Sample records for activatable cell penetrating

  1. Angiopep-2 and activatable cell penetrating peptide dual modified nanoparticles for enhanced tumor targeting and penetrating. (United States)

    Mei, Ling; Zhang, Qianyu; Yang, Yuting; He, Qin; Gao, Huile


    Delivering chemotherapeutics by nanoparticles into tumor was influenced by at least two factors: specific targeting and highly efficient penetrating of the nanoparticles. In this study, two targeting ligands, angiopep-2 and activatable cell penetrating peptide (ACP), were functionalized onto nanoparticles for tumor targeting delivery. In this system, angiopep-2 is a ligand of low-density lipoprotein receptor-related protein-1 (LRP1) which was highly expressed on tumor cells, and the ACP was constructed by the conjugation of RRRRRRRR (R8) with EEEEEEEE through a matrix metalloproteinase-2 (MMP-2) sensitive linker, enabling the ACP with tumor microenvironment-responsive cell penetrating property. 4h incubation of ACP with MMP-2 leads to over 80% cleavage of ACP, demonstrating ACP indeed possessed MMP-2 responsive property. The constructed dual targeting nanoparticles (AnACNPs) were approximately 110 nm with a polydispersity index of 0.231. In vitro, ACP modification and angiopep-2 modification could both enhance the U-87 MG cell uptake because of the high expression of MMP-2 and LRP-1 on C6 cells. AnACNPs showed higher uptake level than the single ligand modified nanoparticles. The uptake of all particles was time- and concentration-dependent and endosomes were involved. In vivo, AnACNPs showed best tumor targeting efficiency. The distribution of AnACNPs in tumor was higher than all the other particles. After microvessel staining with anti-CD31 antibody, the fluorescent distribution demonstrated AnACNPs could distribute in the whole tumor with the highest intensity. In conclusion, a novel drug delivery system was developed for enhanced tumor dual targeting and elevated cell internalization.

  2. Ratiometric activatable cell-penetrating peptides provide rapid in vivo readout of thrombin activation. (United States)

    Whitney, Michael; Savariar, Elamprakash N; Friedman, Beth; Levin, Rachel A; Crisp, Jessica L; Glasgow, Heather L; Lefkowitz, Roy; Adams, Stephen R; Steinbach, Paul; Nashi, Nadia; Nguyen, Quyen T; Tsien, Roger Y


    In real time: thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold.

  3. In vivo targeting of hydrogen peroxide by activatable cell-penetrating peptides. (United States)

    Weinstain, Roy; Savariar, Elamprakash N; Felsen, Csilla N; Tsien, Roger Y


    A hydrogen peroxide (H2O2)-activated cell-penetrating peptide was developed through incorporation of a boronic acid-containing cleavable linker between polycationic cell-penetrating peptide and polyanionic fragments. Fluorescence labeling of the two ends of the molecule enabled monitoring its reaction with H2O2 through release of the highly adhesive cell-penetrating peptide and disruption of fluorescence resonance energy transfer. The H2O2 sensor selectively reacts with endogenous H2O2 in cell culture to monitor the oxidative burst of promyelocytes and in vivo to image lung inflammation. Targeting H2O2 has potential applications in imaging and therapy of diseases related to oxidative stress.

  4. Tumor Detection at 3 Tesla with an Activatable Cell Penetrating Peptide Dendrimer (ACPPD-Gd, a T1 Magnetic Resonance (MR Molecular Imaging Agent.

    Directory of Open Access Journals (Sweden)

    Christopher D Malone

    Full Text Available The ability to detect small malignant lesions with magnetic resonance imaging (MRI is limited by inadequate accumulations of Gd with standard chelate agents. To date, no T1-targeted agents have proven superiority to Gd chelates in their ability to detect small tumors at clinically relevant field strengths. Activatable cell-penetrating peptides and their Gd-loaded dendrimeric form (ACPPD-Gd have been shown to selectively accumulate in tumors. In this study we compared the performance of ACPPD-Gd vs. untargeted Gd chelates to detect small tumors in rodent models using a clinical 3T-MR system.This study was approved by the Institutional-Animal Care-and-Use Committee. 2 of 4 inguinal breast fat pads of 16 albino-C57BL/6 mice were inoculated with tumor Py8119 cells and the other 2 with saline at random. MRI at 3T was performed at 4, 9, and 14 days after inoculation on 8 mice 24-hours after injection of 0.036mmol Gd/kg (ACPPD-Gd, and before and 2-3 minutes after 0.1 mmol/kg gadobutrol on the other 8 mice. T1-weighted (T1w tumor signal normalized to muscle, was compared among the non-contrast, gadobutrol, and ACPPD-Gd groups using ANOVA. Experienced and trainee readers blinded to experimental conditions assessed for the presence of tumor in each of the 4 breast regions. Receiver operator characteristic (ROC curves and area-under-curve (AUC values were constructed and analyzed.Tumors ≥1mm3 were iso-intense to muscle without contrast on T1w sequences. They enhanced diffusely and homogeneously by 57±20% (p5mm3 were removed from the ROC analysis for both the experienced observer (0.96 vs. 0.86 and more so for the trainee (0.86 vs. 0.69, p = 0.04.ACPPD-Gd enhances MMP-expressing tumors of any size at 3T 24 hours after administration, improving their detection by blinded observers when compared to non-contrast and contrast groups given commercial Gd-chelates and imaged during the equilibrium phase.

  5. Polypeptide micelles with dual pH activatable dyes for sensing cells and cancer imaging. (United States)

    Gong, Ping; Yang, Yueting; Yi, Huqiang; Fang, Shengtao; Zhang, Pengfei; Sheng, Zonghai; Gao, Guanhui; Gao, Duyang; Cai, Lintao


    pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence in an alkaline environment. Hence, DPNs exhibited a dual response signal with strong red fluorescence and weak green fluorescence under acidic conditions; in contrast, they showed strong green fluorescence and almost no red fluorescence under alkaline and neutral conditions. The favorable inverse pH responses of the two fluorescent dyes resulted in ratiometric pH determination for DPNs with an optimized pH-sensitive range of pH 4.5-7.5. Quantitative analysis of the intracellular pH of intact MCF-7 cells has been successfully demonstrated with our nanosensor. Moreover, single acid activatable fluorescent dye doped polypeptide nanoparticles that only contained RBLC can distinguish tumor tissue from normal tissue by monitoring the acidic extracellular environment.

  6. Activatable photodynamic destruction of cancer cells by NIR dye/photosensitizer loaded liposomes. (United States)

    Yuan, Ahu; Tang, Xiaolei; Qiu, Xuefeng; Jiang, Ke; Wu, Jinhui; Hu, Yiqiao


    The phototoxicity of Chlorin e6 (Ce6) for photodynamic therapy (PDT) was found to be effectively suppressed by indocyanine green (ICG), a near infrared (NIR) dye. Upon NIR laser irradiation at 808 nm, ICG in the liposomes containing ICG and Ce6 could be degraded, while the phototoxicity of Ce6 could be recovered. In addition, we demonstrate that this newly developed liposomal component can be successfully used for activatable PDT to destroy cancer cells in vitro.

  7. Activatable molecular MRI nanoprobe for tumor cell imaging based on gadolinium oxide and iron oxide nanoparticle. (United States)

    Li, Jingjing; Wang, Shan; Wu, Chen; Dai, Yue; Hou, Pingfu; Han, Cuiping; Xu, Kai


    Activatable molecular MRI nanoprobe for intracellular GSH sensing was designed. As an alternative to "always on" nanoprobe, activatable imaging nanoprobes which are designed to amplify or boost imaging signals only in response to the targets have attracted more and more attention. In this paper, we designed a novel activatable molecular magnetic resonance imaging (MRI) nanoprobe for tumor cell recognization based on a MRI signal variation induced by the distance change between T1 and T2 contrast agents (CAs) in the presence of glutathione (GSH). To achieve this aim, carboxyl group functionalized iron oxide nanoparticles (Fe3O4 NPs) and polyethylene glycol-coated gadolinium oxide (PEG-Gd2O3) NPs as T2 and T1 MRI CA were connected by cystamine which contains a disulfide linkage. Transmission electron microscopic (TEM), X-ray photoelectron spectroscopy (XPS), energy dispersive spectrometer (EDS), fourier transform infrared spectroscopy (FT-IR), mass spectra and (1)H nuclear magnetic resonance spectroscopy ((1)H NMR) were introduced for their characterizations. The formation of Fe3O4-cystamine-Gd2O3 (Fe3O4-SS-Gd2O3) nanocomplex resulted in a quenched T1 signal due to the near proximity of PEG-Gd2O3 NPs to Fe3O4 NPs and a "light-up" T1 signal with the cleavage of disulfide bond in the presence of GSH. These results provide not only an easy way to realize MRI of tumor cells based on the overexpressed intracellular GSH level, but also a new insight for the design of activatable MRI nanoprobe.

  8. Molecular Imaging with Activatable Reporter Systems

    Directory of Open Access Journals (Sweden)

    Gang Niu, Xiaoyuan Chen


    Full Text Available Molecular imaging is a newly emerged multiple disciplinary field that aims to visualize, characterize and quantitatively measure biological processes at cellular and molecular levels in humans and other living systems. A reporter gene is a piece of DNA encoding reporter protein, which presents as a readily measurable phenotype that can be distinguished easily from the background of endogenous protein. After being transferred into cells of organ systems (transgenes, the reporter gene can be utilized to visualize transcriptional and posttranscriptional regulation of gene expression, protein-protein interactions, or trafficking of proteins or cells in living subjects. Herein, we review previous classification of reporter genes and regroup the reporter gene based imaging as basic, inducible and activatable, based on the regulation of reporter gene transcription and post-translational modification of reporter proteins. We then focus on activatable reporters, in which the signal can be activated at the posttranslational level for visualizing protein-protein interactions, protein phosphorylation or tertiary structure changes. The applications of several types of activatable reporters will also be summarized. We conclude that activatable reporter imaging can benefit both basic biomedical research and drug development.

  9. Magnetic resonance imaging of stem cell apoptosis in arthritic joints with a caspase activatable contrast agent. (United States)

    Nejadnik, Hossein; Ye, Deju; Lenkov, Olga D; Donig, Jessica S; Martin, John E; Castillo, Rostislav; Derugin, Nikita; Sennino, Barbara; Rao, Jianghong; Daldrup-Link, Heike


    About 43 million individuals in the U.S. encounter cartilage injuries due to trauma or osteoarthritis, leading to joint pain and functional disability. Matrix-associated stem cell implants (MASI) represent a promising approach for repair of cartilage defects. However, limited survival of MASI creates a significant bottleneck for successful cartilage regeneration outcomes and functional reconstitution. We report an approach for noninvasive detection of stem cell apoptosis with magnetic resonance imaging (MRI), based on a caspase-3-sensitive nanoaggregation MRI probe (C-SNAM). C-SNAM self-assembles into nanoparticles after hydrolysis by caspase-3, leading to 90% amplification of (1)H MR signal and prolonged in vivo retention. Following intra-articular injection, C-SNAM causes significant MR signal enhancement in apoptotic MASI compared to viable MASI. Our results indicate that C-SNAM functions as an imaging probe for stem cell apoptosis in MASI. This concept could be applied to a broad range of cell transplants and target sites.

  10. Chemical engineering of cell penetrating antibodies. (United States)

    Zhao, Y; Lou, D; Burkett, J; Kohler, H


    Antibodies, being exquisitely specific tools in biology, are routinely used to detect and identify intra-cellular structures. However, current intra-cellular application of antibodies requires that the membrane be rendered leaky, resulting in the death of cells. Here, we present a novel method to allow antibodies to penetrate the cellular membrane of living cells without affecting cell viability. A peptide (MTS, membrane transport sequence) that facilitates transport across membranes has been site-specifically attached to antibodies. MTS-antibodies enter the living cells in culture and can be detected by immunofluorescence and ELISA after extraction. Cellular structures are visualized in living cells using a specific MTS-antibody. Antibodies with membrane penetrating properties can become an important tool for the study of intra-cellular processes in living cells. Furthermore, such membrane penetrating antibodies can be used to selectively stimulate or suppress functions of the cellular machinery.

  11. Penetration of Bdellovibrio bacteriovorus into host cells. (United States)

    Abram, D; Castro e Melo, J; Chou, D


    Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite.

  12. Activatable iRGD-based peptide monolith: Targeting, internalization, and fluorescence activation for precise tumor imaging. (United States)

    Cho, Hong-Jun; Lee, Sung-Jin; Park, Sung-Jun; Paik, Chang H; Lee, Sang-Myung; Kim, Sehoon; Lee, Yoon-Sik


    A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis.

  13. Lysosomal pH Decrease in Inflammatory Cells Used To Enable Activatable Imaging of Inflammation with a Sialic Acid Conjugated Profluorophore. (United States)

    Yu, Mingzhu; Wu, Xuanjun; Lin, Bijuan; Han, Jiahuai; Yang, Liu; Han, Shoufa


    Inflammation causes significant morbidity and mortality, necessitating effective in vivo imaging of inflammation. Prior approaches often rely on combination of optical agents with entities specific for proteinaceous biomarkers overexpressed in inflammatory tissues. We herein report a fundamentally new approach to image inflammation by targeting lysosomes undergoing acidification in inflammatory cells with a sialic acid (Sia) conjugated near-infrared profluorophore (pNIR). Sia-pNIR contains a sialic acid domain for in vivo targeting of inflamed tissues and a pNIR domain which isomerizes into fluorescent and optoacoustic species in acidic lysosomes. Sia-pNIR displays high inflammation-to-healthy tissue signal contrasts in mice treated with Escherichia coli, Staphylococcus aureus, or lipopolysaccharide. In addition, inflammation-associated fluorescence is switched off upon antibiotics treatment in mice. This report shows the potentials of Sia-pNIR for activatable dual-modality inflammation imaging, and particularly the use of lysosomes of inflamed cells as a previously unappreciated biomarker for inflammation imaging.

  14. Market penetration scenarios for fuel cell vehicles

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, C.E.; James, B.D.; Lomax, F.D. Jr. [Directed Technologies, Inc., Arlington, VA (United States)


    Fuel cell vehicles may create the first mass market for hydrogen as an energy carrier. Directed Technologies, Inc., working with the US Department of Energy hydrogen systems analysis team, has developed a time-dependent computer market penetration model. This model estimates the number of fuel cell vehicles that would be purchased over time as a function of their cost and the cost of hydrogen relative to the costs of competing vehicles and fuels. The model then calculates the return on investment for fuel cell vehicle manufacturers and hydrogen fuel suppliers. The model also projects the benefit/cost ratio for government--the ratio of societal benefits such as reduced oil consumption, reduced urban air pollution and reduced greenhouse gas emissions to the government cost for assisting the development of hydrogen energy and fuel cell vehicle technologies. The purpose of this model is to assist industry and government in choosing the best investment strategies to achieve significant return on investment and to maximize benefit/cost ratios. The model can illustrate trends and highlight the sensitivity of market penetration to various parameters such as fuel cell efficiency, cost, weight, and hydrogen cost. It can also illustrate the potential benefits of successful R and D and early demonstration projects. Results will be shown comparing the market penetration and return on investment estimates for direct hydrogen fuel cell vehicles compared to fuel cell vehicles with onboard fuel processors including methanol steam reformers and gasoline partial oxidation systems. Other alternative fueled vehicles including natural gas hybrids, direct injection diesels and hydrogen-powered internal combustion hybrid vehicles will also be analyzed.

  15. Cell-penetrating peptides: From mammalian to plant cells


    Eudes, François; Chugh, Archana


    Internalization of cell-penetrating peptides, well described in mammalian cell system, has recently been reported in a range of plant cells by three independent groups. Despite fundamental differences between animal cell and plant cell composition, the CPP uptake pattern between the mammalian system and the plant system is very similar. Tat, Tat-2 pVEC and transportan internalisation is concentration dependent and non saturable, enhanced at low temperature (4°C), and receptor independent. The...

  16. XTEN as Biological Alternative to PEGylation Allows Complete Expression of a Protease-Activatable Killin-Based Cytostatic.

    Directory of Open Access Journals (Sweden)

    Akvile Haeckel

    Full Text Available Increased effectiveness and reduced side effects are general goals in drug research, especially important in cancer therapy. The aim of this study was to design a long-circulating, activatable cytostatic drug that is completely producible in E. coli. Crucial for this goal was the novel unstructured polypeptide XTEN, which acts like polyethylene glycol (PEG but has many important advantages. Most importantly, it can be produced in E. coli, is less immunogenic, and is biodegradable. We tested constructs containing a fragment of Killin as cytostatic/cytotoxic element, a cell-penetrating peptide, an MMP-2 cleavage site for specific activation, and XTEN for long blood circulation and deactivation of Killin. One of three sequence variants was efficiently expressed in E. coli. As typical for XTEN, it allowed efficient purification of the E. coli lysate by a heat step (10 min 75°C and subsequent anion exchange chromatography using XTEN as purification tag. After 24 h XTEN-Killin reduced the number of viable cells of HT-1080 tumor cell line to 3.8 ±2.0% (p<0.001 compared to untreated controls. In contrast, liver derived non-tumor cells (BRL3A did not show significant changes in viability. Our results demonstrate the feasibility of completely producing a complex protease-activatable, potentially long-circulating cytostatic/cytotoxic prodrug in E. coli-a concept that could lead to efficient production of highly multifunctional drugs in the future.

  17. Cell Penetration Properties of a Highly Efficient Mini Maurocalcine Peptide

    Directory of Open Access Journals (Sweden)

    Michel De Waard


    Full Text Available Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one such peptide, MCaUF1-9, whose sequence matches that of the hydrophobic face of maurocalcine. This peptide shows very favorable cell-penetration efficacy compared to Tat, penetratin or polyarginine. The peptide appears so specialized in cell penetration that it seems hard to improve by site directed mutagenesis. A comparative analysis of the efficacies of similar peptides isolated from other toxin members of the same family leads to the identification of hadrucalcin’s hydrophobic face as an even better CPP. Protonation of the histidine residue at position 6 renders the cell penetration of MCaUF1-9 pH-sensitive. Greater cell penetration at acidic pH suggests that MCaUF1-9 can be used to specifically target cancer cells in vivo where tumor masses grow in more acidic environments.

  18. A Miniature Probe for Ultrasonic Penetration of a Single Cell

    Directory of Open Access Journals (Sweden)

    Mingfei Xiao


    Full Text Available Although ultrasound cavitation must be avoided for safe diagnostic applications, the ability of ultrasound to disrupt cell membranes has taken on increasing significance as a method to facilitate drug and gene delivery. A new ultrasonic resonance driving method is introduced to penetrate rigid wall plant cells or oocytes with springy cell membranes. When a reasonable design is created, ultrasound can gather energy and increase the amplitude factor. Ultrasonic penetration enables exogenous materials to enter cells without damaging them by utilizing instant acceleration. This paper seeks to develop a miniature ultrasonic probe experiment system for cell penetration. A miniature ultrasonic probe is designed and optimized using the Precise Four Terminal Network Method and Finite Element Method (FEM and an ultrasonic generator to drive the probe is designed. The system was able to successfully puncture a single fish cell.

  19. Prediction of cell penetrating peptides by support vector machines.

    Directory of Open Access Journals (Sweden)

    William S Sanders


    Full Text Available Cell penetrating peptides (CPPs are those peptides that can transverse cell membranes to enter cells. Once inside the cell, different CPPs can localize to different cellular components and perform different roles. Some generate pore-forming complexes resulting in the destruction of cells while others localize to various organelles. Use of machine learning methods to predict potential new CPPs will enable more rapid screening for applications such as drug delivery. We have investigated the influence of the composition of training datasets on the ability to classify peptides as cell penetrating using support vector machines (SVMs. We identified 111 known CPPs and 34 known non-penetrating peptides from the literature and commercial vendors and used several approaches to build training data sets for the classifiers. Features were calculated from the datasets using a set of basic biochemical properties combined with features from the literature determined to be relevant in the prediction of CPPs. Our results using different training datasets confirm the importance of a balanced training set with approximately equal number of positive and negative examples. The SVM based classifiers have greater classification accuracy than previously reported methods for the prediction of CPPs, and because they use primary biochemical properties of the peptides as features, these classifiers provide insight into the properties needed for cell-penetration. To confirm our SVM classifications, a subset of peptides classified as either penetrating or non-penetrating was selected for synthesis and experimental validation. Of the synthesized peptides predicted to be CPPs, 100% of these peptides were shown to be penetrating.

  20. Cell-penetrating peptides for drug delivery across membrane barriers

    DEFF Research Database (Denmark)

    Foged, Camilla; Nielsen, Hanne Moerck


    During the last decade, cell-penetrating peptides have been investigated for their ability to overcome the plasma membrane barrier of mammalian cells for the intracellular or transcellular delivery of cargoes as diverse as low molecular weight drugs, imaging agents, oligonucleotides, peptides......-penetrating peptides as transmembrane drug delivery agents, according to the recent literature, and discusses critical issues and future challenges in relation to fully understanding the fundamental principles of the cell-penetrating peptide-mediated membrane translocation of cargoes and the exploitation......, proteins and colloidal carriers such as liposomes and polymeric nanoparticles. Their ability to cross biological membranes in a non-disruptive way without apparent toxicity is highly desired for increasing drug bioavailability. This review provides an overview of the application of cell...

