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Tunable Microwave Dielectric Qualities of Ca0.6La0.8/3TiO3 as well as Ca0.8Sm0.4/3TiO3-Modified (Mg0.6Zn0.Several)0.95Ni0.05TiO3 Ceramics having a Near-Zero Temperature Coefficient.
Using near-infrared fluorophore Alexa Fluor 680 labeled peptide nucleic acids (PNAs) the biodistribution of such antisense agents can be analyzed in real time in live mice using in vivo imaging. Using the fluorescence intensity emitted from the mouse at different time points following administration, the systemic distribution and organ accumulation of PNA can be tracked. In addition, an estimation of the body half-life of the compound can be obtained by the change in fluorescence intensity over time. With this technique, the distribution of compounds can be monitored real time, while reducing the number of animals and amount of compounds required.Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy and is caused by gene mutations that abolish production of functional dystrophin muscle protein. A promising new treatment exploits specifically targeted RNA-acting drugs that are able to partially restore the dystrophin protein. The mdx mouse model (animal model of DMD) serves as a good in vivo model for testing these antisense drugs. The simplest in vivo test, which circumvents the systemic circulation, is intramuscular administration of the compound. After 7 days it is possible to detect exon skipping by reverse transcriptase PCR, and newly synthesized dystrophin-positive fibers by immunohistochemistry and western blotting. All muscles, including the heart, are affected by the disease and must be treated. Therefore the use of antisense therapy for treatment of DMD requires systemic administration, and the model is also useful for systemic administration.Antisense PNA-peptide conjugates targeting essential bacterial genes have shown interesting potential for discovery of novel precision antibiotics. In this context, the minimal inhibitory concentration (MIC) assay is used to assess and compare the antimicrobial activity of natural as well as synthetic antimicrobial compounds. Here, we describe the determination of the minimal inhibitory concentration of peptide-PNA conjugates against Escherichia coli. This method can be expanded to include minimal bactericidal concentration (MBC) determination and kill-curve kinetics.Fluorescence in situ hybridization (FISH) is a 30-year-old technology that has evolved continuously and is now one of the most well-established molecular biology techniques. Traditionally, DNA probes are used for in situ hybridization. However, synthetic molecules are emerging as very promising alternatives, providing better hybridization performance and making FISH procedures easier and more efficient. In this chapter, we describe a universal FISH protocol, using nucleic acid probes, for the detection of bacteria. This protocol should be easily applied to different microorganisms as a way of identifying in situ relevant microorganisms (including pathogens) and their distribution patterns in different types of samples.The involvement of microRNAs in human pathologies is firmly established. Accordingly, the pharmacological modulation of microRNA activity appears to be a very interesting approach in the development of new types of drugs (miRNA therapeutics). One important research area is the possible development of miRNA therapeutics in the field of rare diseases. In this respect, appealing molecules are based on peptide nucleic acids (PNAs), displaying, in their first description, a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units, and found to be excellent candidates for antisense and antigene therapies. The aim of the present article is to describe methods for determining the activity of PNAs designed to target microRNAs involved in cystic fibrosis, using as model system miR-145-5p and its target cystic fibrosis transmembrane conductance regulator (CFTR) mRNA. The methods employed to study the effects of PNAs targeting miR-145-5p are presented here by discussing data obtained using as cellular model system the human lung epithelial Calu-3 cell line.Oligonucleotide-templated reactions (OTRs) between two reactive hybridization probes allow for the detection of a DNA or RNA of interest by exploiting the target molecule as a catalyst of chemical reactions. The product of such a reaction commonly exhibits distinct fluorescence properties and can be detected by the means of fluorescence spectroscopy. The vast majority of OTR systems utilize organic dyes as fluorescent reporters. However, the use of brighter emitters, such as semiconductor quantum dots (QDs), has potential to improve the sensitivity of detection by providing brighter signals and permitting the use of probes at very low concentrations. Here we report an RNA-templated reaction between two fluorescently labeled peptide nucleic acid (PNA)-based probes, which proceeds on the surface of a QD. The QD-bound PNA probe bears a cysteine functionality, while the other PNA is functionalized with an organic dye as a thioester. OTR between these probes proceeds through a transfer of the organic dye to the QD and can be conveniently monitored via fluorescence resonance energy transfer (FRET) from the QD to the Cy5. The reaction was performed in a conventional fluorescence microplate reader and permits the detection of RNA in the picomolar range.Cellular delivery methods are a prerequisite for cellular studies with PNA. This chapter describes PNA cellular delivery using cell-penetrating peptide (CPP)-PNA conjugates and transfection of PNA-ligand conjugates mediated by cationic lipids. Furthermore, two endosomolytic procedures employing chloroquine treatment or photochemical internalization (PCI) for significantly improving PNA delivery efficacy are described.Because of the important roles noncoding RNAs play in gene expression, their sequence-specific recognition is important for both fundamental science and the pharmaceutical industry. However, most noncoding RNAs fold in complex helical structures that are challenging problems for molecular recognition. Herein, we describe a method for sequence-specific recognition of double-stranded RNA using peptide nucleic acids (PNAs) that form triple helices in the major grove of RNA under physiologically relevant conditions. We also outline methods for solid-phase conjugation of PNA with cell-penetrating peptides and fluorescent dyes. compound 78c purchase Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.
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