influenced by municipal wastewater (organic pollution) and hemosiderin in fish spleen from water influenced by mining activity (heavy metals pollution).     

Characterisation of flavour compounds in Biska – a herbal spirit produced with mistletoe

Mistletoe (Viscum album) is mostly used in its herbal form owing to its content of diverse biologically active substances. In Croatia's Adriatic region, it is used for production of Biska, a strong alcoholic beverage produced by maceration of mistletoe in spirit or brandy, with or without the addition of honey. In this work, Biska was characterised by analysis of aroma compounds and physicochemical parameters and is the first study of the aroma profile of a strong alcoholic beverage produced with mistletoe. The aroma of 14 samples of Biska – five commercial and nine homemade – was analysed using GC/MS with solid‐phase microextraction. A total of 166 aroma compounds were detected. Major components were ethyl esters (medium and long chain fatty acids), fatty alcohols, isopropyl myristate, aldehyde decanal and some terpenes. The distribution of aroma compounds was evaluated by principal component analysis. Some significant differences in physicochemical properties were observed including ethanol concentration (28–44% v/v) and pH (4.4–6.5). Although the colour of Biska was light brown with greenish reflections, there were differences in chromatic parameters. The commercial samples were darker and were less transparent than homemade Biska, perhaps reflecting more extensive maceration/extraction or the addition of caramel. © 2018 The Institute of Brewing & Distilling