  1. Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections

    DEFF Research Database (Denmark)

    Bahnsen, Jesper S; Franzyk, Henrik; Sayers, Edward J;


    . TPk showed the highest antibacterial activity. SA-3 exhibited selective disruption of liposomes mimicking Gram-positive and Gram-negative membranes. CONCLUSION: PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected...

  2. Discovery and characterization of a new cell-penetrating protein. (United States)

    Simeon, Rudo L; Chamoun, Ana Maria; McMillin, Thomas; Chen, Zhilei


    We describe a new cell-penetrating protein, B1, capable of delivering conjugated proteins and nucleic acids into mammalian cells. B1 is a 244-amino-acid product of a single-base frameshift in the gene encoding enhanced green fluorescent protein (eGFP). The molecule has a net positive charge of 43 and a very high charge-to-mass ratio of 1.5. eGFP-fused B1 potently penetrates both adherent and suspension cells with >80% of cells taking up the protein when exposed to concentrations as low as 1 μM. The protein was found to cluster in the paranuclear region of TZM-bl cells. Most importantly, we show that B1 not only facilitates cellular uptake but allows biomolecular cargo to reach sites of biological relevance. For example, baby hamster kidney cells underwent DNA recombination when exposed to B1-tagged Cre recombinase at protein concentrations as low as 2.5 μM, indicating potent nuclear delivery of functional protein cargos. Additionally, B1 delivers noncovalently conjugated RNA and DNA across the cell membrane to cytosolic and nuclear sites accessible to the cellular translation and transcription machinery, as gauged by detection of encoded reporter functions, with efficiency comparable to commercially available cationic lipid reagents. B1 appears to utilize cell-surface glycans and multiple competing endocytic pathways to enter and traffic through cells. These studies provide both a new tool for intracellular delivery of biomolecules and insights that could aid in the design of more effective cell penetrating proteins.

  3. Chemical-functional diversity in cell-penetrating peptides.

    Directory of Open Access Journals (Sweden)

    Sofie Stalmans

    Full Text Available Cell-penetrating peptides (CPPs are a promising tool to overcome cell membrane barriers. They have already been successfully applied as carriers for several problematic cargoes, like e.g. plasmid DNA and (siRNA, opening doors for new therapeutics. Although several hundreds of CPPs are already described in the literature, only a few commercial applications of CPPs are currently available. Cellular uptake studies of these peptides suffer from inconsistencies in used techniques and other experimental conditions, leading to uncertainties about their uptake mechanisms and structural properties. To clarify the structural characteristics influencing the cell-penetrating properties of peptides, the chemical-functional space of peptides, already investigated for cellular uptake, was explored. For 186 peptides, a new cell-penetrating (CP-response was proposed, based upon the scattered quantitative results for cellular influx available in the literature. Principal component analysis (PCA and a quantitative structure-property relationship study (QSPR, using chemo-molecular descriptors and our newly defined CP-response, learned that besides typical well-known properties of CPPs, i.e. positive charge and amphipathicity, the shape, structure complexity and the 3D-pattern of constituting atoms influence the cellular uptake capacity of peptides.

  4. Penetration of living cell membranes with fortified carbon nanotube tips. (United States)

    Vakarelski, Ivan U; Brown, Scott C; Higashitani, Ko; Moudgil, Brij M


    We have fabricated robust nanosurgical needles suitable for single cell operations by modifying multiwalled carbon nanotube (MCNT)-terminated atomic force microscopy (AFM) tips. Extra-long MCNT AFM tips were prepared and fortified with molecular layers of carbon to overcome mechanical instabilities and then coated with an outer shell of gold to promote chemical versatility. The terminal diameters of the final fabricated tips were approximately 30-40 nm, and the MCNT probes were several micrometers in length. We illustrate the capability of these modified MCNT tips to carry nanoparticulate payloads and to penetrate the plasma membrane of living pleural mesothelial cells at the smallest indentation depths (100-200 nm) and lowest penetration forces (100-200 pN) currently reported for these procedures.

  5. Activatable Multifunctional Persistent Luminescence Nanoparticle/Copper Sulfide Nanoprobe for in Vivo Luminescence Imaging-Guided Photothermal Therapy. (United States)

    Chen, Li-Jian; Sun, Shao-Kai; Wang, Yong; Yang, Cheng-Xiong; Wu, Shu-Qi; Yan, Xiu-Ping


    Multifunctional nanoprobes that provide diagnosis and treatment features have attracted great interest in precision medicine. Near-infrared (NIR) persistent luminescence nanoparticles (PLNPs) are optimal materials due to no in situ excitation needed, deep tissue penetration, and high signal-to-noise ratio, while activatable optical probes can further enhance signal-to-noise ratio for the signal turn-on nature. Here, we show the design of an activatable multifunctional PLNP/copper sulfide (CuS)-based nanoprobe for luminescence imaging-guided photothermal therapy in vivo. Matrix metalloproteinases (MMPs)-specific peptide substrate (H2N-GPLGVRGC-SH) was used to connect PLNP and CuS to build a MMP activatable system. The nanoprobe not only possesses ultralow-background for in vivo luminescence imaging due to the absence of autofluorescence and optical activatable nature but also offers effective photothermal therapy from CuS nanoparticles. Further bioconjugation of c(RGDyK) enables the nanoprobe for cancer-targeted luminescence imaging-guided photothermal therapy. The good biocompatibility and the multiple functions of highly sensitive tumor-targeting luminescence imaging and effective photothermal therapy make the nanoprobe promising for theranostic application.

  6. Antimicrobial and cell-penetrating properties of penetratin analogs

    DEFF Research Database (Denmark)

    Bahnsen, Jesper Søborg; Franzyk, Henrik; Sandberg-Schaal, Anne;


    Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well as octaar......Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) show great potential as drug delivery vectors and new antibiotic drug entities, respectively. The current study deals with the properties of a variety of peptide analogs derived from the well-known CPP penetratin as well...... as octaarginine and different Tat sequences. The effects of peptide length, guanidinium content, and sequence of non-cationic residues were assessed in mammalian and bacterial cells. The arginine (Arg) content in the penetratin analogs was found to influence eukaryotic cell uptake efficiency, antimicrobial...... activity towards both Gram-positive and Gram-negative bacteria as well as eukaryotic cell viability. All examined analogs retained the ability to cross eukaryotic membranes giving rise to a distribution within the vacuolar apparatus. Interestingly, a series of shuffled analogs of penetratin...

  7. Cell-penetrating peptides modulate the vascular action of phenylephrine. (United States)

    Kocić, Ivan; Ruczyński, Jarosław; Szczepańska, Renata; Olkowicz, Mariola; Parfianowicz, Brygida; Rusiecka, Izabela; Rekowski, Piotr


    Cell-penetrating peptides (CPP) are a family of peptides able to penetrate the cell membrane. This group of compounds has attracted consideration as potential therapeutic tools for the delivery of various substances into cells. Here, we investigated possible interactions between several CPP synthesized in our laboratory and the vascular action of phenylephrine. We used isolated rat tail artery and examined the influence of pretreatment by seven different CPP on the concentration-response curve induced by the α1 receptor agonist phenylephrine. Peptides were synthesized by solid-phase peptide synthesis (SPPS) using the 9-fluorenylmethoxycarbonyl (Fmoc) method. Among the seven different polypeptides, i.e., TP10 (transportan-10), [Lys(AAc)13]TP, [Lys(CAc)13]TP, [Lys(GAc)13]TP, [Lys(TAc)13]TP, [Lys(UAc)13]TP and [Lys(Ac)13]TP, only TP10 and [Lys(AAc)13]TP, both at a concentration of 1 μM (the lowest concentration inducing a significant change in the contraction of isolated rat stomach in our pilot study), rendered rat tail artery more sensitive to phenylephrine; the relative potency increased significantly. Conversely, [Lys(Ac)13]TP strongly decreased the efficacy of phenylephrine.

  8. Investigation of penetration force of living cell using an atomic force microscope

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Eun Young; Kim, Young Tae; Kim, Dae Eun [Yonsei University, Seoul (Korea, Republic of)


    Recently, the manipulation of a single cell has been receiving much attention in transgenesis, in-vitro fertilization, individual cell based diagnosis, and pharmaceutical applications. As these techniques require precise injection and manipulation of cells, issues related to penetration force arise. In this work the penetration force of living cell was studied using an atomic force microscope (AFM). L929, HeLa, 4T1, and TA3 HA II cells were used for the experiments. The results showed that the penetration force was in the range of 2{approx}22 nN. It was also found that location of cell penetration and stiffness of the AFM cantilever affected the penetration force significantly. Furthermore, double penetration events could be detected, due to the multi-membrane layers of the cell. The findings of this work are expected to aid in the development of precision micro-medical instruments for cell manipulation and treatment

  9. Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Kaljot, K.T.; Shaw, R.D.; Greenberg, H.B. (Stanford Univ. School of Medicine, CA (USA) Palo Alto Veterans Administration Medical Center, CA (USA)); Rubin, D.H. (Univ. of Pennsylvania, Philadelphia (USA))


    Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 (VP3); 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. To determine whether trypsin treatment affected rotavirus internalization, the authors studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 mediated {sup 51}Cr, ({sup 14}C)choline, and ({sup 3}H)inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.

  10. Membrane adsorption and binding, cellular uptake and cytotoxicity of cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone

    DEFF Research Database (Denmark)

    Jing, Xiaona; Yang, Mingjun; Kasimova, Marina Robertovna


    Cell-penetrating peptides (CPPs) provide a promising approach for enhancing intracellular delivery of therapeutic biomacromolecules by increasing transport through membrane barriers. Here, proteolytically stable cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone were studied to ev...

  11. Active penetration of Trypanosoma cruzi into host cells: historical considerations and current concepts (United States)

    de Souza, Wanderley; de Carvalho, Tecia M. Ulisses


    In the present short review, we analyze past experiments that addressed the interactions of intracellular pathogenic protozoa (Trypanosoma cruzi, Toxoplasma gondii, and Plasmodium) with host cells and the initial use of the term active penetration to indicate that a protozoan “crossed the host cell membrane, penetrating into the cytoplasm.” However, the subsequent use of transmission electron microscopy showed that, for all of the protozoans and cell types examined, endocytosis, classically defined as involving the formation of a membrane-bound vacuole, took place during the interaction process. As a consequence, the recently penetrated parasites are always within a vacuole, designated the parasitophorous vacuole (PV). PMID:23355838

  12. Thrombin activatable fibrinolysis inhibitor as a bleeding predictor in liver transplantation: a pilot observational study (United States)

    Nedel, Wagner Luis; Rodrigues Filho, Edison Moraes; Pasqualotto, Alessandro Comarú


    Objective To correlate the levels of thrombin activatable fibrinolysis inhibitor in the immediate postoperative period and at 24 hours postoperatively with the volume of intraoperative bleeding. Methods Twenty-one patients allocated immediately before (elective or emergency) liver transplantation were analyzed. Blood samples were collected for thrombin activatable fibrinolysis inhibitor analysis at three different time points: immediately before liver transplantation (preoperative thrombin activatable fibrinolysis inhibitor), immediately after the surgical procedure (immediate postoperative thrombin activatable fibrinolysis inhibitor), and 24 hours after surgery (thrombin activatable fibrinolysis inhibitor 24 hours after surgery). The primary outcome of the study was to correlate the preoperative and immediate postoperative levels of thrombin activatable fibrinolysis inhibitor with intraoperative blood loss. Results There was a correlation between the preoperative thrombin activatable fibrinolysis inhibitor levels and bleeding volume (ρ = -0.469; p = 0.05) but no correlation between the immediate postoperative thrombin activatable fibrinolysis inhibitor and bleeding volume (ρ = -0.062; p = 0.79). No variable included in the linear regression analysis (prehemoglobin, prefibrinogen and preoperative thrombin activatable fibrinolysis inhibitor) was a bleeding predictor. There was a similar trend in the variation between the levels of thrombin activatable fibrinolysis inhibitor at the three different time points and fibrinogen levels. Patients who died within 6 months (14.3%) showed decreased preoperative and immediate postoperative levels of thrombin activatable fibrinolysis compared with survivors (preoperative: 1.3 ± 0.15 versus 2.55 ± 0.53, p = 0.06; immediate postoperative: 1.2 ± 0.15 versus 2.5 ± 0.42, p = 0.007). Conclusion There was a moderate correlation between preoperative thrombin activatable fibrinolysis inhibitor and intraoperative bleeding in liver

  13. Potential of activatable FAP-targeting immunoliposomes in intraoperative imaging of spontaneous metastases. (United States)

    Tansi, Felista L; Rüger, Ronny; Böhm, Claudia; Kontermann, Roland E; Teichgraeber, Ulf K; Fahr, Alfred; Hilger, Ingrid


    Despite intensive research and medical advances met, metastatic disease remains the most common cause of death in cancer patients. This results from late diagnosis, poor therapeutic response and undetected micrometastases and tumor margins during surgery. One approach to overcome these challenges involves fluorescence imaging, which exploits the properties of fluorescent probes for diagnostic detection of molecular structures at the onset of transformation and for intraoperative detection of metastases and tumor margins in real time. Considering these benefits, many contrast agents suitable for fluorescence imaging have been reported. However, most reports only demonstrate the detection of primary tumors and not the detection of metastases or their application in models of image-guided surgery. In this work, we demonstrate the influence of fibroblast activation protein (FAP) on the metastatic potential of fibrosarcoma cells and elucidate the efficacy of activatable FAP-targeting immunoliposomes (FAP-IL) for image-guided detection of the spontaneous metastases in mice models. Furthermore, we characterized the biodistribution and cellular localization of the liposomal fluorescent components in mice organs and traced their excretion over time in urine and feces. Taken together, activatable FAP-IL enhances intraoperative imaging of metastases. Their high accumulation in metastases, subsequent localization in the bile canaliculi and liver kupffer cells and suitable excretion in feces substantiates their potency as contrast agents for intraoperative imaging.

  14. Sensing lymphoma cells based on a cell-penetrating/apoptosis-inducing/electron-transfer peptide probe

    Energy Technology Data Exchange (ETDEWEB)

    Sugawara, Kazuharu, E-mail: [Maebashi Institute of Technology, Gunma 371-0816 (Japan); Shinohara, Hiroki; Kadoya, Toshihiko [Maebashi Institute of Technology, Gunma 371-0816 (Japan); Kuramitz, Hideki [Department of Environmental Biology and Chemistry, Graduate School of Science and Engineering for Research, University of Toyama, Toyama 930-8555 (Japan)


    To electrochemically sense lymphoma cells (U937), we fabricated a multifunctional peptide probe that consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. Electron-transfer peptides derive from cysteine residue combined with the C-terminals of four tyrosine residues (Y{sub 4}). A peptide whereby Y{sub 4}C is bound to the C-terminals of protegrin 1 (RGGRLCYCRRRFCVCVGR-NH{sub 2}) is known to be an apoptosis-inducing agent against U937 cells, and is referred to as a peptide-1 probe. An oxidation response of the peptide-1 probe has been observed due to a phenolic hydroxyl group, and this response is decreased by the uptake of the peptide probe into the cells. To improve the cell membrane permeability against U937 cells, the RGGR at the N-terminals of the peptide-1 probe was replaced by RRRR (peptide-2 probe). In contrast, RNRCKGTDVQAWY{sub 4}C (peptide-3 probe), which recognizes ovalbumin, was constructed as a control. Compared with the other probes, the change in the peak current of the peptide-2 probe was the greatest at low concentrations and occurred in a short amount of time. Therefore, the cell membrane permeability of the peptide-2 probe was increased based on the arginine residues and the apoptosis-inducing peptides. The peak current was linear and ranged from 100 to 1000 cells/ml. The relative standard deviation of 600 cells/ml was 5.0% (n = 5). Furthermore, the membrane permeability of the peptide probes was confirmed using fluorescent dye. - Highlights: • We constructed a multifunctional peptide probe for the electrochemical sensing of lymphoma cells. • The peptide probe consists of cell-penetrating/apoptosis-inducing/electron-transfer peptides. • The electrode response of the peptide probe changes due to selective uptake into the cells.

  15. Chemically-activatable alkyne-tagged probe for imaging microdomains in lipid bilayer membranes (United States)

    Yamaguchi, Satoshi; Matsushita, Taku; Izuta, Shin; Katada, Sumika; Ura, Manami; Ikeda, Taro; Hayashi, Gosuke; Suzuki, Yuta; Kobayashi, Koya; Tokunaga, Kyoya; Ozeki, Yasuyuki; Okamoto, Akimitsu


    A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the lipid raft membrane domains by Raman microscopy. The Raman probe, in which ring A of its steroid backbone is replaced with an alkynyl group, was designed to enable activation of the alkyne signal through the Eschenmoser-Tanabe fragmentation reaction of the oxidized cholesterol precursor in lipid bilayer membranes. The alkynyl steroid analogue was observed to form liquid-ordered raft-like domains on a model giant-liposome system in a similar manner as cholesterol, and the large alkyne signal of the accumulated probe at 2120 cm−1 was mapped on the microdomains with a Raman microscope. The alkyne moiety of the probe was confirmed to be converted from the α,β-epoxy ketone group of its precursor by reaction with p-toluensulfonyl hydrazine under a mild condition. Through the reaction, the alkyne signal of the probe was activated on the lipid bilayer membrane of liposomes. Furthermore, the signal activation of the probe was also detected on living cells by stimulated Raman scattering microscopy. The ring-A-opened alkyne steroid analogue, thus, provides a first chemically-activatable Raman probe as a promising tool for potentially unravelling the intracellular formation and trafficking of cholesterol-rich microdomains. PMID:28117375

  16. Data supporting the structural and functional characterization of Thrombin‐Activatable Fibrinolysis Inhibitor in breast cancer (United States)

    Fawzy, Manal S.; Toraih, Eman A.


    The data in this paper is related to the research article entitled “Thrombin-activatable fibrinolysis inhibitor Thr325Ile polymorphism and plasma level in breast cancer: A pilot study” (Fawzy et al., 2015) [1]. Many emerging studies have begun to unravel the pathophysiologic role of the fibrinolytic system in breast cancer (BC) progression (Zorio et al., 2008) [2]. Activation of the fibrinolytic plasminogen/plasmin system results in degradation of protein barriers, thereby mediating cell migration essential for tumor growth, angiogenesis, and dissemination (Castellino and Ploplis, 2005) [3]. In the current study, in silico data analysis of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) gene and protein has been done. Data have been retrieved from several databases mentioned in details in the text. Determination and analysis of the structural and functional impact of TAFI and its expression could help elucidate the contribution of the TAFI pathway to acquired hemostatic dysfunction and will form the basis of potential therapeutic strategies to manipulate this pathway. An inhibition of TAFI (e.g. by FXI inhibitors) will offer the therapeutic possibilities to improve the decreased fibrinolysis and increase the efficiency of fibrinolytic therapy in thrombotic disorders including cancer. PMID:26740968

  17. Data supporting the structural and functional characterization of Thrombin‐Activatable Fibrinolysis Inhibitor in breast cancer

    Directory of Open Access Journals (Sweden)

    Manal S. Fawzy


    Full Text Available The data in this paper is related to the research article entitled “Thrombin-activatable fibrinolysis inhibitor Thr325Ile polymorphism and plasma level in breast cancer: A pilot study” (Fawzy et al., 2015 [1]. Many emerging studies have begun to unravel the pathophysiologic role of the fibrinolytic system in breast cancer (BC progression (Zorio et al., 2008 [2]. Activation of the fibrinolytic plasminogen/plasmin system results in degradation of protein barriers, thereby mediating cell migration essential for tumor growth, angiogenesis, and dissemination (Castellino and Ploplis, 2005 [3]. In the current study, in silico data analysis of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI gene and protein has been done. Data have been retrieved from several databases mentioned in details in the text. Determination and analysis of the structural and functional impact of TAFI and its expression could help elucidate the contribution of the TAFI pathway to acquired hemostatic dysfunction and will form the basis of potential therapeutic strategies to manipulate this pathway. An inhibition of TAFI (e.g. by FXI inhibitors will offer the therapeutic possibilities to improve the decreased fibrinolysis and increase the efficiency of fibrinolytic therapy in thrombotic disorders including cancer.

  18. Visualization of the Nucleolus in Living Cells with Cell-Penetrating Fluorescent Peptides. (United States)

    Martin, Robert M; Herce, Henry D; Ludwig, Anne K; Cardoso, M Cristina


    The nucleolus is the hallmark of nuclear compartmentalization and has been shown to exert multiple roles in cellular metabolism besides its main function as the place of ribosomal RNA synthesis and assembly of ribosomes. The nucleolus plays also a major role in nuclear organization as the largest compartment within the nucleus. The prominent structure of the nucleolus can be detected using contrast light microscopy providing an approximate localization of the nucleolus, but this approach does not allow to determine accurately the three-dimensional structure of the nucleolus in cells and tissues. Immunofluorescence staining with antibodies specific to nucleolar proteins albeit very useful is time consuming, normally antibodies recognize their epitopes only within a small range of species and is applicable only in fixed cells. Here, we present a simple method to selectively and accurately label this ubiquitous subnuclear compartment in living cells of a large range of species using a fluorescently labeled cell-penetrating peptide.

