Gregersenkonradsen2230

So far energy independent direct membrane penetration and energy-dependent endocytosis have been suggested as two main mechanisms of cellular entry for CPPs, and both may be applicable for the same CPP-complex, depending on the conditions.In order to understand which mechanism is associated with a particular CPP 's cellular uptake in a particular cell (sometimes including endosomal escape), different biological and biophysical methods and strategies have been applied. In this chapter, we will address several biophysical methods, such as fluorescence spectroscopy, circular dichroism (CD) spectroscopy, dynamic light scattering, and NMR .We also review different membrane model systems which are suitable for the biophysical studies. These include large unilamellar phospholipid vesicles (LUVs ), which are the most commonly used in the lipid-peptide interaction studies. Detergent micelles and mixed micelles (bicelles) are also suitable membrane model systems, particularly in high-resolution NMR studies.Fluorescence-based methods are widely used to detect crossing of peptides across model or biological membranes. For membrane-active peptides, i.e., peptides that have strong membrane tropism, fluorescence experiments must be accompanied by relevant controls, otherwise they can lead to inconsistent interpretation and underestimation of their limitations. Here we describe how to prepare samples to study fluorescent peptide crossing droplet interface bilayer (model membrane) or cell membrane (biological membrane) and the pitfalls that can affect observational qualitative and quantitative data.Extracellular vesicles are lipid-bilayer-enclosed nanoparticles present in the majority of biological fluids that mediate intercellular communication. EVs are able to transfer their contents (including nucleic acids, proteins, lipids, and small molecules) to recipient cells, and thus hold great promise as drug delivery vehicles. However, their therapeutic application is limited by lack of efficient cargo loading strategies, a need to improve EV tissue-targeting capabilities and a requirement to improve escape from the endolysosomal system. These challenges can be effectively addressed by modifying EVs with peptides which confer specific advantageous properties, thus enhancing their therapeutic potential. Here we provide an overview of the applications of peptide technology with respect to EV therapeutics. We focus on the utility of EV-modifying peptides for the purposes of promoting cargo loading, tissue-targeting and endosomal escape, leading to enhanced delivery of the EV cargo to desired cells/tissues and subcellular target locations. Both endogenous and exogenous methods for modifying EVs with peptides are considered.The surface decoration of nanoparticles with cell-penetrating peptides (CPPs) represents a common technique for intracellular delivery of nanotherapeutics. Conjugate formation can be performed via covalent or non-covalent strategies. Here, we describe on the synthesis of silica nanoparticles, a well-known inorganic drug delivery vehicle type, and their surface modification with cell-penetrating peptides using sC18 and derivatives thereof. Moreover, physicochemical as well as biological characterization methods, including cellular uptake measurements, of particle-peptide conjugates are described.The cell-penetrating peptides (CPPs) are characterized by the ability of internalization into cells in vitro and in vivo, and the ability of these peptides can rely on a high content of positive charges, as it is the case of the native CPP crotamine. Crotamine is a polypeptide of about 42 amino acid residues with high content of basic residues as Arg and Lys. Although most of known CPPs are linear peptides, native crotamine from the venom of a South American rattlesnake has a well-defined 3D structure stabilized by three disulfide bonds which guarantee the exposure of side chains of basic amino acids. This 3D structure also protects this amphipathic polypeptide from the degradation even if administered by oral route, therefore, protecting also the biological activities of crotamine. As several different biological properties of crotamine are dependent of cell penetration, the methods mainly employed for analyzing crotamine properties as anthelminthic and antimalarial activities, antimicrobial and antitumor acmediation of several pathogenic conditions are presented here.Rational design and optimization of cell penetrating peptides (CPPs) is difficult to accomplish because of the lack of quantitative sequence-structure-function rules describing the activity and because of the complex, poorly understood mechanisms of CPPs. Synthetic molecular evolution is a powerful method to identify gain-of-function cell penetrating peptide variants in this situation. Synthetic molecular evolution requires the design and synthesis of iterative, knowledge-based peptide libraries and the screening of such libraries in complex orthogonal cell-based screens for improved activity. In this chapter, we describe methods for synthesizing powerful combinatorial peptide libraries for synthetic molecular evolution.In the past few decades, a large number of cell-penetrating peptides (CPPs) have been discovered. These CPPs have a wide range of applications including drug delivery vehicles. Numerous in silico tools have been developed over the years to design and predict the cell-penetrating peptides that contain natural amino acids. The majority of natural cell-penetrating peptides have several limitations including stability, immunogenicity as well as got entrapped in the cell's endosomes. The chemical modification is commonly used to most of these limitations. An in silico tool called CellPPDMod have been developed by our group to predict cell-penetration potential of chemically modified peptides. This chapter is dedicated for designing therapeutically important cell-penetrating peptides using CellPPDMod ( http//webs.iiitd.edu.in/raghava/cellppdmod/ ).Crossing cellular membranes is a versatile molecular property that allows for a wide variety of peptides with cell penetrating capabilities. This broadness complicates identification of candidates suited best for a specific application. To facilitate the screening of this enormous molecular space in a supervised manner we here present a method to "breed" the desired molecules by applying the rules of Darwinian evolution. With this mate-and-check protocol, which combines an in silico evolution step with an in vitro performance test, cell penetrating peptides that are optimized for a specific task can be achieved in a few rounds of breeding. The procedure is simple and straightforward on the synthetic site but requires robust, highly reproducible and close-to-reality biological assays to yield realistic functional output. With this technology even top-performing peptides can be further improved and functionally adjusted.The transfer of homeoprotein transcription factors is at the origin of the discovery of Penetratin, one of the first transduction peptides allowing for the addressing of hydrophilic cargoes to the cell cytoplasm and nucleus. Beyond this important technological application, homeoprotein transduction has now been confirmed for more than 150 members of this family, and represents an intriguing mode of signaling for which actual in vivo functions are known for a handful of these proteins. Because homeoproteins are expressed in all eukaryotes, and their intercellular transfer occurs both in plants and animals, it is likely that this signaling activity appeared before the separation between plants, fungi, and animals, and is therefore very ancient. These aspects are discussed in the present review, with an accent placed on evolution and on the comparison of homeoprotein signaling between species belonging to distinct phyla.In this introductory chapter, we first define cell-penetrating peptides (CPPs), give short overview of CPP history and discuss several aspects of CPP classification. Next section is devoted to the mechanism of CPP penetration into the cells, where direct and endocytic internalization of CPP is explained. HS148 Kinetics of internalization is discussed more extensively, since this topic is not discussed in other chapters of this book. At the end of this section some features of the thermodynamics of CPP interaction with the membrane is also presented. Finally, we present different cargoes that can be transferred into the cells by CPPs and briefly discuss the effect of cargo on the rate and efficiency of penetration into the cells.

