Spray-dried voriconazole–cyclodextrin complexes: Solubility,dissolution rate and chemical stability

The present work investigates the effect of complexation with hydroxypropyl-beta-cyclodextrin (HPBCD) and 2-O-methyl-beta-cyclodextrin (2-O-MBCD), on voriconazole solubility, dissolution rate and chemical stability. Drug–cyclodextrin complexes were prepared as aqueous solutions, which were spray-dried, and their properties were compared to wet ground samples and physical mixtures. DSC analysis revealed absence of crystalline voriconazole from spray-dried complexes. FTIR spectroscopy indicated changes in the H-bonding network of the hydroxyl groups of cyclodextrin following drug inclusion. Dissolution rate of voriconazole was significantly higher from spray-dried complexes with either cyclodextrin in comparison with free drug, physical mixtures, or wet ground mixtures. However, two degradation impurities were found in aged samples, with slightly higher impurity level with HPBCD. Performed solubility studies suggested that 2-O-MBCD is more efficient solubilizer. Molecular docking simulations showed a difference in the 1:1 binding affinities and sites, with HPBCD surprisingly forming complexes of much lower energy, thus suggesting a multiple rather than a 1:1 complexation.     

Metformin inhibits melanoma development through autophagy and apoptosis mechanisms

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.     

Phytochemical Investigation of Cuscuta campestris Yunck. Stem Extract and Evaluation of Its Bioherbicidal Effect on Amaranthus retroflexus L. and Portulaca oleracea L

This study focused on characterizing chemically and evaluating in vitro allelopathic and bioherbicidal potential of secondary metabolites extracted from the stem of Cuscuta campestris in seed germination, early seedling growth and early plant growth of Amaranthus retroflexus and Portulaca oleracea. The combined effects of stem extract and a reduced dose of herbicide metribuzin were also examined. Plant extract contained 17 phenolic compounds and the most abundant phenols were flavonoids: quercetin, (+)-catechin, daidzin, luteolin, and rutin. The seeds of P. oleracea were less sensitive than the seeds of A. retroflexus. The seed bioassay confirmed the inhibitory effect of stem extract on germination and early growth of both weed seedlings at concentrations of 0.75 % and 1 %, and a minor inhibitory effect in the plant bioassay. On the other hand, a synergy of C. campestris stem extract and metribuzin was revealed, as their combination achieved better results in the control of both weed species. Based on obtained data C. campestris stem extract could be a potential source of natural-based weed control molecules.     

Adaptive spatio-temporal denoising of fluoroscopic X-ray sequences

Lowering the cumulative radiation dose to a patient undergoing fluoroscopic examination requires efficient denoising algorithms. We propose a method, which extensively utilizes temporal dimension in order to maximize denoising efficiency. A set of subsequent images is processed and two estimates of denoised images are calculated. One is based on a special implementation of an adaptive edge preserving wavelet transform, while the other is based on the statistical method intersection of confidence intervals (ICI) rule. Wavelet transform is thought to produce high quality denoised images and ICI estimate can be used to further improve denoising performance about object edges. The estimates are fused to produce the final denoised image. We show that the proposed method performs very well and do not suffer from blurring in clinically important parts of images. As a result, its application could allow for significant lowering of the fluoroscope single frame dose.     

Developmental shaping of dendritic arbors in Drosophila relies on tightly regulated intra-neuronal activity of protein kinase A (PKA)

Dendrites develop morphologies characterized by multiple levels of complexity that involve neuron type specific dendritic length and particular spatial distribution. How this is developmentally regulated and in particular which signaling molecules are crucial in the process is still not understood. Using Drosophila class IV dendritic arborization (da) neurons we test in vivo the effects of cell-autonomous dose-dependent changes in the activity levels of the cAMP-dependent Protein Kinase A (PKA) on the formation of complex dendritic arbors. We find that genetic manipulations of the PKA activity levels affect profoundly the arbor complexity with strongest impact on distal branches. Both decreasing and increasing PKA activity result in a reduced complexity of the arbors, as reflected in decreased dendritic length and number of branching points, suggesting an inverted U-shape response to PKA. The phenotypes are accompanied by changes in organelle distribution: Golgi outposts and early endosomes in distal dendritic branches are reduced in PKA mutants. By using Rab5 dominant negative we find that PKA interacts genetically with the early endosomal pathway. We test if the possible relationship between PKA and organelles may be the result of phosphorylation of the microtubule motor dynein components or Rab5. We find that Drosophila cytoplasmic dynein components are direct PKA phosphorylation targets in vitro, but not in vivo, thus pointing to a different putative in vivo target. Our data argue that tightly controlled dose-dependent intra-neuronal PKA activity levels are critical in determining the dendritic arbor complexity, one of the possible ways being through the regulation of organelle distribution.     

Heterologous Escherichia coli Expression, Purification and Characterization of the GrmA Aminoglycoside-Resistance Methyltransferase

The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6× histidine tag with and without an enterokinase recognition producing proteins His₆-EK-GrmA and His₆-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.