  19. Mitochondrial transit peptide exhibits cell penetration ability and efficiently delivers macromolecules to mitochondria. (United States)

    Jain, Aastha; Chugh, Archana


    Mitochondrial malfunction under various circumstances can lead to a variety of disorders. Effective targeting of macromolecules (drugs) is important for restoration of mitochondrial function and treatment of related disorders. We have designed a novel cell-penetrating mitochondrial transit peptide (CpMTP) for delivery of macromolecules to mitochondria. Comparison between properties of cell-penetrating peptides (CPPs) and mitochondrial signal sequences enabled prediction of peptides with dual ability for cellular translocation and mitochondrial localization. Among the predicted peptides, CpMTP translocates across HeLa cells and shows successful delivery of noncovalently conjugated cargo molecules to mitochondria. CpMTP may have applications in transduction and transfection of mitochondria for therapeutics.

  20. Methods to Study the Role of the Glycocalyx in the Uptake of Cell-Penetrating Peptides

    NARCIS (Netherlands)

    Schmidt, S.; Wallbrecher, R.; Kuppevelt, T.H. van; Brock, R.E.


    Cells are covered by a layer of negatively charged oligo- and polysaccharides, the glycocalyx. Cell-penetrating peptides and other drug delivery vehicles first encounter these polyanions before contacting the lipid bilayer of the plasma membrane. While a large body of data supports the notion that i

  1. The uptake of arginine-rich cell-penetrating peptides: putting the puzzle together

    NARCIS (Netherlands)

    Brock, R.E.


    Over the past 20 years, cell-penetrating peptides (CPPs) have captured the attention of biomedical researchers, biophysicists, and (bio)organic chemists. These molecules efficiently enter cells and mediate entry of (macro)molecules that by themselves do not cross the plasma membrane. Since their dis

  2. Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.

    Directory of Open Access Journals (Sweden)

    Yoshinori Ohno

    Full Text Available Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.

  3. SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides

    Institute of Scientific and Technical Information of China (English)

    ZHAI Xiao-hui; LIU Min; GUO Xiao-juan; WANG Si-cen; ZHANG Hong-xia; GUO You-min


    Background The hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines,SKOV-3.Methods hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid gadolinium (Ⅲ) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM).Results The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r=0.990, P <0.01) and concentration-dependent manner (r=0.964, P <0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium.Conclusions hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

  4. Liposome-based engineering of cells to package hydrophobic compounds in membrane vesicles for tumor penetration. (United States)

    Lee, Junsung; Kim, Jiyoung; Jeong, Moonkyoung; Lee, Hyoungjin; Goh, Unbyeol; Kim, Hyaeyeong; Kim, Byungji; Park, Ji-Ho


    Natural membrane vesicles (MVs) derived from various types of cells play an essential role in transporting biological materials between cells. Here, we show that exogenous compounds are packaged in the MVs by engineering the parental cells via liposomes, and the MVs mediate autonomous intercellular migration of the compounds through multiple cancer cell layers. Hydrophobic compounds delivered selectively to the plasma membrane of cancer cells using synthetic membrane fusogenic liposomes were efficiently incorporated into the membrane of MVs secreted from the cells and then transferred to neighboring cells via the MVs. This liposome-mediated MV engineering strategy allowed hydrophobic photosensitizers to significantly penetrate both spheroids and in vivo tumors, thereby enhancing the therapeutic efficacy. These results suggest that innate biological transport systems can be in situ engineered via synthetic liposomes to guide the penetration of chemotherapeutics across challenging tissue barriers in solid tumors.

  5. Active penetration of Trypanosoma cruzi into host cells: historical considerations and current concepts

    Directory of Open Access Journals (Sweden)

    Tecia Maria Ulisses Carvalho


    Full Text Available A significant number of scientific groups working on several countries have made efforts to better understand the process of invasion of several types of host cells by Trypanosoma cruzi, the etiologic agent of Chagas disease. In this mini-review we analyze the two mechanisms of invasion considered to be relevant: active penetration and endocytosis. The term active penetration is considered in view of its original description by Dvorak and co-workers. Taking into consideration all results obtained we conclude that endocytosis, with its many variations, is the only mechanism used by T. cruzi to invade host cells.

  6. Effects of antiphagocytic agents on penetration of Eimeria magna sporozoites into cultured cells. (United States)

    Jensen, J B; Edgar, S A


    Madin-Darby Bovine Kidney cells were treated with sodium flouride, iodoacetate, and 2-deosyglucose, reagents that block glycolysis, and thus reduce phagocytosis. Sporozoites readily entered cells whose ATP stores were largely depleted. They also entered cells treated with colchicine, colcemid, and vinblastine. These latter agents did not inhibit sporozite motility after 6 hr incubation. Cytochalasin B prevented penetration of cells by inhibiting the motility of sporozoites. This effect was reversible. Warm sporozoites entered cold cells 4 times more radily than cold sporozoites into warm cells. The above findings suggest that phagocytosis is not the mechanism for entry of E. magna sporozoites into cultured cells, but that sporozoite motility is of primary importance.

  7. Structure of Phage P22 Cell Envelope-Penetrating Needle

    Energy Technology Data Exchange (ETDEWEB)

    Olia, A.S.; Casjens, S.; Cingolani, G.


    Bacteriophage P22 infects Salmonella enterica by injecting its genetic material through the cell envelope. During infection, a specialized tail needle, gp26, is injected into the host, likely piercing a hole in the host cell envelope. The 2.1-{angstrom} crystal structure of gp26 reveals a 240-{angstrom} elongated protein fiber formed by two trimeric coiled-coil domains interrupted by a triple {beta}-helix. The N terminus of gp26 plugs the portal protein channel, retaining the genetic material inside the virion. The C-terminal tip of the fiber exposes {beta}-hairpins with hydrophobic tips similar to those seen in class II fusion peptides. The {alpha}-helical core connecting these two functionally polarized tips presents four trimerization octads with consensus sequence IXXLXXXV. The slender conformation of the gp26 fiber minimizes the surface exposed to solvent, which is consistent with the idea that gp26 traverses the cell envelope lipid bilayers.

  8. Nanoformulated cell-penetrating survivin mutant and its dual actions

    Directory of Open Access Journals (Sweden)

    Sriramoju B


    Full Text Available Bhasker Sriramoju, Rupinder K Kanwar, Jagat R Kanwar Nanomedicine Laboratory of Immunology and Molecular Biomedical Research (NLIMBR, School of Medicine, Faculty of Health, Deakin University, Geelong, Australia Abstract: In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells, and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome. Keywords: survivin mutant, neurological disorders, protein therapeutics, inhibitor of apoptosis protein family, poly(lactic-co-glycolic acid

  9. Polymeric pH nanosensor with extended measurement range bearing octaarginine as cell penetrating peptide

    DEFF Research Database (Denmark)

    Ke, Peng; Sun, Honghao; Liu, Mingxing;


    A synthetic peptide octaarginine which mimics human immunodeficiency virus-1, Tat protein is used as cell penetrating moiety for new pH nanosensors which demonstrate enhanced cellular uptake and expanded measurement range from pH 3.9 to pH 7.3 by simultaneously incorporating two complemental pH-s...

  10. Application of Cell Penetrating Peptide in Magnetic Resonance Imaging of Bone Marrow Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Min LIU; You-Min GUO; Jun-Le YANG; Peng WANG; Lin-Yu ZHAO; Nian SHEN; Si-Cen WANG; Xiao-Juan GUO; Qi-Fei WU


    Tracking the distribution and differentiation of stem cells by high-resolution imaging techniques would have significant clinical and research implications. In this study, a model cell-penetrating peptide was used to carry gadolinium particles for magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs).MSCs were isolated from rat bone marrow and identified by osteogenic differentiation in vitro. The cellpenetrating peptide labeled with fluorescein-5-isothiocyanate (FITC) and gadolinium was synthesized by a solid-phase peptide synthesis method. Fluorescein imaging analysis confirmed that this new peptide could internalize into the cytoplasm and nucleus at room temperature, 4℃ and 37℃. Gadolinium were efficiently internalized into mesenchymal stem cells by the peptide in a time or concentration-dependent manner,resulting in intercellular shortening of longitudinal relaxation enhancements, which were obviously detected by 1.5 Tesla Magnetic Resonance Imaging. Cytotoxicity assay and flow cytometric analysis showed that the intercellular contrast medium incorporation did not affect cell viability at the tested concentrations. The in vitro experiment results suggested that the new constructed peptides could be a vector for tracking MSCs.

  11. Cell penetrating peptide-modified poly(lactic-co-glycolic acid) nanoparticles with enhanced cell internalization. (United States)

    Steinbach, Jill M; Seo, Young-Eun; Saltzman, W Mark


    The surface modification of nanoparticles (NPs) can enhance the intracellular delivery of drugs, proteins, and genetic agents. Here we studied the effect of different surface ligands, including cell penetrating peptides (CPPs), on the cell binding and internalization of poly(lactic-co-glycolic) (PLGA) NPs. Relative to unmodified NPs, we observed that surface-modified NPs greatly enhanced cell internalization. Using one CPP, MPG (unabbreviated notation), that achieved the highest degree of internalization at both low and high surface modification densities, we evaluated the effect of two different NP surface chemistries on cell internalization. After 2h, avidin-MPG NPs enhanced cellular internalization by 5 to 26-fold relative to DSPE-MPG NP formulations. Yet, despite a 5-fold increase in MPG density on DSPE compared to Avidin NPs, both formulations resulted in similar internalization levels (48 and 64-fold, respectively) after 24h. Regardless of surface modification, all NPs were internalized through an energy-dependent, clathrin-mediated process, and became dispersed throughout the cell. Overall both Avidin- and DSPE-CPP modified NPs significantly increased internalization and offer promising delivery options for applications in which internalization presents challenges to efficacious delivery.

  12. Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides.

    Directory of Open Access Journals (Sweden)

    Caghan Kizil

    Full Text Available Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI, RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

  13. Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs


    Ru, Renli; Yao, Yongchao; Yu, Songlin; Yin, Benpeng; Xu, Wanwan; Zhao, Siting; Li QIN; Chen, Xiaoping


    Background Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed. Results In this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional...

  14. Topical Delivery of Protein and Peptide Using Novel Cell Penetrating Peptide IMT-P8 (United States)

    Gautam, Ankur; Nanda, Jagpreet Singh; Samuel, Jesse S.; Kumari, Manisha; Priyanka, Priyanka; Bedi, Gursimran; Nath, Samir K.; Mittal, Garima; Khatri, Neeraj; Raghava, Gajendra Pal Singh


    Skin, being the largest organ of the body, is an important site for drug administration. However, most of the drugs have poor permeability and thus drug delivery through the skin is very challenging. In this study, we examined the transdermal delivery capability of IMT-P8, a novel cell-penetrating peptide. We generated IMT-P8-GFP and IMT-P8-KLA fusion constructs and evaluated their internalization into mouse skin after topical application. Our results demonstrate that IMT-P8 is capable of transporting green fluorescent protein (GFP) and proapoptotic peptide, KLA into the skin and also in different cell lines. Interestingly, uptake of IMT-P8-GFP was considerably higher than TAT-GFP in HeLa cells. After internalization, IMT-P8-KLA got localized to the mitochondria and caused significant cell death in HeLa cells signifying an intact biological activity. Further in vivo skin penetration experiments revealed that after topical application, IMT-P8 penetrated the stratum corneum, entered into the viable epidermis and accumulated inside the hair follicles. In addition, both IMT-P8-KLA and IMT-P8-GFP internalized into the hair follicles and dermal tissue of the skin following topical application. These results suggested that IMT-P8 could be a potential candidate to be used as a topical delivery vehicle for various cosmetic and skin disease applications. PMID:27189051

  15. Conformational analysis of Infectious bursal disease virus (IBDV derived cell penetrating peptide (CPP analogs

    Directory of Open Access Journals (Sweden)

    Vinay G. Joshi


    Full Text Available Aim: This study was designed to develop peptide analogs of Infectious Bursal Disease (IBD virus VP5 protein segment having cell penetrating ability to improve their interaction with cargo molecule (Nucleic acid without affecting the backbone conformation. Materials and Methods: IBDV VP5 protein segment designated as RATH peptide were synthesized using solid phase peptide synthesis and their solution conformation was elucidated using CD spectroscopy in polar (water and apolar (TFE solvents. Cell penetrating ability of RATH-CONH2 was observed using FITC labeled peptide internalization in to HeLa cells under fluorescent microscopy. The efficacy of RATH analog interactions with nucleic acids was evaluated using FITC labeled oligonucleotides by fluorescence spectroscopy and plasmid constructs in gel retardation assay. Results: CD spectra of RATH analogs in water and apolar trifluroethanol (TFE helped to compare their secondary structures which were almost similar with dominant beta conformations suggesting successful induction of positive charge in the analogs without affecting back bone conformation of CPP designed. Cell penetrating ability of RATH CONH2 in HeLa cell was more than 90%. The fluorescence spectroscopy and plasmid constructs in gel retardation assay demonstrated successful interaction of amide analogs with nucleic acid. Conclusion: Intentional changes made in IBDV derived peptide RATH COOH to RATH CONH2 did not showed major changes in backbone conformation and such modifications may help to improve the cationic charge in most CPPs to interact with nucleic acid. [Vet World 2013; 6(6.000: 307-312

  16. MMP-2/9-Specific Activatable Lifetime Imaging Agent

    Directory of Open Access Journals (Sweden)

    Marcus T.M. Rood


    Full Text Available Optical (molecular imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable lifetime imaging agent. Important in the design of this imaging agent is the use of two luminophores that are tethered by a specific peptide with a hairpin-motive that ensured close proximity of the two while also having a specific amino acid sequence available for enzymatic cleavage by tumor-related MMP-2/9. Ir(ppy3 and Cy5 were used because in close proximity the emission intensities of both luminophores were quenched and the influence of Cy5 shortens the Ir(ppy3 luminescence lifetime from 98 ns to 30 ns. Upon cleavage in vitro, both effects are undone, yielding an increase in Ir(ppy3 and Cy5 luminescence and a restoration of Ir(ppy3 luminescence lifetime to 94 ns. As a reference for the luminescence activation, a similar imaging agent with the more common Cy3-Cy5 fluorophore pair was used. Our findings underline that the combination of enzymatic signal activation with lifetime imaging is possible and that it provides a promising method in the design of future disease specific imaging agents.

  17. The bacteriophage ϕ29 tail possesses a pore-forming loop for cell membrane penetration. (United States)

    Xu, Jingwei; Gui, Miao; Wang, Dianhong; Xiang, Ye


    Most bacteriophages are tailed bacteriophages with an isometric or a prolate head attached to a long contractile, long non-contractile, or short non-contractile tail. The tail is a complex machine that plays a central role in host cell recognition and attachment, cell wall and membrane penetration, and viral genome ejection. The mechanisms involved in the penetration of the inner host cell membrane by bacteriophage tails are not well understood. Here we describe structural and functional studies of the bacteriophage ϕ29 tail knob protein gene product 9 (gp9). The 2.0 Å crystal structure of gp9 shows that six gp9 molecules form a hexameric tube structure with six flexible hydrophobic loops blocking one end of the tube before DNA ejection. Sequence and structural analyses suggest that the loops in the tube could be membrane active. Further biochemical assays and electron microscopy structural analyses show that the six hydrophobic loops in the tube exit upon DNA ejection and form a channel that spans the lipid bilayer of the membrane and allows the release of the bacteriophage genomic DNA, suggesting that cell membrane penetration involves a pore-forming mechanism similar to that of certain non-enveloped eukaryotic viruses. A search of other phage tail proteins identified similar hydrophobic loops, which indicates that a common mechanism might be used for membrane penetration by prokaryotic viruses. These findings suggest that although prokaryotic and eukaryotic viruses use apparently very different mechanisms for infection, they have evolved similar mechanisms for breaching the cell membrane.

  18. Cell-Penetrating Peptides—Mechanisms of Cellular Uptake and Generation of Delivery Systems

    Directory of Open Access Journals (Sweden)

    Sara Trabulo


    Full Text Available The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, mandatory. Since the seminal observations two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides (CPPs have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial. Over the last years, our research group has been focused on the S413-PV cell-penetrating peptide, a prototype of this class of peptides that results from the combination of 13-amino-acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal. By performing an extensive biophysical and biochemical characterization of this peptide and its analogs, we have gained important insights into the mechanisms governing the interaction of CPPs with cells and their translocation across biological membranes. More recently, we have started to explore this peptide for the intracellular delivery of nucleic acids (plasmid DNA, siRNA and oligonucleotides. In this review we discuss the current knowledge of the mechanisms responsible for the cellular uptake of cell-penetrating peptides, including the S413-PV peptide, and the potential of peptide-based formulations to mediate nucleic acid delivery.

  19. Dimerization of a cell-penetrating peptide leads to enhanced cellular uptake and drug delivery

    Directory of Open Access Journals (Sweden)

    Jan Hoyer


    Full Text Available Over the past 20 years, cell-penetrating peptides (CPPs have gained tremendous interest due to their ability to deliver a variety of therapeutically active molecules that would otherwise be unable to cross the cellular membrane due to their size or hydrophilicity. Recently, we reported on the identification of a novel CPP, sC18, which is derived from the C-terminus of the 18 kDa cationic antimicrobial protein. Furthermore, we demonstrated successful application of sC18 for the delivery of functionalized cyclopentadienyl manganese tricarbonyl (cymantrene complexes to tumor cell lines, inducing high cellular toxicity. In order to increase the potential of the organometallic complexes to kill tumor cells, we were looking for a way to enhance cellular uptake. Therefore, we designed a branched dimeric variant of sC18, (sC182, which was shown to have a dramatically improved capacity to internalize into various cell lines, even primary cells, using flow cytometry and fluorescence microscopy. Cell viability assays indicated increased cytotoxicity of the dimer presumably caused by membrane leakage; however, this effect turned out to be dependent on the specific cell type. Finally, we could show that conjugation of a functionalized cymantrene with (sC182 leads to significant reduction of its IC50 value in tumor cells compared to the respective sC18 conjugate, proving that dimerization is a useful method to increase the drug-delivery potential of a cell-penetrating peptide.

  20. 31P Solid-state NMR based monitoring of permeation of cell penetrating peptides into skin (United States)

    Desai, Pinaki R.; Cormier, Ashley R.; Shah, Punit P.; Patlolla, Ram R.; Paravastu, Anant K.; Singh, Mandip


    The main objective of the current study was to investigate penetration of cell penetrating peptides (CPPs: TAT, R8, R11 and YKA) through skin intercellular lipids using 31P magic angle spinning (MAS) solid-state NMR. In vitro skin permeation studies were performed on rat skin, sections (0–60, 61–120 and 121–180 µm) were collected and analyzed for 31P NMR signal. The concentration dependent shift of 0, 25, 50, 100 and 200 mg/ml of TAT on skin layers, diffusion of TAT, R8, R11 and YKA in the skin and time dependent permeation of R11 was measured on various skin sections using 31P solid-state NMR. Further, CPPs and CPP-tagged fluorescent dye encapsulate liposomes (FLip) in skin layers were tagged using confocal microscopy. The change in 31P NMR chemical shift was found to depend monotonically on the amount of CPP applied on skin, with saturation behavior above 100 mg/ml CPP concentration. R11 and TAT caused more shift in solid-state NMR peaks compared to other peptides. Furthermore, NMR spectra showed R11 penetration up to 180 µm within 30 min. The results of the solid-state NMR study were in agreement with confocal microscopy studies. Thus, 31P solid-state NMR can be used to track CPP penetration into different skin layers. PMID:23702274

  1. Cell-Penetrating Ability of Peptide Hormones: Key Role of Glycosaminoglycans Clustering

    Directory of Open Access Journals (Sweden)

    Armelle Tchoumi Neree


    Full Text Available Over the last two decades, the potential usage of cell-penetrating peptides (CPPs for the intracellular delivery of various molecules has prompted the identification of novel peptidic identities. However, cytotoxic effects and unpredicted immunological responses have often limited the use of various CPP sequences in the clinic. To overcome these issues, the usage of endogenous peptides appears as an appropriate alternative approach. The hormone pituitary adenylate-cyclase-activating polypeptide (PACAP38 has been recently identified as a novel and very efficient CPP. This 38-residue polycationic peptide is a member of the secretin/glucagon/growth hormone-releasing hormone (GHRH superfamily, with which PACAP38 shares high structural and conformational homologies. In this study, we evaluated the cell-penetrating ability of cationic peptide hormones in the context of the expression of cell surface glycosaminoglycans (GAGs. Our results indicated that among all peptides evaluated, PACAP38 was unique for its potent efficiency of cellular uptake. Interestingly, the abilities of the peptides to reach the intracellular space did not correlate with their binding affinities to sulfated GAGs, but rather to their capacity to clustered heparin in vitro. This study demonstrates that the uptake efficiency of a given cationic CPP does not necessarily correlate with its affinity to sulfated GAGs and that its ability to cluster GAGs should be considered for the identification of novel peptidic sequences with potent cellular penetrating properties.