To investigate and compare the cytotoxicity and bioactivity of CMCR agents on stem cells derived from exfoliated deciduous teeth.

MTT assay, flow cytometry, Alizarin Red staining and scratch assay were used to assess the cellular viability, apoptosis, calcium matrix deposits and cell migration, respectively. The gene expression of ALP and BMP-2 was measured with RT-PCR. One-way ANOVA and Bonferroni post-test was used for statistical analysis.

0.5% Carisolv showed highest cell proliferation and calcium matrix formation, whereas 0.5% Papacarie reported the highest% live cells and cell migration. The highest mRNA expression of ALP and BMP-2 was reported in SHEDs cultured in 0.5% Papacarie (after 72h incubation) and 0.5% Carisolv (after 24h incubation), respectively.

CMCR agents are biocompatible and bioactive when cultured in stem cells derived from exfoliated primary teeth.

CMCR agents are biocompatible and bioactive when cultured in stem cells derived from exfoliated primary teeth.Understanding the genomic landscape of cancer in single cells can be valuable for the characterization of molecular events that drive evolution of tumorigenesis and fostering progress in identifying druggable regimens for patient treatment scenarios. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using microfluidics paired with next-generation sequencing. Our procedure determines targeted protein levels, mRNA transcript levels, and somatic gDNA sequence variations including copy number variants. This approach can resolve over 20 proteins, 100s of targeted transcripts, and DNA amplicons.Single-cell RNA sequencing (sc-RNAseq) has become a critical approach for the analysis of immune cell function and heterogeneity. So far, the immune cell isolation, based on surface marker expression predicted by the RNA expression profiles, is often limited by the poor correlation between transcript and protein expression patterns. To overcome these difficulties, novel single-cell multi-omic approaches based on the combined analysis of transcript and surface protein expression have been developed. One of the major benefits of these technologies is the possibility to use a high number of antibodies conjugated with oligonucleotide (AbOs) for the surface marker detection, thus overcoming the limit of using few surface markers as occurs in flow cytometry. Here we describe the BD Rhapsody single-cell analysis system protocol for 3' mRNA whole transcriptome analysis (WTA), combined with AbO- and Sample Tag library preparation.Understanding the principles of gene regulation at single-cell resolution would require measurement and integration of multiple methods such as DNA mutation profiling, open chromatin profiling, RNA expression, protein quantification, and DNA methylation. Recent developments in single-cell multi-omic technologies have enabled integration of these modes in various combinations.With the advent of RNA expression and protein sequencing assay (REAP-seq), researchers can simultaneously analyze protein and gene expression within the same cell. In REAP-seq , cells are labeled with antibodies conjugated to unique DNA sequences. A barcode of 8 nucleotides can allow up to 65,536 unique barcodes for multiplex analysis of proteins, circumventing the limitations of fluorescence (~17 targets). Here, we describe the implementation of REAP-seq assay in the Fluidigm® C1™ mRNA Seq HT (high-throughput) v2 system.