Generation of carrier peptides for the delivery of nucleic acid drugs in primary cells

Now that the human genome has been decoded, the demand for novel therapeutic concepts, such as gene and stem cell therapy, is higher than ever before. Although new and better pharmaceutical agents are available, their efficient delivery to the intracellular site of action is still a serious challenge. A possible solution to this problem is the use of cell-penetrating peptides as delivery vectors, including derivatives of human calcitonin (hCT). The aim of this study was to synthesise novel branched hCT-derived peptides for the noncovalent delivery of nucleic acids. The uptake of the resulting oligocationic peptides into various cell lines as well as primary cells was monitored by fluorescence microscopy. To determine the appropriate peptide-plasmid charge ratios for efficient cell transfection, electromobility shift assays were carried out. Finally, flow cytometric and fluorescence microscopic studies of gene expression highlighted two novel hCT-derived peptides as highly effective in the delivery of noncovalently complexed plasmid DNA. Thus, the absence of cytotoxicity paired with highly efficient cell internalisation and transfection rates, in primary cells as well, make both peptides powerful candidates as drug delivery vectors, especially for plasmid DNA, for both in vivo and ex vivo therapeutic applications.     

Loliolide,a New Therapeutic Option for Neurological Diseases? In Vitro Neuroprotective and Anti-Inflammatory Activities of a Monoterpenoid Lactone Isolated from Codium tomentosum

Parkinsons Disease (PD) is the second most common neurodegenerative disease worldwide, and is characterized by a progressive degeneration of dopaminergic neurons. Without an effective treatment, it is crucial to find new therapeutic options to fight the neurodegenerative process, which may arise from marine resources. Accordingly, the goal of the present work was to evaluate the ability of the monoterpenoid lactone Loliolide, isolated from the green seaweed Codium tomentosum, to prevent neurological cell death mediated by the neurotoxin 6-hydroxydopamine (6-OHDA) on SH-SY5Y cells and their anti-inflammatory effects in RAW 264.7 macrophages. Loliolide was obtained from the diethyl ether extract, purified through column chromatography and identified by NMR spectroscopy. The neuroprotective effects were evaluated by the MTT method. Cells’ exposure to 6-OHDA in the presence of Loliolide led to an increase of cells’ viability in 40%, and this effect was mediated by mitochondrial protection, reduction of oxidative stress condition and apoptosis, and inhibition of the NF-kB pathway. Additionally, Loliolide also suppressed nitric oxide production and inhibited the production of TNF-α and IL-6 pro-inflammatory cytokines. The results suggest that Loliolide can inspire the development of new neuroprotective therapeutic agents and thus, more detailed studies should be considered to validate its pharmacological potential.     

Vancomycin dimerization and chiral recognition studied by high-performance liquid chromatography

The retention and separation of D,L-dansylvaline enantiomers (used as test solutes) were investigated using silica gel as stationary phase and vancomycin as chiral mobile-phase additive. A retention model was developed to describe the mechanistic aspects of the interaction between solute and vancomycin in the chromatographic system. It considered the formation of vancomycin dimers both "free" in the mobile phase and adsorbed on silica. By fitting the model equation to experimental data, it appeared clearly that the approach taking into account the vancomycin dimerization described accurately the retention behavior of the compounds. The examination of the model equation parameters showed that the glycopeptide dimerization increased the enantioselectivity by a factor of approximately 3.7. This study demonstrated the preponderant role of the vancomycin dimerization on the chiral recognition process of D,L-dansylvaline. Also, an additional analysis on a vancomycin chiral stationary phase indicated that the addition of vancomycin in the mobile phase promoted a greater enantioselectivity mediated by the formation of dimers in the stationary phase.     

Bax-induced cell death in Candida albicans

Bax is a pro-apoptotic member of the Bcl-2 family of proteins involved in the regulation of genetically programmed cell death in mammalian cells. It has been shown that heterologous expression of Bax in several yeast species, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris, also induces cell death. In this study we investigated the effects of Bax expression in the pathogenic yeast Candida albicans. Cell death inducing expression of Bax required a synthetic BAX gene that was codon-optimized for expression in Candida albicans. Expression of this BAX gene resulted in growth inhibition and cell death. By fusing Bax with the yeast enhanced green fluorescent protein of Aequoria victoria, the cell death-inducing effect of Bax was increased due to reduced proteolytic degradation of Bax. Using this fusion protein we showed that, upon expression in C. albicans, Bax co-localizes with the mitochondria. Furthermore, we showed for the first time that expression of Bax in yeast causes the mitochondria, which are normally distributed throughout the cell, to cluster in the perinuclear region.     

Soil hydraulic parameters estimated from satellite information through data assimilation

Leaf area index (LAI) and actual evapotranspiration (ET_a) from satellite observations were used to estimate simultaneously the soil hydraulic parameters of four soil layers down to 60 cm depth using the combined soil water atmosphere plant and genetic algorithm (SWAP–GA) model. This inverse model assimilates the remotely sensed LAI and/or ET_a by searching for the most appropriate sets of soil hydraulic parameters that could minimize the difference between the observed and simulated LAI (LAI_sim) or simulated ET_a (ET_asim). The simulated soil moisture estimates derived from soil hydraulic parameters were validated using values obtained from soil moisture sensors installed in the field. Results showed that the soil hydraulic parameters derived from LAI alone yielded good estimations of soil moisture at 3 cm depth; LAI and ET_a in combination at 12 cm depth, and ET_a alone at 28 cm depth. There appeared to be no match with measurement at 60 cm depth. Additional information would therefore be needed to better estimate soil hydraulic parameters at greater depths. Despite this inability of satellite data alone to provide reliable estimates of soil moisture at the lowest depth, derivation of soil hydraulic parameters using remote sensing methods remains a promising area for research with significant application potential. This is especially the case in areas of water management for agriculture and in forecasting of floods or drought on the regional scale.     