  2. Evaluation of a cell penetrating prenylated peptide lacking an intrinsic fluorophore via in situ click reaction. (United States)

    Ochocki, Joshua D; Mullen, Daniel G; Wattenberg, Elizabeth V; Distefano, Mark D


    Protein prenylation involves the addition of either a farnesyl (C(15)) or geranylgeranyl (C(20)) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These peptides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group is still present [Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Wattenberg, E. V.; Barany, G.; Distefano, M. D. Multifunctional Prenylated Peptides for Live Cell Analysis. J. Am. Chem. Soc.2009, 131, 7293-7303]. The present study investigates the effect of removing the fluorophore from the peptides and investigating the uptake by confocal microscopy and flow cytometry. Our results show that the fluorophore is not necessary for uptake of these peptides. This information is significant because it indicates that the prenyl group is the major determinant in allowing these peptides to enter cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells.

  3. 可激活的细胞穿透肽:一种应用于疾病诊断和治疗的极具潜力的可活化模式%Activatable cell-penetrating peptides: a potential activatable modality for diseases diagnosis and therapy

    Institute of Scientific and Technical Information of China (English)

    杨少坤; 闫逍; 孙伟红; 杜青; 向柏; 曹德英; 齐宪荣



  4. Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tingting Lin


    Full Text Available Brain delivery of macromolecular therapeutics (e.g., proteins remains an unsolved problem because of the formidable blood–brain barrier (BBB. Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion, a low molecular weight protamine (LMWP cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

  5. Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides. (United States)

    Lin, Tingting; Liu, Ergang; He, Huining; Shin, Meong Cheol; Moon, Cheol; Yang, Victor C; Huang, Yongzhuo


    Brain delivery of macromolecular therapeutics (e.g., proteins) remains an unsolved problem because of the formidable blood-brain barrier (BBB). Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion), a low molecular weight protamine (LMWP) cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP) have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP-proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases.

  6. Antibacterial Effects of a Cell-Penetrating Peptide Isolated from Kefir. (United States)

    Miao, Jianyin; Guo, Haoxian; Chen, Feilong; Zhao, Lichao; He, Liping; Ou, Yangwen; Huang, Manman; Zhang, Yi; Guo, Baoyan; Cao, Yong; Huang, Qingrong


    Kefir is a traditional fermented milk beverage used throughout the world for centuries. A cell-penetrating peptide, F3, was isolated from kefir by Sephadex G-50 gel filtration, DEAE-52 ion exchange, and reverse-phase high-performance liquid chromatography. F3 was determined to be a low molecular weight peptide containing one leucine and one tyrosine with two phosphate radicals. This peptide displayed antimicrobial activity across a broad spectrum of organisms including several Gram-positive and Gram-negative bacteria as well as fungi, with minimal inhibitory concentration (MIC) values ranging from 125 to 500 μg/mL. Cellular penetration and accumulation of F3 were determined by confocal laser scanning microscopy. The peptide was able to penetrate the cellular membrane of Escherichia coli and Staphylococcus aureus. Changes in cell morphology were observed by scanning electron microscopy (SEM). The results indicate that peptide F3 may be a good candidate for use as an effective biological preservative in agriculture and the food industry.

  7. Metabolic cleavage of cell-penetrating peptides in contact with epithelial models

    DEFF Research Database (Denmark)

    Tréhin, Rachel; Nielsen, Hanne Mørck; Jahnke, Heinz-Georg;


    We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47......-57) and penetratin(43-58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin-Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting...

  8. Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection (United States)

    Li, Wenyu; Liu, Yajie; Du, Jianwei; Ren, Kefeng; Wang, Youxiang


    Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 μg mL-1. Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers.Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid

  9. Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration. (United States)

    Park, Hee Ho; Sohn, Youngsoo; Yeo, Ji Woo; Park, Ju Hyun; Lee, Hong Jai; Ryu, Jina; Rhee, Won Jong; Park, Tai Hyun


    Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.

  10. Distinct behaviour of the homeodomain derived cell penetrating peptide penetratin in interaction with different phospholipids.

    Directory of Open Access Journals (Sweden)

    Ofelia Maniti

    Full Text Available BACKGROUND: Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC and phosphatidylethanolamine (PE to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and β-sheet contributions. CONCLUSIONS/SIGNIFICANCE: These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import and on prokaryotes may result from different peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution.

  11. Cationic cell-penetrating peptides as vehicles for siRNA delivery. (United States)

    Beloor, Jagadish; Zeller, Skye; Choi, Chang Seon; Lee, Sang-Kyung; Kumar, Priti


    RNA interference mediated gene silencing has tremendous applicability in fields ranging from basic biological research to clinical therapy. However, delivery of siRNA across the cell membrane into the cytoplasm, where the RNA silencing machinery is located, is a significant hurdle in most primary cells. Cell-penetrating peptides (CPPs), peptides that possess an intrinsic ability to translocate across cell membranes, have been explored as a means to achieve cellular delivery of siRNA. Approaches using CPPs by themselves or through incorporation into other siRNA delivery platforms have been investigated with the intent of improving cytoplasmic delivery. Here, we review the utilization of CPPs for siRNA delivery with a focus on strategies developed to enhance cellular uptake, endosomal escape and cytoplasmic localization of CPP/siRNA complexes.

  12. Poly(NIPAm-AMPS) nanoparticles for targeted delivery of anti-inflammatory cell penetrating peptides (United States)

    Bartlett, Rush Lloyd, II

    Inflammatory diseases such as osteoarthritis and rheumatoid arthritis cause $127.8 billion in US healthcare expenditures each year and are the cause of disability for 27% of disabled persons in the United States. Current treatment options rarely halt disease progression and often result in significant unwanted and debilitating side effects. Our laboratory has previously developed a family of cell penetrating peptides (CPPs) which inhibit the activity of mitogen activated protein kinase activate protein kinase 2 (MK2). MK2 mediates the inflammatory response by activating Tristetraprline (TTP). Once activated, TTP rapidly stabilizes AU rich regions of pro-inflammatory cytokine mRNA which allows translation of pro-inflammatory cytokines to occur. Blocking MK2 with our labs CPPs yields a decrease in inflammatory activity but CPPs by are highly non specific and prone to rapid enzymatic degradation in vivo.. In order to increase the potency of MK2 inhibiting CPPs we have developed a novel nanoparticle drug carrier composed of poly(N-isopropylacrylamide-co-2-acrylamido-2-methyl-1-propanesulfonic acid). This drug carrier has been shown to have preliminary efficacy in vitro and ex vivo for suppressing pro-inflammatory cytokine production when releasing CPPs. This thesis will present progress made on three aims: Specific Aim 1) Create and validate a NIPAm based drug delivery system that mimics the binding and release previously observed between cell penetrating peptides and glycosaminoglycans. Specific Aim 2) Engineer degradability into poly(NIPAm-AMPS) nanoparticles to enable more drug to be released and qualify that system in vitro. Specific Aim 3) Validate poly(NIPAm-AMPS) nanoparticles for targeted drug delivery in an ex vivo inflammatory model. Overall we have developed a novel anionic nanoparticle system that is biocompatible and efficient at loading and releasing cell penetrating peptides to inflamed tissue. Once loaded with a CPP the nanoparticle drug complex is

  13. Cell-penetrating peptides as tools to enhance non-injectable delivery of biopharmaceuticals

    DEFF Research Database (Denmark)

    Kristensen, Mie; Nielsen, Hanne Mørck


    Non-injectable delivery of peptide and protein drugs is hampered by their labile nature, hydrophilicity, and large molecular size; thus limiting their permeation across mucosae, which represent major biochemical and physical barriers to drugs administered via e.g. the oral, nasal, and pulmonary...... routes. However, in recent years cell-penetrating peptides (CPP) have emerged as promising tools to enhance mucosal delivery of co-administered or conjugated peptide and protein cargo and more advanced CPP-cargo formulations are emerging. CPPs act as transepithelial delivery vectors, but the mechanism...

  14. Conjugation with Cationic Cell-Penetrating Peptide Increases Pulmonary Absorption of Insulin


    Patel, Leena N.; Wang, Jeffrey; Kim, Kwang-Jin; Borok, Zea; Crandall, Edward; Shen, Wei-Chiang


    In this study, we determined if cell-penetrating peptides (CPPs) can be used to enhance the absorption rate of insulin (INS) across the alveolar epithelial barrier. Using a heterobifuctional crosslinker, INS was conjugated to a series of cationic CPPs, including Tat peptide, oligoarginine (r9) or oligolysine (k9), via disulfide bridge to a D-isoform cysteine (c) present at the N-terminal of the peptide sequence, yielding INS-cTat, INS-cr9, and INS-ck9, respectively. SDS-PAGE and MALDI-TOF mas...

  15. Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

    DEFF Research Database (Denmark)

    Ndeboko, Bénédicte; Lemamy, Guy Joseph; Nielsen, Peter E


    Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers...... hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its...... against chronic hepatitis B....

  16. SAP(E) - A cell-penetrating polyproline helix at lipid interfaces. (United States)

    Franz, Johannes; Lelle, Marco; Peneva, Kalina; Bonn, Mischa; Weidner, Tobias


    Cell-penetrating peptides (CPPs) are short membrane-permeating amino acid sequences that can be used to deliver cargoes, e.g. drugs, into cells. The mechanism for CPP internalization is still subject of ongoing research. An interesting family of CPPs is the sweet arrow peptides - SAP(E) - which are known to adopt a polyproline II helical secondary structure. SAP(E) peptides stand out among CPPs because they carry a net negative charge while most CPPs are positively charged, the latter being conducive to electrostatic interaction with generally negatively charged membranes. For SAP(E)s, an internalization mechanism has been proposed, based on polypeptide aggregation on the cell surface, followed by an endocytic uptake. However, this process has not yet been observed directly - since peptide-membrane interactions are inherently difficult to monitor on a molecular scale. Here, we use sum frequency generation (SFG) vibrational spectroscopy to investigate molecular interactions of SAP(E) with differently charged model membranes, in both mono- and bi-layer configurations. The data suggest that the initial binding mechanism is accompanied by structural changes of the peptide. Also, the peptide-model membrane interaction depends on the charge of the lipid headgroup with phosphocholine being a favorable binding site. Moreover, while direct penetration has also been observed for some CPPs, the spectroscopy reveals that for SAP(E), its interaction with model membranes remains limited to the headgroup region, and insertion into the hydrophobic core of the lipid layer does not occur.

  17. Cell-Penetrating Cross-β Peptide Assemblies with Controlled Biodegradable Properties. (United States)

    Han, Sanghun; Lee, Mun-Kyung; Lim, Yong-Beom


    Although self-assembled peptide nanostructures (SPNs) have shown potential as promising biomaterials, there is a potential problem associated with the extremely slow hydrolysis rate of amide bonds. Here, we report the development of cell-penetrating cross-β SPNs with a controllable biodegradation rate. The designed self-assembling β-sheet peptide incorporating a hydrolyzable ester bond (self-assembling depsipeptide; SADP) can be assembled into bilayer β-sandwich one-dimensional (1D) fibers similarly to conventional β-sheet peptides. The rate of hydrolysis can be controlled by the pH, temperature, and structural characteristics of the ester unit. The 1D fiber of the SADP transforms into vesicle-like 3D structures when the hydrophilic cell-penetrating peptide segment is attached to the SADP segment. Efficient cell internalization of the 3D nanostructures was observed, and we verified the intracellular degradation and disassembly of the biodegradable nanostructures. This study illustrates the potential of biodegradable cross-β SPNs and provides a valuable toolkit that can be used with self-assembling peptides.

  18. Purification and characterization of a novel cell-penetrating carrier similar to cholera toxin chimeric protein. (United States)

    Lin, Weiping; Zheng, Xi; Wang, Huaqian; Yu, Lin; Zhou, Xiaofen; Sun, Yunxiao; Zhao, Suqing; Du, Zhiyun; Zhang, Kun


    Developing a recombinant vector for noninvasively delivering biological macromolecules into the brain is important. This study constructed and purified a protein complex based on the cholera toxin (CT) molecular structure. Enhanced green fluorescent protein (EGFP)-modified A2 subunits of CT (CTA2) were used as tracer molecules for introduction of transactivator of transcription (TAT) through the A subunit into cells. The protein complex EGFP-CTA2-TAT/(CTB)5 (CTB: B subunit of CT) was obtained using an in vitro recombination method and verified by monosialoganglioside-enzyme-linked immunosorbent assay and high performance liquid chromatography assay. The protein complexes bound more strongly to monosialoganglioside (GM1) than (CTB)5 at low concentrations (0.625-1.25 μg/mL). In vitro assays revealed that the transmembrane function of TAT was also maintained. The GM1-binding activity and cell membrane-penetrating ability suggested that a CT structure-based protein complexes could be used to design a delivery carrier for intranasal administration through GM1 binding. The expression vector introduced in this study provides a feasible expression frame for constructing several new macromolecular protein drugs for effective cell penetration.

  19. Synthesis of cell-penetrating peptides and their application in neurobiology. (United States)

    Dietz, Gunnar P H; Bähr, Mathias


    Short basic amino acid sequences, often called cell-penetrating peptides (CPPs), allow the delivery of proteins and other molecules into cells and across the blood-brain barrier (BBB). Although the ability of basic proteins to facilitate such trafficking is known for a long time, only the application of genetic methods and overexpression of fusion proteins in Escherichia coli has lead to a wide application of CPP in many research areas, including signal transduction, cancer, angiogenesis, apoptosis, bone development, cardioprotection, cell cycle, neurobiology, and many others. For the neuroscientist, CPPs are particularly attractive, as a number of articles in the last 5 years have reported their use for neuronal rescue in a number of models for neurodegenerative diseases in vitro and in vivo in rats, mice, or gerbils. Here, we give a detailed description of the protein purification methodology and applications in neuroscience.

  20. Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available Transcription activator-like (TAL effector nucleases (TALENs have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.

  1. Cell-penetrating peptide-mediated delivery of TALEN proteins via bioconjugation for genome engineering. (United States)

    Liu, Jia; Gaj, Thomas; Patterson, James T; Sirk, Shannon J; Barbas, Carlos F


    Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.

  2. Technique for internal channelling of hydroentangled nonwoven scaffolds to enhance cell penetration. (United States)

    Durham, Elaine R; Ingham, Eileen; Russell, Stephen J


    An important requirement in thick, high-porosity scaffolds is to maximise cellular penetration into the interior and avoid necrosis during culture in vitro. Hitherto, reproducible control of the pore structure in nonwoven scaffolds has proved challenging. A new, channelled scaffold manufacturing process is reported based on water jet entanglement of fibres (hydroentangling) around filamentous template to form a coherent scaffold that is subsequently removed. Longitudinally-oriented channels were introduced within the scaffold in controlled proximity using 220 µm diameter cylindrical templates. In this case study, channelled scaffolds composed of poly(l-lactic acid) were manufactured and evaluated in vitro. Environmental scanning electron microscope and µCT (X-ray microtomography) confirmed channel openings in the scaffold cross-section before and after cell culture with human dermal fibroblasts up to 14 weeks. Histology at week 11 indicated that the channels promoted cell penetration and distribution within the scaffold interior. At week 14, cellular matrix deposition was evident in the internal channel walls and the entrances remained unoccluded by cellular matrix suggesting that diffusion conduits for mass transfer of nutrient to the scaffold interior could be maintained.

  3. Trends in the Binding of Cell Penetrating Peptides to siRNA: A Molecular Docking Study (United States)

    Gunathunge, B. G. C. M.; Wimalasiri, P. N.; Karunaratne, D. N.


    The use of gene therapeutics, including short interfering RNA (siRNA), is limited by the lack of efficient delivery systems. An appealing approach to deliver gene therapeutics involves noncovalent complexation with cell penetrating peptides (CPPs) which are able to penetrate the cell membranes of mammals. Although a number of CPPs have been discovered, our understanding of their complexation and translocation of siRNA is as yet insufficient. Here, we report on computational studies comparing the binding affinities of CPPs with siRNA, considering a variety of CPPs. Specifically, seventeen CPPs from three different categories, cationic, amphipathic, and hydrophobic CPPs, were studied. Molecular mechanics were used to minimize structures, while molecular docking calculations were used to predict the orientation and favorability of sequentially binding multiple peptides to siRNA. Binding scores from docking calculations were highest for amphipathic peptides over cationic and hydrophobic peptides. Results indicate that initial complexation of peptides will likely occur along the major groove of the siRNA, driven by electrostatic interactions. Subsequent binding of CPPs is likely to occur in the minor groove and later on bind randomly, to siRNA or previously bound CPPs, through hydrophobic interactions. However, hydrophobic CPPs do not show this binding pattern. Ultimately binding yields a positively charged nanoparticle capable of noninvasive cellular import of therapeutic molecules.

  4. Selective mono-radioiodination and characterization of a cell-penetrating peptide. L-Tyr-maurocalcine

    Energy Technology Data Exchange (ETDEWEB)

    Ahmadi, Mitra; Bacot, Sandrine; Perret, Pascale; Riou, Laurent; Ghezzi, Catherine [Universite Joseph Fourier, Grenoble (France); INSERM U1039, Grenoble (France). Radiopharmaceutiques Biocliniques; Poillot, Cathy; Cestele, Sandrine [INSERM U836, Grenoble (France). Grenoble Inst. of Neuroscience; Universite Joseph Fourier, Grenoble (France); Desruet, Marie-Dominique [INSERM U1039, Grenoble (France). Radiopharmaceutiques Biocliniques; Couvet, Morgane; Bourgoin, Sandrine; Seve, Michel [CRI-INSERM U823, Grenoble (France). Inst. of Albert Bonniot; Universite Joseph Fourier, Grenoble (France); Waard, Michel de [INSERM U836, Grenoble (France). Grenoble Inst. of Neuroscience; Universite Joseph Fourier, Grenoble (France); Smartox Biotechnologies, Grenoble (France)


    Mono-and poly-iodinated peptides form frequently during radioiodination procedures. However, the formation of a single species in its mono-iodinated form is essential for quantitative studies such as determination of tissue concentration or image quantification. Therefore, the aim of the present study was to define the optimal experimental conditions in order to exclusively obtain the mono-iodinated form of L-maurocalcine (L-MCa). L-MCa is an animal venom toxin which was shown to act as a cell-penetrating peptide. In order to apply the current direct radioiodination technique using oxidative agents including chloramine T, Iodo-Gen {sup registered} or lactoperoxidase, an analogue of this peptide containing a tyrosine residue (Tyr-L-MCa) was synthesized and was shown to fold/oxidize properly. The enzymatic approach using lactoperoxidase/H{sub 2}O{sub 2} was found to be the best method for radioiodination of Tyr-L-MCa. MALDI-TOF mass spectrometry analyses were then used for identification of the chromatographic eluting components of the reaction mixtures. We observed that the production of different radioiodinated species depended upon the reaction conditions. Our results successfully described the experimental conditions of peptide radioiodination allowing the exclusive production of the mono-iodinated form with high radiochemical purity and without the need for a purification step. Mono-radioiodination of L-Tyr-MCa will be crucial for future quantitative studies, investigating the mechanism of cell penetration and in vivo biodistribution.

  5. p53 Aggregates penetrate cells and induce the co-aggregation of intracellular p53.

    Directory of Open Access Journals (Sweden)

    Karolyn J Forget

    Full Text Available Prion diseases are unique pathologies in which the infectious particles are prions, a protein aggregate. The prion protein has many particular features, such as spontaneous aggregation, conformation transmission to other native PrP proteins and transmission from an individual to another. Protein aggregation is now frequently associated to many human diseases, for example Alzheimer's disease, Parkinson's disease or type 2 diabetes. A few proteins associated to these conformational diseases are part of a new category of proteins, called prionoids: proteins that share some, but not all, of the characteristics associated with prions. The p53 protein, a transcription factor that plays a major role in cancer, has recently been suggested to be a possible prionoid. The protein has been shown to accumulate in multiple cancer cell types, and its aggregation has also been reproduced in vitro by many independent groups. These observations suggest a role for p53 aggregates in cancer development. This study aims to test the «prion-like» features of p53. Our results show in vitro aggregation of the full length and N-terminally truncated protein (p53C, and penetration of these aggregates into cells. According to our findings, the aggregates enter cells using macropinocytosis, a non-specific pathway of entry. Lastly, we also show that once internalized by the cell, p53C aggregates can co-aggregate with endogenous p53 protein. Together, these findings suggest prion-like characteristics for p53 protein, based on the fact that p53 can spontaneously aggregate, these aggregates can penetrate cells and co-aggregate with cellular p53.

  6. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells. (United States)

    Kaitsuka, Taku; Tomizawa, Kazuhito


    Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

  7. Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Taku Kaitsuka


    Full Text Available Protein transduction using cell-penetrating peptides (CPPs is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

  8. Emerging landscape of cell penetrating peptide in reprogramming and gene editing. (United States)

    Liu, Huiting; Zeng, Fanhui; Zhang, Ming; Huang, Fajun; Wang, Jiajun; Guo, Jingjing; Liu, Changbai; Wang, Hu


    The plasma membrane remains a major barrier for intracellular drug delivery, to overcome this issue, a variety of approaches have been developed and used to deliver therapeutic cargos. Among these approaches, cell penetrating peptide (CPP) is promising and affords widely used vector for efficient intracellular delivery of cargos. Moreover, the latter findings including iPS reprogramming and direct transdifferentiation as well as gene editing have gradually become hot research topic; because their application in tissue engineering and disease modeling have great potential to advance innovation in precision medicine. Since the beginning, research on these approaches is mainly based on virus transduction system, while, under the consideration for obviating the risk of mutagenic insertion and enables more accurate controlling, CPP-based efficient virus-free delivery strategy has been used recently. In this review, we summarize the existing CPP-based delivery system, emerging landscape of CPP application in stem cell manipulation and reprogramming, along with CPP contributions to gene editing techniques.

