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.     

Microscopic oxygen imaging based on fluorescein bleaching efficiency measurements

Photobleaching of the fluorophore fluorescein in an aqueous solution is dependent on the oxygen concentration. Therefore, the time‐dependent bleaching behavior can be used to measure of dissolved oxygen concentrations. The method can be combined with epi‐fluorescence microscopy. The molecular states of the fluorophore can be expressed by a three‐state energy model. This leads to a set of differential equations which describe the photobleaching behavior of fluorescein. The numerical solution of these equations shows that in a conventional wide‐field fluorescence microscope, the fluorescence of fluorescein will fade out faster at low than at high oxygen concentration. Further simulation showed that a simple ratio function of different time‐points during a fluorescence decay recorded during photobleaching could be used to describe oxygen concentrations in an aqueous solution. By careful choice of dye concentration and excitation light intensity the sensitivity in the oxygen concentration range of interest can be optimized. In the simulations, the estimation of oxygen concentration by the ratio function was very little affected by the pH value in the range of pH 6.5–8.5. Filming the fluorescence decay by a charge‐coupled‐device (ccd) camera mounted on a fluorescence microscope allowed a pixelwise estimation of the ratio function in a microscopic image. Use of a microsensor and oxygen‐consuming bacteria in a sample chamber enabled the calibration of the system for quantification of absolute oxygen concentrations. The method was demonstrated on nitrifying biofilms growing on snail and mussel shells, showing clear effects of metabolic activity on oxygen concentrations. Microsc. Res. Tech. 77:341–347, 2014. © 2014 Wiley Periodicals, Inc.     

Evaluation of a cocktail of three bacteriophages for the biocontrol of Salmonella of wastewater

Salmonella serovars are increasing in importance as significant pathogens of both human and animals. Although water and wastewater are treated to eliminate pathogenic microorganisms, they still play an important role in the transmission of Salmonella spp. In this study, bacteriophages infecting Salmonella spp. were isolated from wastewater and evaluated; for their potential to lyse environmental Salmonella strains in vitro at different MOIs and temperatures; and to control the wastewater bacterial community. Three distinct phages designated sww65, sww275, and sww297; as defined by plaque morphology, electron microscopy and host range; were obtained from wastewater. Challenge tests were performed at 37, and 30 °C with the infection of the Salmonella cultures with individual phage, a mixture of two phages, and cocktail of three phages at MOIs of 10⁰, 10², and 10⁴ PFU/CFU. At 30, and 37 °C, a cocktail of three phages reduced all of the Salmonella cultures tested. These results required a high multiplicity of infection. However, when infected with only one phage or a mixture of two phages at MOIs of 10⁰ or 10 ² PFU/CFU, an emergence of bacterial resistance was observed. The dynamic monitoring of wastewater enterobacterial community was conducted using Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). The number of bands decreased gradually with the use of individual phage or phage cocktails. Moreover, the dynamic monitoring of Salmonella community during wastewater treatment was performed using PCR detection of virulence gene invA. The results correlated with the ERIC-PCR fingerprints, and suggested that Salmonella community was affected by the phage treatment. Indeed, in wastewater, bacteriophages are reducing Salmonella and other members of the Enterobacteriaceae. These results indicated that dynamic changes are closely related with the process of treatment. The introduction of wide host range bacteriophages in wastewater can have a potential impact on the dynamics of the microbial communities, manifested by the reduction or the elimination of microbial species.     

Cross-linked soy protein as material for biodegradable films: Synthesis, characterization and biodegradation

The modification of soy protein isolate (SPI) with different amounts of a naturally occurring cross-linking agent (genipin, Gen) and glycerol used as plasticizer was carried out in this work. The films yielded were cast from heated and alkaline aqueous solution of SPI, glycerol and Gen and then dried in an oven. Total soluble matter, water vapor permeability and mechanical properties were improved by adding small amounts of Gen. These properties were not significantly affected (P ? 0.05) by additions exceeding 2.5% (w/w of SPI). The opacity and cross-linking degree were linearly increased with the addition of Gen, whereas the swelling ratios in water were decreased. All the films were submitted to degradation under indoor soil burial conditions and the weight loss of the films was measured at different times. This study revealed that the film biodegradation time can be controlled or modified from at least 14 to 33 days. The tests performed showed the potential of Gen to improve the SPI film properties, in which the possibility of employing such new films as biodegradable food packaging was raised.     

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.     

Crystallization of polyamide 11 during injection molding

The semicrystalline morphology of injection moldings of polyamide 11 (PA 11) prepared using mold temperatures of 25, 50, and 80°C was investigated. Regardless of the mold temperature, position‐resolved X‐ray diffraction (XRD) and polarized‐light optical microscopy (POM) revealed presence of poor/imperfect α‐crystals with an almost hexagonal arrangement of molecular stems in a nonspherulitic superstructure in the skin, and formation of α‐crystals and spherulites in the core. With increasing mold temperature, the thickness of the skin layer decreased, and the perfection of α‐crystals and the spherulite size in the core increased. The experimental observations are discussed in terms of predicted crystallization temperatures, with the prediction based on cooling‐rate simulations for the various parts of the injection moldings using Moldflow^® and analysis of crystallization of the relaxed melt using fast scanning chip calorimetry, XRD, and POM. It is shown that the structure gradient in PA 11 injection moldings can be forecast without considering the effects of shear for this particular polymer. POLYM. ENG. SCI., 58:1053–1061, 2018. © 2017 Society of Plastics Engineers