Method for short-term prediction of milk yield at the quarter level to improve udder health monitoring

Udder health problems are often associated with milk losses. These losses are different between quarters, as infected quarters are affected both by systemic and pathogen-specific local effects, whereas noninfected quarters are only subject to systemic effects. To gain insight in these losses and the milk yield dynamics during disease, it is essential to have a reliable reference for quarter-level milk yield in an unperturbed state, mimicking its potential yield. We developed a novel methodology to predict this quarter milk yield per milking session, using an historical data set of 504 lactations collected on a test farm by an automated milking system from DeLaval (Tumba, Sweden). Using a linear mixed model framework in which covariates associated with the linearized Wood model and the milking interval are included, we were able to describe quarter-level yield per milking session with a proportional error below 10%. Applying this model enables us to predict the milk yield of individual quarters 1 to 50 d ahead with a mean prediction error ranging between 8 and 20%, depending on the amount of historical data available to estimate the random effect covariates for the predicted lactation. The developed methodology was illustrated using 2 examples for which quarter-level milk losses are calculated during clinical mastitis. These showed that the quarter-level mixed model allows us to gain insight in quarter lactation dynamics and enables to calculate milk losses in different situations.     

Plasmachemisches Ätzen und Beschichten bei Atmosphärendruck

Atmospheric pressure plasma‐chemical etching and deposition. Application in crystalline silicon photovoltaics. For industrial processing of wafer based crystalline silicon solar cells a variety of different technologies are applied. The combination of these requires a complex wafer handling; increasing not only investment costs, but also the risk of wafer breakage. Application of plasma technologies offers the possibility to manufacture crystalline silicon solar cells without any wet chemical or vacuum processes. At Fraunhofer IWS all etching steps necessary for the production of solar cells and the deposition of silicon nitride as passivation and anti‐reflection coating were demonstrated successfully using atmospheric pressure plasma technologies.     

Adolescents' and mothers' perceptions of the cognitive and relational functions of collaboration and adjustment in dealing with type 1 diabetes.

We examined how adolescents' and mothers' perceptions of cognitive and interpersonal functions of collaboration surrounding Type 1 diabetes differed and how they related to adolescent age, psychosocial, and diabetes adjustment. Adolescents (M = 14.16 years) and mothers completed questionnaires assessing the functions of collaboration for the adolescent, quality of the mother-adolescent relationship, emotional adjustment, and adherence to the diabetes regimen. Confirmatory factor analysis (CFA) of perceptions of collaboration confirmed three dimensions: collaboration to compensate for cognitive function, interpersonal enjoyment, and frequency of collaboration. Although adolescents' and mothers' views of interpersonal enjoyment and frequency of collaboration converged, their perceptions of compensation were unrelated. Mothers' perceptions of adolescents' compensation were unrelated to perceptions of enjoyment and frequency. Both adolescents and mothers perceived less compensation and less enjoyment of collaboration with increasing adolescent age, but only adolescents reported collaborating less frequently with higher age. Better emotional adjustment and adherence occurred when mothers and adolescents perceived enjoying collaboration (controlling for mothers' acceptance) regardless of age. Enjoying collaboration at an interpersonal level may be an important avenue for successful diabetes management during adolescence. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Composition and quality attributes of conventionally and organically farmed Pangasius fillets (Pangasius hypophthalmus) on the German market

A range of conventionally and organically farmed Pangasius or sutchi catfish fillets available on the German market were analysed to compare both composition and quality. Differentiation of Pangasius hypophthalmus from Pangasius bocourti was achieved by RFLP-SSCP analysis. The protein content of conventionally farmed fillets ranged between 13.3 and 15.7%, whereas organically produced fillets had significantly higher protein contents of between 17.0 and 17.4%. No difference in the fat content between farming methods was observed, which varied between 1.4 and 3.2%. Polyunsaturated fatty acids represented about 24% of the total fatty acids with a high level of linoleic acid. The comparison of the proximate composition indicated that water was added to most of the conventionally farmed products, in different amounts, as well as water-binding capacity enhancing additives. Differential scanning calorimetry was used to demonstrate the presence of polyphosphate on muscle proteins. Differences in texture, water-binding capacity and colour are discussed.     

Building up PtII−Thiosemicarbazone−Lysine−sC18 Conjugates

Three chiral tridentate N^N^S coordinating pyridine-carbaldehyde (S)-N4-(α-methylbenzyl)thiosemicarbazones (HTSCmB) were synthesised along with lysine-modified derivatives. One of them was selected and covalently conjugated to the cell-penetrating peptide sC18 by solid-phase peptide synthesis. The HTSCmB model ligands, the HTSCLp derivatives and the peptide conjugate rapidly and quantitatively form very stable Pt^II chlorido complexes [Pt(TSC)Cl] when treated with K₂PtCl₄ in solution. The Pt(CN) derivatives were obtained from one TSCmB model complex and the peptide conjugate complex through Cl⁻→CN⁻ exchange. Ligands and complexes were characterised by NMR, IR spectroscopy, HR-ESI-MS and single-crystal XRD. Intriguingly, no decrease in cell viability was observed when testing the biological activity of the lysine-tagged HdpyTSCLp, its sC18 conjugate HdpyTSCL-sC18 or the PtCl and Pt(CN) conjugate complexes in three different cell lines. Thus, given the facile and effective preparation of such Pt-TSC-peptide conjugates, these systems might pave the way for future use in late-stage labelling with Pt radionuclides and application in nuclear medicine.