  9. Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides. (United States)

    Oehlke, J; Birth, P; Klauschenz, E; Wiesner, B; Beyermann, M; Oksche, A; Bienert, M


    The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

  10. Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient. (United States)

    Madani, Fatemeh; Abdo, Rania; Lindberg, Staffan; Hirose, Hisaaki; Futaki, Shiroh; Langel, Ulo; Gräslund, Astrid


    Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

  11. Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo.

    Directory of Open Access Journals (Sweden)

    Sofie Stalmans

    Full Text Available Cell-penetrating peptides (CPPs are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB. However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47-57, transportan 10 (TP10 and TP10-2, were determined. The results of the multiple time regression (MTR analysis revealed that CPPs show divergent BBB influx properties: Tat 47-57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 μl/(g × min, 5.63 μl/(g × min and 6.02 μl/(g × min, respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.

  12. Comparative mechanisms of protein transduction mediated by cell-penetrating peptides in prokaryotes. (United States)

    Liu, Betty Revon; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung


    Bacterial and archaeal cell envelopes are complex multilayered barriers that serve to protect these microorganisms from their extremely harsh and often hostile environments. Import of exogenous proteins and nanoparticles into cells is important for biotechnological applications in prokaryotes. In this report, we demonstrate that cell-penetrating peptides (CPPs), both bacteria-expressed nona-arginine peptide (R9) and synthetic R9 (SR9), are able to deliver noncovalently associated proteins or quantum dots into four representative species of prokaryotes: cyanobacteria (Synechocystis sp. PCC 6803), bacteria (Escherichia coli DH5α and Arthrobacter ilicis D-50), and archaea (Thermus aquaticus). Although energy-dependent endocytosis is generally accepted as a hallmark that distinguishes eukaryotes from prokaryotes, cellular uptake of uncomplexed green fluorescent protein (GFP) by cyanobacteria was mediated by classical endocytosis. Mechanistic studies revealed that macropinocytosis plays a critical and major role in CPP-mediated protein transduction in all four prokaryotes. Membrane damage was not observed when cyanobacterial cells were treated with R9/GFP complexes, nor was cytotoxicity detected when bacteria or archaea were treated with SR9/QD complexes in the presence of macropinocytic inhibitors. These results indicate that the uptake of protein is not due to a compromise of membrane integrity in cyanobacteria, and that CPP can be an effective and safe carrier for membrane trafficking in prokaryotic cells. Our investigation provides important new insights into the transport of exogenous proteins and nanoparticles across the complex membrane systems of prokaryotes.

  13. Porous silicon-cell penetrating peptide hybrid nanocarrier for intracellular delivery of oligonucleotides. (United States)

    Rytkönen, Jussi; Arukuusk, Piret; Xu, Wujun; Kurrikoff, Kaido; Langel, Ulo; Lehto, Vesa-Pekka; Närvänen, Ale


    The largest obstacle to the use of oligonucleotides as therapeutic agents is the delivery of these large and negatively charged biomolecules through cell membranes into intracellular space. Mesoporous silicon (PSi) is widely recognized as a potential material for drug delivery purposes due to its several beneficial features like large surface area and pore volume, high loading capacity, biocompatibility, and biodegradability. In the present study, PSi nanoparticles stabilized by thermal oxidation or thermal carbonization and subsequently modified by grafting aminosilanes on the surface are utilized as an oligonucleotide carrier. Splice correcting oligonucleotides (SCOs), a model oligonucleotide drug, were loaded into the positively charged PSi nanoparticles with a loading degree as high as 14.3% (w/w). Rapid loading was achieved by electrostatic interactions, with the loading efficiencies reaching 100% within 5 min. The nanoparticles were shown to deliver and release SCOs, in its biologically active form, inside cells when formulated together with cell penetrating peptides (CPP). The biological effect was monitored with splice correction assay and confocal microscopy utilizing HeLa pLuc 705 cells. Furthermore, the use of PSi carrier platform in oligonucleotide delivery did not reduce the cell viability. Additionally, the SCO-CPP complexes formed in the pores of the carrier were stabilized against proteolytic digestion. The advantageous properties of protecting and releasing the cargo and the possibility to further functionalize the carrier surface make the hybrid nanoparticles a potential system for oligonucleotide delivery.

  14. Glycosaminoglycans are required for translocation of amphipathic cell-penetrating peptides across membranes. (United States)

    Pae, Janely; Liivamägi, Laura; Lubenets, Dmitri; Arukuusk, Piret; Langel, Ülo; Pooga, Margus


    Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways - endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides.

  15. Virulence Potential of Activatable Shiga Toxin 2d–Producing Escherichia coli Isolates from Fresh Produce (United States)

    Melton-Celsa, Angela R.; O'Brien, Alison D.; Feng, Peter C. H.


    Shiga toxin (Stx)–producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named “activation.” Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice. PMID:26555533

  16. Virulence Potential of Activatable Shiga Toxin 2d-Producing Escherichia coli Isolates from Fresh Produce. (United States)

    Melton-Celsa, Angela R; O'Brien, Alison D; Feng, Peter C H


    Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named "activation." Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice.

  17. Characterization of the cell penetrating properties of a human salivary proline-rich peptide. (United States)

    Radicioni, Giorgia; Stringaro, Annarita; Molinari, Agnese; Nocca, Giuseppina; Longhi, Renato; Pirolli, Davide; Scarano, Emanuele; Iavarone, Federica; Manconi, Barbara; Cabras, Tiziana; Messana, Irene; Castagnola, Massimo; Vitali, Alberto


    Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents.

  18. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease. (United States)

    Dinca, Ana; Chien, Wei-Ming; Chin, Michael T


    Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

  19. Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease

    Directory of Open Access Journals (Sweden)

    Ana Dinca


    Full Text Available Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs, a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa. Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

  20. Graphene microsheets enter cells through spontaneous membrane penetration at edge asperities and corner sites. (United States)

    Li, Yinfeng; Yuan, Hongyan; von dem Bussche, Annette; Creighton, Megan; Hurt, Robert H; Kane, Agnes B; Gao, Huajian


    Understanding and controlling the interaction of graphene-based materials with cell membranes is key to the development of graphene-enabled biomedical technologies and to the management of graphene health and safety issues. Very little is known about the fundamental behavior of cell membranes exposed to ultrathin 2D synthetic materials. Here we investigate the interactions of graphene and few-layer graphene (FLG) microsheets with three cell types and with model lipid bilayers by combining coarse-grained molecular dynamics (MD), all-atom MD, analytical modeling, confocal fluorescence imaging, and electron microscopic imaging. The imaging experiments show edge-first uptake and complete internalization for a range of FLG samples of 0.5- to 10-μm lateral dimension. In contrast, the simulations show large energy barriers relative to kBT for membrane penetration by model graphene or FLG microsheets of similar size. More detailed simulations resolve this paradox by showing that entry is initiated at corners or asperities that are abundant along the irregular edges of fabricated graphene materials. Local piercing by these sharp protrusions initiates membrane propagation along the extended graphene edge and thus avoids the high energy barrier calculated in simple idealized MD simulations. We propose that this mechanism allows cellular uptake of even large multilayer sheets of micrometer-scale lateral dimension, which is consistent with our multimodal bioimaging results for primary human keratinocytes, human lung epithelial cells, and murine macrophages.

  1. Enzyme-activatable imaging probe reveals enhanced neutrophil elastase activity in tumors following photodynamic therapy. (United States)

    Mitra, Soumya; Modi, Kshitij D; Foster, Thomas H


    We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1+ cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1+ cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1+ cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1+ cell population.

  2. Cell membrane penetration and mitochondrial targeting by platinum-decorated ceria nanoparticles (United States)

    Torrano, Adriano A.; Herrmann, Rudolf; Strobel, Claudia; Rennhak, Markus; Engelke, Hanna; Reller, Armin; Hilger, Ingrid; Wixforth, Achim; Bräuchle, Christoph


    In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and focus on their fast uptake and association with mitochondria, the cell's powerhouse. Using live-cell imaging and electron microscopy we clearly show that 46 nm platinum-decorated ceria nanoparticles can rapidly penetrate cell membranes and reach the cytosol. Moreover, if suitably targeted, these particles are able to selectively attach to mitochondria. These results are complemented by cytotoxicity assays, thus providing insights into the biological effects of these particles on cells. Interestingly, no permanent membrane disruption or any other significant adverse effects on cells were observed. The unusual uptake behavior observed for 46 nm nanoparticles was not observed for equivalent but larger 143 nm and 285 nm platinum-decorated particles. Our results demonstrate a remarkable particle size effect in which particles smaller than ~50-100 nm escape the usual endocytic pathway and translocate directly into the cytosol, while particles larger than ~150 nm are internalized by conventional endocytosis. Since the small particles are able to bypass endocytosis they could be explored as drug and gene delivery vehicles. Platinum-decorated nanoparticles are therefore highly interesting in the fields of nanotoxicology and nanomedicine.In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and

  3. The effect of dexamethasone/cell-penetrating peptide nanoparticles on gene delivery for inner ear therapy

    Directory of Open Access Journals (Sweden)

    Yoon JY


    Full Text Available Ji Young Yoon,1 Keum-Jin Yang,2 Shi-Nae Park,3 Dong-Kee Kim,3 Jong-Duk Kim1 1Department of Chemical and Biomolecular Engineering, BK 21 Plus Program, Korea Advanced Institute of Science and Technology, Guseong-Dong, Yuseong-Gu, Daejeon, 2Clinical Research Institute, St Mary’s Hospital, Daejeon, 3Department of Otolaryngology – Head and Neck Surgery, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea Abstract: Dexamethasone (Dex-loaded PHEA-g-C18-Arg8 (PCA nanoparticles (PCA/Dex were developed for the delivery of genes to determine the synergistic effect of Dex on gene expression. The cationic PCA nanoparticles were self-assembled to create cationic micelles containing an octadecylamine (C18 core with Dex and an arginine 8 (Arg8 peptide shell for electrostatic complexation with nucleic acids (connexin 26 [Cx26] siRNA, green fluorescent protein [GFP] DNA or brain-derived neurotrophic factor [BDNF] pDNA. The PCA/Dex nanoparticles conjugated with Arg8, a cell-penetrating peptide that enhances permeability through a round window membrane in the inner ear for gene delivery, exhibited high uptake efficiency in HEI-OC1 cells. This potential carrier co-delivering Dex and the gene into inner ear cells has a diameter of 120–140 nm and a zeta potential of 20–25 mV. Different types of genes were complexed with the Dex-loaded PCA nanoparticle (PCA/Dex/gene for gene expression to induce additional anti-inflammatory effects. PCA/Dex showed mildly increased expression of GFP and lower mRNA expression of inflammatory cytokines (IL1b, IL12, and INFr than did Dex-free PCA nanoparticles and Lipofectamine® reagent in HEI-OC1 cells. In addition, after loading Cx26 siRNA onto the surface of PCA/Dex, Cx26 gene expression was downregulated according to real-time polymerase chain reaction for 24 h, compared with that using Lipofectamine reagent. After loading BDNF DNA into PCA/Dex, increased expression of BDNF was observed for 30

  4. Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application. (United States)

    He, Huining; Ye, Junxiao; Wang, Yinsong; Liu, Quan; Chung, Hee Sun; Kwon, Young Min; Shin, Meong Cheol; Lee, Kyuri; Yang, Victor C


    Red blood cells (RBCs) based drug carrier appears to be the most appealing for protein drugs due to their unmatched biocompatability, biodegradability, and long lifespan in the circulation. Numerous methods for encapsulating protein drugs into RBCs were developed, however, most of them induce partial disruption of the cell membrane, resulting in irreversible alterations in both physical and chemical properties of RBCs. Herein, we introduce a novel method for encapsulating proteins into intact RBCs, which was meditated by a cell penetrating peptide (CPP) developed in our lab-low molecular weight protamine (LMWP). l-asparaginase, one of the primary drugs used in treatment of acute lymphoblastic leukemia (ALL), was chosen as a model protein to illustrate the encapsulation into erythrocytes mediated by CPPs. In addition current treatment of ALL using different l-asparaginase delivery and encapsulation methods as well as their associated problems were also reviewed.

  5. Novel Efficient Cell-Penetrating, Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides. (United States)

    Muñoz-Alarcón, Andrés; Eriksson, Jonas; Langel, Ülo


    At present, there are several therapeutic approaches for targeting telomerase in tumors. One in particular, currently undergoing clinical trials, is based on synthetic lipid-modified oligonucleotide antagonists aimed at inhibiting the ribonucleoprotein subunit of human telomerase. However, while enabling efficient uptake, the lipid modifications reduce the potency of the therapeutic oligonucleotides compared to nonmodified oligonucleotides. Moreover, lipid modification may increase oligonucleotide accumulation in the liver causing undesirable hepatotoxicity. Noncovalent complexation strategies for cell-penetrating peptide (CPP)-mediated delivery present an option to circumvent the need for potency-reducing modifications, while allowing for a highly efficient uptake, and could significantly improve the efficiency of telomerase-targeting cancer therapeutics. Delivery of a nonlipidated locked nucleic acid/2'-O-methyl mixmer significantly inhibits the telomerase activity in treated HeLa cells. The inhibitory effect was further improved through addition of a CPP. Furthermore, calculated IC50-values for the oligonucleotide delivered by CPPs into HeLa cells are more than 20 times lower than telomerase inhibitor Imetelstat, currently undergoing clinical trials. These results emphasize the potential of CPP-mediated delivery of future pharmaceuticals and provide means by which to enhance an already promising therapeutic strategy for cancer treatment.

  6. Intracellular Delivery of Molecular Cargo Using Cell-Penetrating Peptides and the Combination Strategies

    Directory of Open Access Journals (Sweden)

    Hua Li


    Full Text Available Cell-penetrating peptides (CPPs can cross cellular membranes in a non-toxic fashion, improving the intracellular delivery of various molecular cargos such as nanoparticles, small molecules and plasmid DNA. Because CPPs provide a safe, efficient, and non-invasive mode of transport for various cargos into cells, they have been developed as vectors for the delivery of genetic and biologic products in recent years. Most common CPPs are positively charged peptides. While delivering negatively charged molecules (e.g., nucleic acids to target cells, the internalization efficiency of CPPs is reduced and inhibited because the cationic charges on the CPPs are neutralized through the covering of CPPs by cargos on the structure. Even under these circumstances, the CPPs can still be non-covalently complexed with the negatively charged molecules. To address this issue, combination strategies of CPPs with other typical carriers provide a promising and novel delivery system. This review summarizes the latest research work in using CPPs combined with molecular cargos including liposomes, polymers, cationic peptides, nanoparticles, adeno-associated virus (AAV and calcium for the delivery of genetic products, especially for small interfering RNA (siRNA. This combination strategy remedies the reduced internalization efficiency caused by neutralization.

  7. Cell Penetrating Peptide Conjugated Chitosan for Enhanced Delivery of Nucleic Acid

    Directory of Open Access Journals (Sweden)

    Buddhadev Layek


    Full Text Available Gene therapy is an emerging therapeutic strategy for the cure or treatment of a spectrum of genetic disorders. Nevertheless, advances in gene therapy are immensely reliant upon design of an efficient gene carrier that can deliver genetic cargoes into the desired cell populations. Among various nonviral gene delivery systems, chitosan-based carriers have gained increasing attention because of their high cationic charge density, excellent biocompatibility, nearly nonexistent cytotoxicity, negligible immune response, and ideal ability to undergo chemical conjugation. However, a major shortcoming of chitosan-based carriers is their poor cellular uptake, leading to inadequate transfection efficiency. The intrinsic feature of cell penetrating peptides (CPPs for transporting diverse cargoes into multiple cell and tissue types in a safe manner suggests that they can be conjugated to chitosan for improving its transfection efficiency. In this review, we briefly discuss CPPs and their classification, and also the major mechanisms contributing to the cellular uptake of CPPs and cargo conjugates. We also discuss immense improvements for the delivery of nucleic acids using CPP-conjugated chitosan-based carriers with special emphasis on plasmid DNA and small interfering RNA.

  8. On the importance of electrostatic interactions between cell penetrating peptides and membranes: a pathway toward tumor cell selectivity? (United States)

    Jobin, Marie-Lise; Alves, Isabel D


    Cell-penetrating peptides (CPPs) are small molecules of major interest due to their ability to efficiently transport cargos across cell membranes in a receptor- and energy-independent way and without being cytotoxic to cells. Since their discovery 20 years ago their potential interest in drug delivery and diagnosis became undeniable. CPPs are being used to deliver inside cells a large variety of cargos such as proteins, DNA, antibodies, imaging agents and nanoparticle drug carriers. Their cellular uptake mechanisms are still debated and may vary depending on their structure, nature and size of cargo they transport and type of cell line targeted. CPPs are generally rich in positively charged residues, thus they are prone to establish electrostatic interactions with anionic membrane components (sugars and lipids). Understanding the molecular basis of CPP membrane interaction and cellular uptake is crucial to improve their in vivo efficiency target-specificity. A great number of studies demonstrated the high potential of CPPs to translocate efficiently therapeutic cargos into cells and some peptides are even in clinical phase studies. Although these molecules seem perfect for a therapeutic or diagnosis purpose, they still possess a small but non negligible drawback: a complete lack of cell type specificity. Tumor cells have recently been shown to over-express certain glycosaminoglycans at the cell membrane surface and to possess a higher amount of anionic lipids in their outer leaflet than healthy cells. Such molecules confer the cell membrane an enhanced anionic character, property that could be used by CPPs to selectively target these cells. Moreover previous studies demonstrate the importance of electrostatic interactions between basic residues in the peptide, especially Arg, and the lipid headgroups and glycosaminoglycans in the cell membrane. Electrostatic interactions put at stake in this process might be one of the keys to resolve the puzzle of CPP cell type

  9. Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes. (United States)

    Li, Lirong; Shi, Yonghui; Cheserek, Maureen Jepkorir; Su, Guanfang; Le, Guowei


    A number of research have proven that antimicrobial peptides are of greatest potential as a new class of antibiotics. Antimicrobial peptides and cell-penetrating peptides share some similar structure characteristics. In our study, a new peptide analog, APP (GLARALTRLLRQLTRQLTRA) from the cell-penetrating peptide ppTG20 (GLFRALLRLLRSLWRLLLRA), was identified simultaneously with the antibacterial mechanism of APP against Salmonella typhimurium and Streptococcus pyogenes. APP displayed potent antibacterial activity against Gram-negative and Gram-positive strains. The minimum inhibitory concentration was in the range of 2 to 4 μM. APP displayed higher cell selectivity (about 42-fold increase) as compared to the parent peptide for it decreased hemolytic activity and increased antimicrobial activity. The calcein leakage from egg yolk L-α-phosphatidylcholine (EYPC)/egg yolk L-α-phosphatidyl-DL-glycerol and EYPC/cholesterol vesicles demonstrated that APP exhibited high selectivity. The antibacterial mechanism analysis indicated that APP induced membrane permeabilization in a kinetic manner for membrane lesions allowing O-nitrophenyl-β-D-galactoside uptake into cells and potassium release from APP-treated cells. Flow cytometry analysis demonstrated that APP induced bacterial live cell membrane damage. Circular dichroism, fluorescence spectra, and gel retardation analysis confirmed that APP interacted with DNA and intercalated into the DNA base pairs after penetrating the cell membrane. Cell cycle assay showed that APP affected DNA synthesis in the cell. Our results suggested that peptides derived from the cell-penetrating peptide have the potential for antimicrobial agent development, and APP exerts its antibacterial activity by damaging bacterial cell membranes and binding to bacterial DNA to inhibit cellular functions, ultimately leading to cell death.

  10. The targeted transduction of MMP-overexpressing tumor cells by ACPP-HPMA copolymer-coated adenovirus conjugates.

    Directory of Open Access Journals (Sweden)

    Shuhua Li

    Full Text Available We have designed and tested a new way to selectively deliver HPMA polymer-coated adenovirus type 5 (Ad5 particles into matrix metalloproteinase (MMP-overexpressing tumor cells. An activatable cell penetrating peptide (ACPP was designed and attached to the reactive 4-nitrophenoxy groups of HPMA polymers by the C-terminal amino acid (asparagine, N. ACPPs are activatable cell penetrating peptides (CPPs with a linker between polycationic and polyanionic domains, and MMP-mediated cleavage releases the CPP portion and its attached cargo to enable cell entry. Our data indicate that the transport of these HPMA polymer conjugates by a single ACPP molecule to the cytoplasm occurs via a nonendocytotic and concentration-independent process. The uptake was observed to finish within 20 minutes by inverted fluorescence microscopy. In contrast, HPMA polymer-coated Ad5 without ACPPs was internalized solely by endocytosis. The optimal formulation was not affected by the presence of Ad5 neutralizing antibodies during transduction, and ACPP/polymer-coated Ad5 also retained high targeting capability to several MMP-overexpressing tumor cell types. For the first time, ACPP-mediated cytoplasmic delivery of polymer-bound Ad5 to MMP-overexpressing tumor cells was demonstrated. These findings are significant, as they demonstrate the use of a polymer-based system for the targeted delivery into MMP-overexpressing solid tumors and highlight how to overcome major cellular obstacles to achieve intracellular macromolecular delivery.

  11. Cell-penetrating peptide TP10 shows broad-spectrum activity against both Plasmodium falciparum and Trypanosoma brucei brucei. (United States)

    Arrighi, Romanico B G; Ebikeme, Charles; Jiang, Yang; Ranford-Cartwright, Lisa; Barrett, Michael P; Langel, Ulo; Faye, Ingrid


    Malaria and trypanosomiasis are diseases which afflict millions and for which novel therapies are urgently required. We have tested two well-characterized cell-penetrating peptides (CPPs) for antiparasitic activity. One CPP, designated TP10, has broad-spectrum antiparasitic activity against Plasmodium falciparum, both blood and mosquito stages, and against blood-stage Trypanosoma brucei brucei.

  12. Cell-Penetrating Recombinant Peptides for Potential Use in Agricultural Pest Control Applications

    Directory of Open Access Journals (Sweden)

    Eric T. Johnson


    Full Text Available Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs because they possess the ability to translocate across cell membranes. This ability makes these peptides attractive candidates for delivery of therapeutic compounds, especially to the interior of cells. Compounds with characteristics similar to CPPs and that, in addition, have antimicrobial properties are being investigated as antibiotics with a reduced risk of causing resistance. These CPP-like membrane-acting antimicrobial peptides (MAMPs are α-helical amphipathic peptides that interact with and perturb cell membranes to produce their antimicrobial effects. One source of MAMPs is spider venom. Because these compounds are toxic to insects, they also show promise for development as biological agents for control of insecticide-resistant agricultural pests. Spider venom is a potential source of novel insect-specific peptide toxins. One example is the small amphipathic α-helical peptide lycotoxin-1 (Lyt-1 or LCTX from the wolf spider (Lycosa carolinensis. One side of the α-helix has mostly hydrophilic and the other mainly hydrophobic amino acid residues. The positive charge of the hydrophilic side interacts with negatively charged prokaryotic membranes and the hydrophobic side associates with the membrane lipid bilayer to permeabilize it. Because the surface of the exoskeleton, or cuticle, of an insect is highly hydrophobic, to repel water and dirt, it would be expected that amphipathic compounds could permeabilize it. Mutagenized lycotoxin 1 peptides were produced and expressed in yeast cultures that were fed to fall armyworm (Spodoptera frugiperda larvae to identify the most lethal mutants. Transgenic expression of spider venom toxins such as lycotoxin-1 in plants could provide durable insect resistance.

  13. Cell-penetrating recombinant peptides for potential use in agricultural pest control applications. (United States)

    Hughes, Stephen R; Dowd, Patrick F; Johnson, Eric T


    Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes these peptides attractive candidates for delivery of therapeutic compounds, especially to the interior of cells. Compounds with characteristics similar to CPPs and that, in addition, have antimicrobial properties are being investigated as antibiotics with a reduced risk of causing resistance. These CPP-like membrane-acting antimicrobial peptides (MAMPs) are α-helical amphipathic peptides that interact with and perturb cell membranes to produce their antimicrobial effects. One source of MAMPs is spider venom. Because these compounds are toxic to insects, they also show promise for development as biological agents for control of insecticide-resistant agricultural pests. Spider venom is a potential source of novel insect-specific peptide toxins. One example is the small amphipathic α-helical peptide lycotoxin-1 (Lyt-1 or LCTX) from the wolf spider (Lycosa carolinensis). One side of the α-helix has mostly hydrophilic and the other mainly hydrophobic amino acid residues. The positive charge of the hydrophilic side interacts with negatively charged prokaryotic membranes and the hydrophobic side associates with the membrane lipid bilayer to permeabilize it. Because the surface of the exoskeleton, or cuticle, of an insect is highly hydrophobic, to repel water and dirt, it would be expected that amphipathic compounds could permeabilize it. Mutagenized lycotoxin 1 peptides were produced and expressed in yeast cultures that were fed to fall armyworm (Spodoptera frugiperda) larvae to identify the most lethal mutants. Transgenic expression of spider venom toxins such as lycotoxin-1 in plants could provide durable insect resistance.

  14. Dataset on FAP-induced emergence of spontaneous metastases and on the preparation of activatable FAP-targeting immunoliposomes to detect the metastases. (United States)

    Tansi, Felista L; Rüger, Ronny; Böhm, Claudia; Kontermann, Roland E; Teichgraeber, Ulf K; Fahr, Alfred; Hilger, Ingrid


    The underlying data demonstrates that fibroblast activation protein (FAP) paves the way for fibrosarcoma cells, which require the proteolysis of the extracellular matrix (ECM) and basement membranes to intravasate from implanted subcutaneous primary tumors into blood vessels, be transported to distant organs where they extravasate from the blood vessels, reattach and proliferate to metastases. The data additionally shows that FAP, when overexpressed on fibrosarcoma cells induces their invasion and formation of spontaneous metastases in multiple organs, particularly after subcutaneous co-implantation of the FAP-expressing and wildtype fibrosarcoma. The raw and processed data presented herein is related to a research article entitled "Potential of activatable FAP-targeting immunoliposomes in intraoperative imaging of spontaneous metastases" (F.L. Tansi, R. Rüger, C. Böhm, R.E. Kontermann, U.K. Teichgraeber, A. Fahr, I. Hilger, 2016) [1]. Furthermore, evidence for the detection of FAP-expressing tumor cells and cells of the tumor stroma by activatable FAP-targeting liposomes is presented in this dataset.

  15. The role of thrombin-activatable fibrinolysis inhibitor in diabetic wound healing

    NARCIS (Netherlands)

    C.J.N. Verkleij; J.J.T.H. Roelofs; S.R. Havik; J.C.M. Meijers; P.F. Marx


    Introduction: One of the major complications in patients with diabetes mellitus is impaired wound healing. The fibrinolytic system is involved in parts of the wound healing process and deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI) results in delayed wound closure. Moreover, levels

  16. An activatable, polarity dependent, dual-luminescent imaging agent with a long luminescence lifetime. (United States)

    Rood, Marcus T M; Oikonomou, Maria; Buckle, Tessa; Raspe, Marcel; Urano, Yasuteru; Jalink, Kees; Velders, Aldrik H; van Leeuwen, Fijs W B


    In this proof-of-concept study, a new activatable imaging agent based on two luminophores and two different quenching mechanisms is reported. Both partial and total activation of the luminescence signal can be achieved, either in solution or in vitro. Bond cleavage makes the compound suitable for luminescence lifetime imaging.

  17. A two-photon activatable amino acid linker for the induction of fluorescence. (United States)

    Friedrich, Felix; Klehs, Kathrin; Fichte, Manuela A H; Junek, Stephan; Heilemann, Mike; Heckel, Alexander


    A new one- and two-photon activatable fluorophore based on ATTO565 was developed using a photolabile linker that simultaneously acts as a quencher. It is especially interesting for protein and peptide applications because it can be incorporated by standard peptide chemistry. The application of the new fluorogenic construct in super-resolution microscopy of antibody conjugates is shown.

  18. Activatable molecular systems using homologous near-infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in vivo. (United States)

    Zhang, Zongren; Fan, Jinda; Cheney, Philip P; Berezin, Mikhail Y; Edwards, W Barry; Akers, Walter J; Shen, Duanwen; Liang, Kexian; Culver, Joseph P; Achilefu, Samuel


    We have developed a generic approach to determine enzyme activities in vitro and monitor their functional status in vivo. Specifically, a method to generate donor (CbOH)-acceptor (Me2NCp) near-infrared (NIR) fluorescent dye pairs for preparing enzyme activatable molecular systems were developed based on the structural template of heptamethine cyanine dyes. Using caspase-3 as a model enzyme, we prepared two new caspase-3 sensitive compounds with high fluorescence quenching efficiency: Me2NCp-DEVD-K(CbOH)-OH (4) and AcGK(Me2NCp)-DEVD-APK(CbOH)-NH2 (5). The mechanism of quenching was based on combined effects of direct (classical) and reverse fluorescence resonance energy transfer (FRET). Caspase-3 cleavage of the scissile DEVD amide bond regenerated the NIR fluorescence of both donor and acceptor dyes. While both compounds were cleaved by caspase-3, substrate 5 was cleaved more readily than 4, yielding k(cat) and K(M), values of 1.02 +/- 0.06 s(-1) and 15 +/- 3 microM, respectively. Treatment of A549 tumor cells with paclitaxel resulted in > 2-fold increase in the fluorescence intensity by NIR confocal microscopy, suggesting the activation of pro-caspase-3 to caspase-3. A similar trend was observed in a mouse model, where the fluorescence intensity was nearly twice the value in caspase-3-rich tissue relative to the control. These results demonstrate the use of the same NIR activatable molecular systems for monitoring the activities of enzymes across a wide spatial scale ranging from in vitro kinetics measurements to in cellulo and in vivo localization of caspase-3 activation. The NIR activatable molecular probes provide an effective strategy to screen new drugs in vitro and monitor treatment response in living organisms.

  19. Cell-penetrating compounds preferentially bind glycosaminoglycans over plasma membrane lipids in a charge density- and stereochemistry-dependent manner. (United States)

    Prevette, Lisa E; Benish, Nicolas C; Schoenecker, Amber R; Braden, Kristin J


    Cell-penetrating compounds (CPCs) are often conjugated to drugs and genes to facilitate cellular uptake. We hypothesize that the electrostatic interaction between the positively charged amines of the cell-penetrating compounds and the negatively charged glycosaminoglycans (GAGs) extending from cell surfaces is the initiating step in the internalization process. The interactions of generation 5 PAMAM dendrimer, Tat peptide and 25 kDa linear PEI with four different GAGs have been studied using isothermal titration calorimetry to elucidate structure-function relationships that could lead to improved drug and gene delivery methods to a wide variety of cell types. Detailed thermodynamic analysis has determined that CPC-GAG binding constants range from 8.7×10(3) to 2.4×10(6)M(-1) and that affinity is dependent upon GAG charge density and stereochemistry and CPC molecular weight. The effect of GAG composition on affinity is likely due to hydrogen bonding between CPC amines and amides and GAG hydroxyl and amine groups. These results were compared to the association of CPCs with lipid vesicles of varying composition as model plasma membranes to finally clarify the relative importance of each cell surface component in initial cell recognition. CPC-lipid affinity increases with anionic lipid content, but GAG affinity is higher for all cell-penetrating compounds, confirming the role these heterogeneous polysaccharides play in cellular association and clustering.

  20. Enhanced penetration into 3D cell culture using two and three layered gold nanoparticles

    Directory of Open Access Journals (Sweden)



    Full Text Available Christopher G England,1 Thomas Priest,2 Guandong Zhang,2 Xinghua Sun,2 Dhruvinkumar N Patel,2 Lacey R McNally,3,4 Victor van Berkel,4,5 André M Gobin,2 Hermann B Frieboes1,2,41Department of Pharmacology and Toxicology, 2Department of Bioengineering, 3Department of Medicine, 4James Graham Brown Cancer Center, 5Department of Surgery, University of Louisville, KY, USAAbstract: Nano-scale particles sized 10–400 nm administered systemically preferentially extravasate from tumor vasculature due to the enhanced permeability and retention effect. Therapeutic success remains elusive, however, because of inhomogeneous particle distribution within tumor tissue. Insufficient tumor vascularization limits particle transport and also results in avascular hypoxic regions with non-proliferating cells, which can regenerate tissue after nanoparticle-delivered cytotoxicity or thermal ablation. Nanoparticle surface modifications provide for increasing tumor targeting and uptake while decreasing immunogenicity and toxicity. Herein, we created novel two layer gold-nanoshell particles coated with alkanethiol and phosphatidylcholine, and three layer nanoshells additionally coated with high-density-lipoprotein. We hypothesize that these particles have enhanced penetration into 3-dimensional cell cultures modeling avascular tissue when compared to standard poly(ethylene glycol (PEG-coated nanoshells. Particle uptake and distribution in liver, lung, and pancreatic tumor cell cultures were evaluated using silver-enhancement staining and hyperspectral imaging with dark field microscopy. Two layer nanoshells exhibited significantly higher uptake compared to PEGylated nanoshells. This multilayer formulation may help overcome transport barriers presented by tumor vasculature, and could be further investigated in vivo as a platform for targeted cancer therapies.Keywords: cancer nanotherapy, tumor hypoxia, nanovector transport

  1. Cellular uptake but low permeation of human calcitonin-derived cell penetrating peptides and Tat(47-57) through well-differentiated epithelial models

    DEFF Research Database (Denmark)

    Tréhin, Rachel; Krauss, Ulrike; Beck-Sickinger, Annette G;


    To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers.......To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers....

  2. Biofunctionalized nanoparticles with pH-responsive and cell penetrating blocks for gene delivery (United States)

    Gaspar, V. M.; Marques, J. G.; Sousa, F.; Louro, R. O.; Queiroz, J. A.; Correia, I. J.


    Bridging the gap between nanoparticulate delivery systems and translational gene therapy is a long sought after requirement in nanomedicine-based applications. However, recent developments regarding nanoparticle functionalization have brought forward the ability to synthesize materials with biofunctional moieties that mimic the evolved features of viral particles. Herein we report the versatile conjugation of both cell penetrating arginine and pH-responsive histidine moieties into the chitosan polymeric backbone, to improve the physicochemical characteristics of the native material. Amino acid coupling was confirmed by 2D TOCSY NMR and Fourier transform infrared spectroscopy. The synthesized chitosan-histidine-arginine (CH-H-R) polymer complexed plasmid DNA biopharmaceuticals, and spontaneously assembled into stable 105 nm nanoparticles with spherical morphology and positive surface charge. The functionalized delivery systems were efficiently internalized into the intracellular compartment, and exhibited remarkably higher transfection efficiency than unmodified chitosan without causing any cytotoxic effect. Additional findings regarding intracellular trafficking events reveal their preferential escape from degradative lysosomal pathways and nuclear localization. Overall, this assembly of nanocarriers with bioinspired moieties provides the foundations for the design of efficient and customizable materials for cancer gene therapy.

  3. Design of aromatic-containing cell-penetrating peptide mimics with structurally modified π electronics. (United States)

    deRonde, Brittany M; Birke, Alexander; Tew, Gregory N


    Cell-penetrating peptides (CPPs) and their synthetic mimics (CPPMs) represent a class of molecules that facilitate the intracellular delivery of various cargo. Previous studies indicated that the presence of aromatic functionalities improved CPPM activity. Given that aromatic functionalities play prominent roles in membrane biology and participate in various π interactions, we explored whether these interactions could be optimized for improved CPPM activity. CPPMs were synthesized by ring-opening metathesis polymerization by using monomers that contained aromatic rings substituted with electron-donating and electron-withdrawing groups and covered an electrostatic potential range from -29.69 to +15.57 kcal mol(-1) . These groups altered the quadrupole moments of the aromatic systems and were used to test if such structural modifications changed CPPM activity. CPPMs were added to dye-loaded vesicles and the release of carboxyfluorescein was monitored as a function of polymer concentration. Changes in the effective polymer concentration to release 50% of the dye (effective concentration, EC50 ) were monitored. Results from this assay showed that the strength of the electron-donating and electron-withdrawing groups incorporated in the CPPMs did not alter polymer EC50 values or activity. This suggests that other design parameters may have a stronger impact on CPPM activity. In addition, these results indicate that a wide range of aromatic groups can be incorporated without negatively impacting polymer activity.

  4. Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides

    KAUST Repository

    Rydberg, Hanna A.


    Cell-penetrating peptides (CPPs) are able to traverse cellular membranes and deliver macromolecular cargo. Uptake occurs through both endocytotic and nonendocytotic pathways, but the molecular requirements for efficient internalization are not fully understood. Here we investigate how the presence of tryptophans and their position within an oligoarginine influence uptake mechanism and efficiency. Flow cytometry and confocal fluorescence imaging are used to estimate uptake efficiency, intracellular distribution and toxicity in Chinese hamster ovarian cells. Further, membrane leakage and lipid membrane affinity are investigated. The peptides contain eight arginine residues and one to four tryptophans, the tryptophans positioned either at the N-terminus, in the middle, or evenly distributed along the amino acid sequence. Our data show that the intracellular distribution varies among peptides with different tryptophan content and backbone spacing. Uptake efficiency is higher for the peptides with four tryptophans in the middle, or evenly distributed along the peptide sequence, than for the peptide with four tryptophans at the N-terminus. All peptides display low cytotoxicity except for the one with four tryptophans at the N-terminus, which was moderately toxic. This finding is consistent with their inability to induce efficient leakage of dye from lipid vesicles. All peptides have comparable affinities for lipid vesicles, showing that lipid binding is not a decisive parameter for uptake. Our results indicate that tryptophan content and backbone spacing can affect both the CPP uptake efficiency and the CPP uptake mechanism. The low cytotoxicity of these peptides and the possibilities of tuning their uptake mechanism are interesting from a therapeutic point of view. © 2012 American Chemical Society.

  5. Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration

    Directory of Open Access Journals (Sweden)

    Brown Theodore J


    Full Text Available Abstract Background The invasive potential of cancer cells is usually assessed in vitro using Matrigel as a surrogate basement membrane. Yet cancer cell interaction with collagen I matrices is critical, particularly for the peritoneal metastatic route undertaken by several cancer types including ovarian. Matrix metalloprotease (MMP activity is important to enable cells to overcome the barrier constraints imposed by basement membranes and stromal matrices in vivo. Our objective was to compare matrices reconstituted from collagen I and Matrigel as representative barriers for ovarian cancer cell invasion. Methods The requirement of MMP activity for ovarian cancer cell penetration of Matrigel and collagen matrices was assessed in 2D transwell and 3D spheroid culture systems. Results The broad range MMP inhibitor GM6001 completely prevented cell perforation of polymerised collagen I-coated transwell membranes. In contrast, GM6001 decreased ES-2 cell penetration of Matrigel by only ~30% and had no effect on HEY cell Matrigel penetration. In 3D culture, ovarian cancer cells grown as spheroids also migrated into surrounding Matrigel matrices despite MMP blockade. In contrast, MMP activity was required for invasion into 3D matrices of collagen I reconstituted from acid-soluble rat-tail collagen I, but not from pepsin-extracted collagen I (Vitrogen/Purecol, which lacks telopeptide regions. Conclusion Matrigel does not form representative barriers to ovarian cancer cells in either 2D or 3D culture systems. Our findings support the use of collagen I rather than Matrigel as a matrix barrier for invasion studies to better approximate critical interactions and events associated with peritoneal metastasis.

  6. Development of a Cell-penetrating Peptide that Exhibits Responsive Changes in its Secondary Structure in the Cellular Environment (United States)

    Yamashita, Hiroko; Kato, Takuma; Oba, Makoto; Misawa, Takashi; Hattori, Takayuki; Ohoka, Nobumichi; Tanaka, Masakazu; Naito, Mikihiko; Kurihara, Masaaki; Demizu, Yosuke


    Cell-penetrating peptides (CPP) are received a lot of attention as an intracellular delivery tool for hydrophilic molecules such as drugs, proteins, and DNAs. We designed and synthesized nona-arginine analogues 1–5 [FAM-β-Ala-(l-Arg-l-Arg-l-Pro)3-(Gly)3-NH2 (1), FAM-β-Ala-(l-Arg-l-Arg-l-ProNH2)3-(Gly)3-NH2 (2), FAM-β-Ala-(l-Arg-l-Arg-l-ProGu)3-(Gly)3-NH2 (3), FAM-β-Ala-(l-Arg)2-(l-ProGu)2-(l-Arg)4-l-ProGu-(Gly)3-NH2 (4), and FAM-β-Ala-(l-Arg)6-(l-ProGu)3-(Gly)3-NH2 (5)] containing l-proline (l-Pro) or cationic proline derivatives (l-ProNH2 and l-ProGu), and investigated their cell-penetrating abilities. Interestingly, only peptide 3 having the side-chain guanidinyl l-ProGu exhibited a secondary structural change in cellular environment. Specifically, peptide 3 formed a random structure in hydrophilic conditions, whereas it formed a helical structure under amphipathic conditions. Furthermore, during cellular permeability tests, peptide 3 demonstrated greater cell-penetrating activity than other peptides and effectively transported plasmid DNA into HeLa cells. Thus, l-ProGu-containing peptide 3 may be a useful candidate as a gene delivery carrier. PMID:27609319

  7. Controlling Factors of Cell Design on Large-format Li-ion Battery Safety During Nail Penetration

    Directory of Open Access Journals (Sweden)

    Qing eWang


    Full Text Available In this paper we investigate the controlling design parameters of large-format Li-ion batteries on safety while undergoing nail penetration. We have identified three critical design parameters that control the safety during the nail penetration process: nail diameter, single sheet foil area, and cell capacity.Using commercial AutoLion software, we have investigated two typical design problems related to the selection of cell thickness and aspect ratio, namely: (1 the safety ramifications of increasing cell capacity via greater cell thickness for a fixed footprint, and (2 the effect of aspect ratio, or single sheet foil size, on safety at a given capacity. For a fixed footprint, our results indicate that the safety of the cell can be predicted by (Qcell Dnail^-0.5. For a given cell capacity, our results indicate that typically a larger single sheet foil area leads to a greater likelihood for thermal runaway due to its effect of making the heating more local in nature; however, for small cells (~ 5Ah and large nails (~ 20mm, the greater aspect ratio can lead to a safer cell, as the greater surface area strongly cools the global heating of the cell.

  8. Follicular penetration and targeting. (United States)

    Lademann, Jürgen; Otberg, Nina; Jacobi, Ute; Hoffman, Robert M; Blume-Peytavi, Ulrike


    In the past, intercellular penetration was assumed to be the most important penetration pathway of topically applied substances. First hints that follicular penetration needs to be taken into consideration were confirmed by recent investigations, presented during the workshop "Follicular Penetration and Targeting" at the 4th Intercontinental Meeting of Hair Research Societies", in Berlin 2004. Hair follicles represent an efficient reservoir for the penetration of topically applied substances with subsequent targeting of distinct cell populations, e.g., nestin-expressing follicular bulge cells. The volume of this reservoir can be determined by differential stripping technology. The follicular penetration processes are significantly influenced by the state of the follicular infundibulum; recent experimental investigations could demonstrate that it is essential to distinguish between open and closed hair follicles. Topically applied substances can only penetrate into open hair follicle. Knowledge of follicular penetration is of high clinical relevance for functional targeting of distinct follicular regions. Human hair follicles show a hair-cycle-dependent variation of the dense neuronal and vascular network. Moreover, during hair follicle cycling with initiation of anagen, newly formed vessels occur. Thus, the potential of nestin-expressing hair follicle stem cells to form neurons and blood vessels was investigated.

  9. Conjugation of a cell-penetrating peptide to parathyroid hormone affects its structure, potency, and transepithelial permeation

    DEFF Research Database (Denmark)

    Kristensen, Mie; de Groot, Anne Marit; Berthelsen, Jens;


    hormone, i.e. PTH(1-34), and to evaluate the effect with regards to secondary structure, potency in Saos-2 cells, immunogenicity, safety as well as the transepithelial permeation across monolayers by using the Caco-2 cell culture model. Further, co-administration of CPP and PTH(1-34) as an alternative......Delivery of therapeutic peptides and proteins by the use of cell-penetrating peptides (CPPs) as carriers has been suggested as a feasible strategy. The aim of the present study was to investigate the effect of conjugating a series of well-known CPPs to the biologically active part of parathyroid...

  10. Effect of the infectious laryngotracheitis virus (ILTV) glycoprotein G on virus attachment, penetration, growth curve and direct cell-to-cell spread

    Institute of Scientific and Technical Information of China (English)

    SUN Zhaogang; ZHANG Manfu


    The secreted alphaherpesvirus glycoprotein G (gG) works differently from other proteins. Analysis of the role of ILTV gG in virus attachment, penetration, direct cell-to-cell spread (CTCS) and the growth curve showed that gG or its antibody had no effect on ILTV attachment and penetration and that the gG antibody reduced the virus plaque size and the one-step growth curve on chicken embryo liver (CEL) cells, but gG did not affect the virus plaque size or the one-step growth curve on CEL cells. Laser scanning confocal microscopy (LSCM) detection showed that ILTV gG is located in the perinuclear region and the membrane of the CEL cells. These results suggested that ILTV gG might contribute to direct cell-to-cell transmission.

  11. Multi-walled Сarbon Nanotubes Penetrate into Plant Cells and Affect the Growth of Onobrychis arenaria Seedlings. (United States)

    Smirnova, E A; Gusev, A A; Zaitseva, O N; Lazareva, E M; Onishchenko, G E; Kuznetsova, E V; Tkachev, A G; Feofanov, A V; Kirpichnikov, M P


    Engineered nanoparticles (ENPs) are now being used in many sectors of industry; however, the impact of ENPs on the environment still requires further study, since their use, recycling, and accidental spill can result in the accumulation of nanoparticles in the atmosphere, soil, and water. Plants are an integral part of ecosystems; hence their interaction with ENPs is inevitable. It is important to understand the consequences of this interaction and assess its potential effects. The present research is focused on studying the effects of the industrial material Taunit, containing multi-walled carbon nanotubes (MWNTs), on plants, and testing of its ability to penetrate into plant cells and tissues. Taunit has been found to stimulate the growth of roots and stems and cause an increase in peroxidase activity inOnobrychis arenariaseedlings. Peroxidase activity increases with decreasing concentration of Taunit from 1,000 to 100 mg/l. MWNTs from Taunit were detected in the cells and tissues of seedling roots and leaves, implying the ability of MWNTs to penetrate into roots and accumulate there, as well as their ability to be transported into seedling leaves. Thus, the changes in the physiological parameters of plants are associated not only with MWNT adsorption on the root surface, as previously believed, but also with their penetration, uptake and accumulation in the plant cells and tissues.

  12. riDOM, a cell-penetrating peptide. Interaction with DNA and heparan sulfate. (United States)

    Québatte, Gabriela; Kitas, Eric; Seelig, Joachim


    DNA condensation in the presence of polycationic molecules is a well-known phenomenon exploited in gene delivery. riDOM (retro-inverso dioleoylmelittin) is a cell-penetrating peptide with excellent transporter properties for DNA. It is a chimeric molecule where ri-melittin is fused to dioleoylphosphoethanolamine. The physical-chemical properties of riDOM in solution and in the presence of DNA and heparan sulfate were investigated with spectroscopic and thermodynamic methods. Dynamic light scattering shows that riDOM in solution aggregates to well-defined nanoparticles with a diameter of ∼13 nm and a ζ-potential of 22 mV, composed of about 220-270 molecules. Binding of riDOM to DNA was studied with dynamic light scattering, ζ-potential measurements, and isothermal titration calorimetry and was compared with authentic melittin-DNA interaction. riDOM binds tightly to DNA with a microscopic binding constant of 5 × 10(7) M(-1) and a stoichiometry of 12 riDOM per 10 DNA base pairs. In the complex the DNA double strand is completely shielded by the more hydrophobic riDOM molecules. Authentic melittin binds to DNA with a much lower binding constant of 5 × 10(6) M(-1) and lower stoichiometry of 5 melittin per 10 DNA base pairs. The binding enthalpies for riDOM and melittin are small and the binding reactions are entropy-driven. Sulfated glycosaminoglycans such as heparan sulfate are also linear molecules with a negative charge. riDOM binding to heparan sulfate on cell surfaces can therefore interfere with DNA-riDOM binding. riDOM-heparan sulfate complex formation was characterized by isothermal titration calorimetry and spectroscopic methods. The binding constant of riDOM for heparan sulfate is K ≈ 2 × 10(6) M(-1). Authentic melittin has a similar binding constant but riDOM shows a 3-fold higher packing density on heparan sulfate than the distinctly smaller melittin.

  13. S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization. (United States)

    Cardoso, Ana M S; Trabulo, Sara; Cardoso, Ana L; Lorents, Annely; Morais, Catarina M; Gomes, Paula; Nunes, Cláudia; Lúcio, Marlene; Reis, Salette; Padari, Kärt; Pooga, Margus; Pedroso de Lima, Maria C; Jurado, Amália S


    The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

  14. Quantitative & qualitative analysis of endothelial cells of donor cornea before & after penetrating keratoplasty in different pathological conditions

    Directory of Open Access Journals (Sweden)

    Aruna K.R. Gupta


    Full Text Available Background & objectives: Endothelial cells of the donor cornea are known to be affected quantitatively and qualitatively in different pathological conditions after penetrating keratoplasty (PK and this has direct effect on the clarity of vision obtained after PK. This study was undertaken to analyze the qualitative and quantitative changes in donor endothelial cells before and after PK in different pathological conditions. Methods: A prospective investigational analysis of 100 consecutive donor corneas used for penetrating keratoplasty between June 2006 and June 2008, was conducted. The patients were evaluated on the first day, at the end of first week, first month, third and six months and one year. Results: A decrease was observed in endothelial cell count in all pathological conditions. After one year of follow up the loss was 33.1 per cent in corneal opacity, 45.9 per cent in acute infective keratitis (AIK, 58.5 per cent in regrafts, 28.5 per cent in pseudophakic bullous keratopathy (PBK, 37 per cent in descemetocele, 27 per cent in keratoconus and 35.5 per cent in aphakic bullous keratopathy (ABK cases. Interpretation & conclusions: The endothelial cell loss was highest in regraft cases which was significant (P<0.05, while the least endothelial cell loss was seen in keratoconus cases. The cell loss was associated with increase in coefficient of variation (CV, i.e. polymegathism and pleomorphism. Inspite of this polymegathism and pleomorphism, the clarity of the graft was maintained.

  15. Enhanced Cellular Uptake of Albumin-Based Lyophilisomes when Functionalized with Cell-Penetrating Peptide TAT in HeLa Cells

    NARCIS (Netherlands)

    van Bracht, Etienne; Versteegden, Luuk R. M.; Stolle, Sarah; Verdurmen, Wouter P. R.; Woestenenk, Rob; Raave, Rene; Hafmans, Theo; Oosterwijk, Egbert; Brock, Roland; van Kuppevelt, Toin H.; Daamen, Willeke F.


    Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various

  16. Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells

    NARCIS (Netherlands)

    Bracht, E. van; Versteegden, L.R.M.; Stolle, S.; Verdurmen, W.P.R.; Woestenenk, R.M.; Raave, R.; Hafmans, T.; Oosterwijk, E.; Brock, R.E.; Kuppevelt, T.H. van; Daamen, W.F.


    Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various

  17. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity. (United States)

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun


    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area.

  18. Cell penetrable humanized-VH/V(HH that inhibit RNA dependent RNA polymerase (NS5B of HCV.

    Directory of Open Access Journals (Sweden)

    Kanyarat Thueng-in

    Full Text Available NS5B is pivotal RNA dependent RNA polymerase (RdRp of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V(HH that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V(HH from a humanized-camel VH/V(HH display library. VH/V(HH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3'di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V(HH hallmark and were designated V(HH6 and V(HH24; other clones were conventional VH, designated VH9 and VH13. All VH/V(HH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V(HH6 and V(HH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V(HH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

  19. Cell penetrable humanized-VH/V(H)H that inhibit RNA dependent RNA polymerase (NS5B) of HCV. (United States)

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Srimanote, Potjanee; Bangphoomi, Kunan; Poungpair, Ornnuthchar; Maneewatch, Santi; Choowongkomon, Kiattawee; Chaicumpa, Wanpen


    NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V(H)H) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V(H)H from a humanized-camel VH/V(H)H display library. VH/V(H)H from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3'di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V(H)H hallmark and were designated V(H)H6 and V(H)H24; other clones were conventional VH, designated VH9 and VH13. All VH/V(H)H were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V(H)H6 and V(H)H24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V(H)H mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

  20.   Cell Penetrating Peptoids (CPPos: Synthesis of a Small Combinatorial Library by Using IRORI MiniKans

    Directory of Open Access Journals (Sweden)

    Dominik K. Kölmel


    Full Text Available Cell penetrating peptoids (CPPos are potent mimics of the corresponding cell penetrating peptides (CPPs. The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used to either target different cellular organelles or even different tissues and organs. In this study we established the submonomer-based solid phase synthesis of a “proof of principle” peptoid library in IRORI MiniKans to expand the amount for phenotypic high throughput screens of CPPos. The library consisting of tetrameric peptoids [oligo(N-alkylglycines] was established on Rink amide resin in a split and mix approach with hydrophilic and hydrophobic peptoid side chains. All CPPos of the presented library were labeled with rhodamine B to allow for the monitoring of cellular uptake by fluorescent confocal microscopy. Eventually, all the purified peptoids were subjected to live cell imaging to screen for CPPos with organelle specificity. While highly charged CPPos enter the cells by endocytosis with subsequent endosomal release, critical levels of lipophilicity allow other CPPos to specifically localize to mitochondria once a certain lipophilicity threshold is reached.

  1. Cell-penetrating peptide-doxorubicin conjugate loaded NGR-modified nanobubbles for ultrasound triggered drug delivery. (United States)

    Lin, Wen; Xie, Xiangyang; Deng, Jianping; Liu, Hui; Chen, Ying; Fu, Xudong; Liu, Hong; Yang, Yang


    A new drug-targeting system for CD13(+) tumors has been developed, based on ultrasound-sensitive nanobubbles (NBs) and cell-permeable peptides (CPPs). Here, the CPP-doxorubicin conjugate (CPP-DOX) was entrapped in the asparagine-glycine-arginine (NGR) peptide modified NB (CPP-DOX/NGR-NB) and the penetration of CPP-DOX was temporally masked; local ultrasound stimulation could trigger the CPP-DOX release from NB and activate its penetration. The CPP-DOX/NGR-NBs had particle sizes of about 200 nm and drug entrapment efficiency larger than 90%. In vitro release results showed that over 85% of the encapsulated DOX or CPP-DOX would release from NBs in the presence of ultrasound, while less than 1.5% of that (30 min) without ultrasound. Cell experiments showed the higher cellular CPP-DOX uptake of CPP-DOX/NGR-NB among the various NB formulations in Human fibrosarcoma cells (HT-1080, CD13(+)). The CPP-DOX/NGR-NB with ultrasound treatment exhibited an increased cytotoxic activity than the one without ultrasound. In nude mice xenograft of HT-1080 cells, CPP-DOX/NGR-NB with ultrasound showed a higher tumor inhibition effect (3.1% of T/C%, day 24), longer median survival time (50 days) and excellent body safety compared with the normal DOX injection group. These results indicate that the constructed vesicle would be a promising drug delivery system for specific cancer treatment.

  2. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Directory of Open Access Journals (Sweden)

    Suh JS


    Full Text Available Jin Sook Suh,1,* Jue Yeon Lee,2,* Yoon Jung Choi,1 Hyung Keun You,3 Seong-Doo Hong,4 Chong Pyoung Chung,2 Yoon Jeong Park1,2 1Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 2Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Seoul, 3Department of Periodontology, College of Dentistry, Wonkwang University, Iksan, 4Department of Oral Pathology, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP, and a transcriptional coactivator with a PDZ-binding motif (TAZ protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in

  3. 细胞穿透肽的研究进展%Recent advances in research on cell penetrating peptides

    Institute of Scientific and Technical Information of China (English)

    李凤英; 何京; 钟海军


    细胞穿透肽(cell penetrating peptides,CPP)是一类能够通过生物膜进入细胞的短肽,它具有一些其他运载系统无法比拟的优点:低浓度条件下,可以穿过细胞膜进入细胞并且不会对膜造成明显破坏和损伤;能介导各种物质包括小分子、核酸、蛋白多肽以及纳米粒子等入胞;高效、低毒.本文就CPP的分类、与载物的连接方式、穿膜机制、应用和常用研究方法等方面进行系统的综述,并对CPP的临床应用前景进行展望.%Cell penetrating peptides (CPP) are a class of peptides of less than 35 amino acids with a remarkable capacity for membrane transportation.Compared with other delivery systems,the CPP-based system has several advantages.These peptides are able to penetrate into cells at low micromolar concentrations in vitro and in vivo without causing significant membrane damage.Furthermore,they constitute very promising tools for cellular import of various substances including small molecules,nucleic acids,proteins and nanoparticles.In this review,the categories,chemical linkage with cargoes,cellular uptake mechanism,applications and research methods of CPP are systematically summarized.The research future of CPP,especially the future of its clinical application,is also introduced.

  4. Development of helix-stabilized cell-penetrating peptides containing cationic α,α-disubstituted amino acids as helical promoters. (United States)

    Yamashita, Hiroko; Misawa, Takashi; Oba, Makoto; Tanaka, Masakazu; Naito, Mikihiko; Kurihara, Masaaki; Demizu, Yosuke


    Cell-penetrating peptides (CPP) have attracted many scientists' attention as intracellular delivery tools due to their high cargo molecule transportation efficiency and low cytotoxicity. Therefore, in many research fields CPP, such as HIV-Tat and oligoarginine (Rn), are used to deliver hydrophilic drugs and biomolecules, including proteins, DNA, and RNA. We designed four types of CPP that contained cationic α,α-disubstituted amino acids (Api(C2Gu) and Api(C4Gu)) as helical promoters; i.e., 1-4 [FAM-β-Ala-(l-Arg-l-Arg-Xaa)3-(Gly)3-NH2 (1: Xaa=Api(C2Gu), 2: Xaa=Api(C4Gu)), 3: FAM-β-Ala-(l-Arg)8-Api(C2Gu)-(Gly)3-NH2, and 4: FAM-β-Ala-(l-Arg)5-Api(C2Gu)-(l-Arg)2-Api(C2Gu)-(Gly)3-NH2], and investigated their preferred secondary structures and cell membrane-penetrating ability. As a result, we found that the permeation efficiency of the CPP was affected by the number of helical promoters in their sequences. Specially, peptide 1, which contained three Api(C2Gu) residues, formed a stable helical structure and passed through the cell membrane more efficiently than the other peptides. Moreover, it was demonstrated that the spatial arrangement of the peptides' side chains also influenced their permeability and the helical stabilization of their main chains.

  5. Transduction of proteins into leishmania tarentolae by formation of non-covalent complexes with cell-penetrating peptides. (United States)

    Keller, Andrea-Anneliese; Breitling, Reinhard; Hemmerich, Peter; Kappe, Katarina; Braun, Maria; Wittig, Berith; Schaefer, Buerk; Lorkowski, Stefan; Reissmann, Siegmund


    Cell-penetrating peptides (CPPs) are used to transport peptides, proteins, different types of ribonucleic acids (or mimics of these molecules), and DNA into live cells, both plant and mammalian. Leishmania belongs to the class of protozoa having, in comparison to mammalian cells, a different lipid composition of the membrane, proteoglycans on the surface, and signal pathways. We investigated the uptake of two different and easily detectable proteins into the non-pathogenic strain Leishmania tarentolae. From the large number of CPPs available, six and a histone were chosen specifically for their ability to form non-covalent complexes. For Leishmania we used the enzyme β-galactosidase and fluorescent labeled bovine serum albumin as cargoes. The results are compared to similar internalization studies using mammalian cells [Mussbach et al., ]. Leishmania cells can degrade CPPs by a secreted and membrane-bound chymotrypsin-like protease. Both cargo proteins were internalized with sufficient efficiency and achieved intramolecular concentrations similar to mammalian cells. The transport efficiencies of the CPPs differed from each other, and showed a different rank order for both cargoes. The intracellular distribution of fluorescent-labeled bovine serum albumin showed highest concentrations in the nucleus and kinetoplast. Leishmania are susceptible to high concentrations of some CPPs, although comparably dissimilar to mammalian cells. MPG-peptides are more cytotoxic in Leishmania than in mammalian cells, acting as antimicrobial peptides. Our results contribute to a better understanding of molecular interactions in Leishmania cells and possibly to new treatments of leishmaniasis.

  6. Cell- and nuclear-penetrating anti-dsDNA autoantibodies have multiple arginines in CDR3 of VH and increase cellular level of pERK and Bcl-2 in mesangial cells. (United States)

    Im, Sae-Ran; Im, Sun-Woo; Chung, Hee-Yong; Pravinsagar, Pavithra; Jang, Young-Ju


    Investigation of characteristics of cell- and nuclear-penetrating anti-double stranded (ds)DNA autoantibodies (autoAbs) is important to understand pathogenesis of lupus nephritis, but has not been clearly explored. The present study reports that three anti-dsDNA monoclonal autoAbs, which contain more than two arginine residues in their CDR3s of variable heavy domain (VH), penetrated into living murine mesangial cells and the cell nuclei. However, an anti-dsDNA monoclonal Ab (mAb) having only one arginine in the CDR3-VH did not penetrate cells. To assess the contribution of antigen-binding sites, especially the VH, in cell- and nuclear-penetration, we evaluated the characteristics of recombinant single chain Fv(scFv), VH, and variable light domain (VL) of a penetrating mAb. The scFv and VH domain, containing arginine in CDR3-VH maintained the ability to penetrate cells and the cell nuclei, whereas the VL domain, having no arginine in CDR3, did not penetrate cells. The penetratingm Abs, scFv, and VH activated ERK and increased cellular protein levels of Bcl-2, whereas the non-penetrating Ab and VL did not. The cell survival was decreased by the penetrating mAbs, scFv and VH, not by the non-penetrating mAb and VL. The data indicate that an antigen-binding site is required for cell-penetration and that positively-charged arginine residues in CDR3-VH contribute to the cell- and nuclear-penetrating ability of a subset of anti-dsDNA autoAbs. Furthermore,the nuclear-penetrating anti-dsDNA autoAbs could possibly function as a pathogenic factor for lupus nephritis by up-regulating ERK activation and Bcl-2 production in mesangial cells. The cell- and nuclear-penetrating VH domain may be exploited as a vehicle for the intra cellular delivery of various useful molecules.

  7. A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems

    Directory of Open Access Journals (Sweden)

    Mariana H. Massaoka


    Full Text Available The Wilms tumor protein 1 (WT1 transcription factor has been associated in malignant melanoma with cell survival and metastasis, thus emerging as a candidate for targeted therapy. A lysine–arginine rich peptide, WT1-pTj, derived from the ZF domain of WT1 was evaluated as an antitumor agent against A2058 human melanoma cells and B16F10-Nex2 syngeneic murine melanoma. Peptide WT1-pTj quickly penetrated human melanoma cells and induced senescence, recognized by increased SA-β-galactosidase activity, enhanced transcriptional activity of p53, and induction of the cell cycle inhibitors p21 and p27. Moreover, the peptide bound to p53 and competed with WT1 protein for binding to p53. WT1-pTj treatment led to sustained cell growth suppression, abrogation of clonogenicity and G2/M cell cycle arrest. Notably, in vivo studies showed that WT1-pTj inhibited both the metastases and subcutaneous growth of murine melanoma in syngeneic mice, and prolonged the survival of nude mice challenged with human melanoma cells. The 27-amino acid cell-penetrating WT1-derived peptide, depends on C3 and H16 for effective antimelanoma activity, inhibits proliferation of WT1-expressing human tumor cell lines, and may have an effective role in the treatment of WT1-expressing malignancies.

  8. Peptide based DNA nanocarriers incorporating a cell-penetrating peptide derived from neurturin protein and poly-L-lysine dendrons. (United States)

    Rosli, Nurlina; Christie, Michelle P; Moyle, Peter M; Toth, Istvan


    Multicomponent gene delivery systems incorporating cell-penetrating peptides (CPP) from the human neurturin protein (NRTN-30, NRTN(132-161); NRTN-17, NRTN(145-161)) and a poly-l-lysine (PLL) dendron, were synthesized and characterized for plasmid DNA (pDNA) delivery. Acetylated NRTN peptides (Ac-CPP) and peptides conjugated to a PLL dendron (DEN-CPP) efficiently condensed and stabilized pDNA. Complexes between pDNA and DEN-CPP formed smaller and more stable nanoparticles. Flow cytometry experiments showed that pDNA-DEN-CPPs were taken up more efficiently into HeLa cells. There was also no significant difference between NRTN-30 and NRTN-17 for pDNA uptake, indicating that the truncated peptide alone is sufficient as a CPP for pDNA delivery.

  9. Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E


    We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting...... splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA...... that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP...

  10. Dual-wavelength imaging of tumor progression by activatable and targeting near-infrared fluorescent probes in a bioluminescent breast cancer model.

    Directory of Open Access Journals (Sweden)

    Bang-Wen Xie

    Full Text Available Bioluminescence imaging (BLI has shown its appeal as a sensitive technique for in vivo whole body optical imaging. However, the development of injectable tumor-specific near-infrared fluorescent (NIRF probes makes fluorescence imaging (FLI a promising alternative to BLI in situations where BLI cannot be used or is unwanted (e.g., spontaneous transgenic tumor models, or syngeneic mice to study immune effects.In this study, we addressed the questions whether it is possible to detect tumor progression using FLI with appropriate sensitivity and how FLI correlates with BLI measurements. In addition, we explored the possibility to simultaneously detect multiple tumor characteristics by dual-wavelength FLI (~700 and ~800 nm in combination with spectral unmixing. Using a luciferase-expressing 4T1-luc2 mouse breast cancer model and combinations of activatable and targeting NIRF probes, we showed that the activatable NIRF probes (ProSense680 and MMPSense680 and the targeting NIRF probes (IRDye 800CW 2-DG and IRDye 800CW EGF were either activated by or bound to 4T1-luc2 cells. In vivo, we implanted 4T1-luc2 cells orthotopically in nude mice and were able to follow tumor progression longitudinally both by BLI and dual-wavelength FLI. We were able to reveal different probe signals within the tumor, which co-localized with immuno-staining. Moreover, we observed a linear correlation between the internal BLI signals and the FLI signals obtained from the NIRF probes. Finally, we could detect pulmonary metastases both by BLI and FLI and confirmed their presence histologically.Taken together, these data suggest that dual-wavelength FLI is a feasible approach to simultaneously detect different features of one tumor and to follow tumor progression with appropriate specificity and sensitivity. This study may open up new perspectives for the detection of tumors and metastases in various experimental models and could also have clinical applications, such as image

  11. Far-Red Light Activatable, Multifunctional Prodrug for Fluorescence Optical Imaging and Combinational Treatment


    Bio, Moses; Rajaputra, Pallavi; Nkepang, Gregory; You, Youngjae


    We recently developed “photo-unclick chemistry”, a novel chemical tool involving the cleavage of aminoacrylate by singlet oxygen, and demonstrated its application to visible light-activatable prodrugs. In this study, we prepared an advanced multifunctional prodrug, Pc-(L-CA4)2, composed of the fluorescent photosensitizer phthalocyanine (Pc), an SO-labile aminoacrylate linker (L), and a cytotoxic drug combretastatin A-4 (CA4). Pc-(L-CA4)2 had reduced dark toxicity compared with CA4. However, o...

  12. Cell-penetrating peptide derived from human eosinophil cationic protein inhibits mite allergen Der p 2 induced inflammasome activation.

    Directory of Open Access Journals (Sweden)

    Sheng-Jie Yu

    Full Text Available Newly discovered cell penetration peptides derived from human eosinophil cationic proteins (CPPecp have the characteristic of cell internalization, but the effect of CPPecp on immunomodulation has not been clarified. House dust mite (HDM major allergen, Der p 2, can induce proinflammatory cytokine production which contributes to airway inflammation and allergic asthma. However, the mechanism of Der p 2 on NLRP3 inflammasome activation remains unclear. The aim of this study was to investigate the immunomodulatory effect of CPPecp on inhibition of Der p 2 induced inflammasome activation. We showed that proinflammatory cytokines IL-1β, IL-6 and IL-8 were significantly upregulated in peripheral blood mononuclear cells (PBMCs derived from HDM allergic patients after Der p 2 stimulation. Expression of NLRP3, ASC, Caspase-1, IL-1β and Caspase-1 activity was upregulated in THP-1 cells after Der p 2 stimulation. Proinflammatory cytokine production, NLRP3 inflammasome activation and caspase-1 activity were downregulated in THP-1 cells and CD14+ cells co-cultured with Der p 2 and CPPecp. The immunomodulatory effect of CPPecp was through upregulation of IFN-α production but not induction of autophagy. These results suggested Der p 2 plays an important role in NLRP3 inflammasome activation and CPPecp has the potential to be a novel anti-inflammatory agent for allergic inflammation treatment in the future.

  13. Massive glycosaminoglycan-dependent entry of Trp-containing cell-penetrating peptides induced by exogenous sphingomyelinase or cholesterol depletion. (United States)

    Bechara, Chérine; Pallerla, Manjula; Burlina, Fabienne; Illien, Françoise; Cribier, Sophie; Sagan, Sandrine


    Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.

  14. Using the peptide BP100 as a cell-penetrating tool for the chemical engineering of actin filaments within living plant cells. (United States)

    Eggenberger, Kai; Mink, Christian; Wadhwani, Parvesh; Ulrich, Anne S; Nick, Peter


    The delivery of externally applied macromolecules or nanoparticles into living cells still represents a critically limiting step before the full capabilities of chemical engineering can be explored. Molecular transporters such as cell-penetrating peptides, peptoids, and other mimetics can be used to carry cargo across the cellular membrane, but it is still difficult to find suitable sequences that operate efficiently for any particular type of cell. Here we report that BP100 (KKLFKKILKYL-amide), originally designed as an antimicrobial peptide against plant pathogens, can be employed as a fast and efficient cell-penetrating agent to transport fluorescent test cargoes into the cytosol of walled plant cells. The uptake of BP100 proceeds slightly more slowly than the endocytosis of fluorescent dextranes, but BP100 accumulates more efficiently and to much higher levels (by an order of magnitude). The entry of BP100 can be efficiently blocked by latrunculin B; this suggests that actin filaments are essential to the uptake mechanism. To test whether this novel transporter can also be used to deliver functional cargoes, we designed a fusion construct of BP100 with the actin-binding Lifeact peptide (MGVADLIKKFESISKEE). We demonstrated that the short BP100 could transport the attached 17-residue sequence quickly and efficiently into tobacco cells. The Lifeact construct retained its functionality as it successfully labeled the actin bundles that tether the nucleus in the cell center.

  15. Effect of chirality and length on the penetrability of single-walled carbon nanotubes into lipid bilayer cell membranes. (United States)

    Skandani, A Alipour; Zeineldin, R; Al-Haik, M


    The ability of carbon nanotubes to enter the cell membrane acting as drug-delivery vehicles has yielded a plethora of experimental investigations, mostly with inconclusive results because of the wide spectra of carbon nanotube structures. Because of the virtual impossibility of synthesizing CNTs with distinct chirality, we report a parametric study on the use of molecular dynamics to provide better insight into the effect of the carbon nanotube chirality and the aspect ratio on the interaction with a lipid bilayer membrane. The simulation results indicated that a single-walled carbon nanotube utilizes different time-evolving mechanisms to facilitate their internalization within the membrane. These mechanisms comprise both penetration and endocytosis. It was observed that carbon nanotubes with higher aspect ratios penetrate the membrane faster whereas shorter nanotubes undergo significant rotation during the final stages of endocytosis. Furthermore, nanotubes with lower chiral indices developed significant adhesion with the membrane. This adhesion is hypothesized to consume some of the carbon nanotube energy, thus resulting in longer times for the nanotube to translocate through the membrane.

  16. A dual functional fluorescent probe for glioma imaging mediated by blood-brain barrier penetration and glioma cell targeting. (United States)

    Ma, Hongwei; Gao, Zhiyong; Yu, Panfeng; Shen, Shun; Liu, Yongmei; Xu, Bainan


    Glioma is a huge threat for human being because it was hard to be completely removed owing to both the infiltrating growth of glioma cells and integrity of blood brain barrier. Thus effectively imaging the glioma cells may pave a way for surgical removing of glioma. In this study, a fluorescent probe, Cy3, was anchored onto the terminal of AS1411, a glioma cell targeting aptamer, and then TGN, a BBB targeting peptide, was conjugated with Cy3-AS1411 through a PEG linker. The production, named AsT, was characterized by gel electrophoresis, (1)H NMR and FTIR. In vitro cellular uptake and glioma spheroid uptake demonstrated the AsT could not only be uptaken by both glioma and endothelial cells, but also penetrate through endothelial cell monolayer and uptake by glioma spheroids. In vivo, AsT could effectively target to glioma with high intensity. In conclusion, AsT could be used as an effective glioma imaging probe.

  17. Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide. (United States)

    Oehlke, Johannes; Wallukat, Gerd; Wolf, Yvonne; Ehrlich, Angelika; Wiesner, Burkhard; Berger, Hartmut; Bienert, Michael


    In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.

  18. Amelioration of penetrating ballistic-like brain injury induced cognitive deficits after neuronal differentiation of transplanted human neural stem cells. (United States)

    Spurlock, Markus S; Ahmed, Aminul Islam; Rivera, Karla N; Yokobori, Shoji; Lee, Stephanie W; Sam, Pingdewinde N; Shear, Deborah A; Hefferan, Michael P; Hazel, Thomas G; Johe, Karl K; Gajavelli, Shyam; Tortella, Frank C; Bullock, Ross


    Penetrating traumatic brain injury (PTBI) is one of the major cause of death and disability worldwide. Previous studies in penetrating ballistic-like brain injury (PBBI), a PTBI rat model revealed widespread peri-lesional neurodegeneration, similar to that seen in humans following gunshot wound to head, which is unmitigated by any available therapies to date. Therefore, we evaluated human neural stem cell (hNSC) engraftment to putatively exploit the potential of cell therapy that has been seen in other central nervous system injury models. Towards this, green fluorescent protein (GFP) labeled hNSCs (400,000 per animal) were transplanted in immunosuppressed Sprague Dawley (SD), Fisher, and athymic (ATN) PBBI rats one week after injury. Tacrolimus (3mg/kg two days prior to transplantation, then 1mg/kg/day), Methylprednisolone (10mg/kg on day of transplant, 1mg/kg/week thereafter), and Mycophenolate mofetil (30mg/kg/day) for seven days following transplantation were used to confer immunosuppression. Engraftment in SD and ATN was comparable at 8-weeks post transplantation. Evaluation of hNSC differentiation and distribution revealed increased neuronal differentiation of transplanted cells with time. At 16-weeks post-transplantation neither cell proliferation nor glial lineage markers expression was detected. Transplanted cell morphology was similar to neighboring host neurons and there was relatively little migration of cells from the peri-transplant site. By 16 weeks, GFP positive processes extended both rostro-caudally and bilaterally into parenchyma, spreading along host white matter tracts, traversing internal capsule, extending ~13 mm caudally from transplantation site reaching into the brain stem. In a Morris water maze test at 8-weeks post-transplantation, animals with transplants had shorter latency to platform compared to vehicle treated animals. However, weak injury-induced cognitive deficits in the control group at the delayed time point confounded benefits

  19. Cell Adhesion Induced Using Surface Modification with Cell-Penetrating Peptide-Conjugated Poly(ethylene glycol)-Lipid: A New Cell Glue for 3D Cell-Based Structures. (United States)

    Teramura, Yuji; Asif, Sana; Ekdahl, Kristina N; Gustafson, Elisabet; Nilsson, Bo


    We synthesized a novel material, cell-penetrating peptide-conjugated poly(ethylene glycol)-lipid (CPP-PEG-lipid), that can induce the adhesion of floating cells. Firm cell adhesion with spreading could be induced by cell surface modification with the CPP-PEG-lipids. Cell adhesion was induced by CPPs but not by any other cationic short peptides we tested. Here, we demonstrated adherence using the floating cell line CCRF-CEM as well as primary human T cells, B cells, erythrocytes, and hepatocytes. As compared to cells grown in suspension, adherent cells were more rapidly induced to attach to substrates with the cell-surface modification. The critical factor for attachment was localization of CPPs at the cell membrane by PEG-lipids with PEG > 20 kDa. These cationic CPPs on PEG chains were able to interact with substrate surfaces such as polystyrene (PS) surfaces, glass surfaces, and PS microfibers that are negatively charged, inducing firm cell adhesion and cell spreading. Also, as opposed to normal cationic peptides that interact strongly with cell membranes, CPPs were less interactive with the cell surfaces because of their cell-penetrating property, making them more available for adhering cells to the substrate surface. No effects on cell viability or cell proliferation were observed after the induction of cell adhesion. With this technique, cells could be easily immobilized onto PS microfibers, an important step in fabricating 3D cell-based structures. Cells immobilized onto 3D PS microfibers were alive, and human hepatocytes showed normal production of urea and albumin on the microfibers. This method is novel in inducing firm cell adhesion via a one-step treatment.

  20. Binding characteristics of thrombin-activatable fibrinolysis inhibitor to streptococcal surface collagen-like proteins A and B

    NARCIS (Netherlands)

    Seron, Mercedes Valls; Plug, Tom; Marquart, J. Arnoud; Marx, Pauline F.; Herwald, Heiko; de Groot, Philip G.; Meijers, Joost C. M.


    Streptococcus pyogenes is the causative agent in a wide range of diseases in humans. Thrombin-activatable fibrinolysis inhibitor (TAFI) binds to collagen-like proteins ScIA and ScIB at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a transient to chronic s

  1. Distal phenylalanine modification for enhancing cellular delivery of fluorophores, proteins and quantum dots by cell penetrating peptides. (United States)

    Sayers, E J; Cleal, K; Eissa, N G; Watson, P; Jones, A T


    For cell penetrating peptides (CPPs) to fulfil their promise as effective delivery vectors we need a better understanding of their mechanisms of cell binding and uptake. This is especially the case when they are linked to different types of cargo. Here we describe new studies based on our previous findings suggesting that, for peptide-CPP chimeras, distal hydrophobic residues upstream of the CPP sequence can have profound effects on the way they interact with cells. We studied peptides bearing an N-terminal Glycine or Phenylalanine linked via a neutral and flexible bridging group, SGSGSGSG, to three well-studied CPPs: octaarginine, penetratin and TP10. Using a combination of flow cytometry, live-cell imaging and image analysis we examined the effects of this single amino acid change on binding and uptake of Alexa488-fluorophore, bovine serum albumin and quantum dot cargoes. The influence of the glycine-phenylalanine switch for fluorophore delivery was most dramatic in TP10, increasing cellular uptake by 4.4 and 9.9 fold in non-adherent and adherent cells, respectively. Only penetratin showed effective uptake of bovine serum albumin with the phenylalanine variant showing an increase of 1.6 fold over the glycine variant. The uptake of quantum dots was most efficiently demonstrated by octaarginine, with the glycine variant increasing uptake 4.8 fold and the phenylalanine variant increasing uptake 9.5 fold over quantum dots alone. Overall the data demonstrate that hydrophobicity distal to the CPP could be utilised to enhance their capacity to bind to the cell membrane and deliver a range of macromolecules to the insides of cells.

  2. Ultra-small lipid nanoparticles promote the penetration of coenzyme Q10 in skin cells and counteract oxidative stress. (United States)

    Lohan, Silke B; Bauersachs, Sonja; Ahlberg, Sebastian; Baisaeng, Nuttakorn; Keck, Cornelia M; Müller, Rainer H; Witte, Ellen; Wolk, Kerstin; Hackbarth, Steffen; Röder, Beate; Lademann, Jürgen; Meinke, Martina C


    UV irradiation leads to the formation of reactive oxygen species (ROS). An imbalance between the antioxidant system and ROS can lead to cell damage, premature skin aging or skin cancer. To counteract these processes, antioxidants such as coenzyme Q10 (CoQ10) are contained in many cosmetics. To improve and optimize cell/tissue penetration properties of the lipophilic CoQ10, ultra-small lipid nanoparticles (usNLC) were developed. The antioxidant effectiveness of CoQ10-loaded usNLC compared to conventional nanocarriers was investigated in the human keratinocyte cell line HaCaT. Using confocal laser scanning microscopy investigations of the carriers additionally loaded with nile red showed a clear uptake into cells and their distribution within the cytoplasm. By use of the XTT cell viability test, CoQ10 concentrations of 10-50 μg/ml were shown to be non-toxic, and the antioxidant potential of 10 μg/ml CoQ10 loaded usNLC in the HaCaT cells was analyzed via electron paramagnetic resonance spectroscopy after cellular exposure to UVA (1J/cm(2)) and UVB (18 mJ/cm(2)) irradiation. In comparison with the CoQ10-loaded conventional carriers, usNLC-CoQ10 demonstrated the strongest reduction of the radical formation; reaching up to 23% compared to control cells without nanocarrier treatment. Therefore, usNLC-CoQ10 are very suitable to increase the antioxidant potential of skin.

  3. PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation. (United States)

    Ezzat, Kariem; Andaloussi, Samir E L; Zaghloul, Eman M; Lehto, Taavi; Lindberg, Staffan; Moreno, Pedro M D; Viola, Joana R; Magdy, Tarek; Abdo, Rania; Guterstam, Peter; Sillard, Rannar; Hammond, Suzan M; Wood, Matthew J A; Arzumanov, Andrey A; Gait, Michael J; Smith, C I Edvard; Hällbrink, Mattias; Langel, Ülo


    Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.

  4. Cell Penetrable Human scFv Specific to Middle Domain of Matrix Protein-1 Protects Mice from Lethal Influenza

    Directory of Open Access Journals (Sweden)

    Fonthip Dong-din-on


    Full Text Available A new anti-influenza remedy that can tolerate the virus antigenic variation is needed. Influenza virus matrix protein-1 (M1 is highly conserved and pivotal for the virus replication cycle: virus uncoating, assembly and budding. An agent that blocks the M1 functions should be an effective anti-influenza agent. In this study, human scFv that bound to recombinant M1 middle domain (MD and native M1 of A/H5N1 was produced. Phage mimotope search and computerized molecular docking revealed that the scFv bound to the MD conformational epitope formed by juxtaposed helices 7 and 9 of the M1. The scFv was linked molecularly to a cell penetrable peptide, penetratin (PEN. The PEN-scFv (transbody, when used to treat the cells pre-infected with the heterologous clade/subclade A/H5N1 reduced the viral mRNA intracellularly and in the cell culture fluids. The transbody mitigated symptom severity and lung histopathology of the H5N1 infected mice and caused reduction of virus antigen in the tissues as well as extricated the animals from the lethal challenge in a dose dependent manner. The transbody specific to the M1 MD, either alone or in combination with the cognate human scFvs specific to other influenza virus proteins, should be an effective, safe and mutation tolerable anti-influenza agent.

  5. Research Progress of Cell -penetrating Peptides Applied in Vaccine%穿膜肽在疫苗中应用的研究进展

    Institute of Scientific and Technical Information of China (English)

    范兴琼; 吴双林; 刘成倩; 易建中


    Since the discovery of the function of protein transduction, cell- penetrating peptides (CPP) provides a novel approach for gene therapy and vaccine development. According to recent studies on the CPP species, structure, membrane -penetrating mechanisms and relative applications, cell -penetrating peptides applied in the DNA vaccine and tumor vaccine were reviewed and prospected.%穿膜肽自发现以来,因其独特的蛋白转导功能,为基因治疗和新型疫苗的开发等开辟了新的途径。通过综述穿膜肽的种类、结构特点、作用机制及其在疫苗中的应用,为进一步研发DNA疫苗和肿瘤疫苗奠定基础。

  6. Spotlight on Human LL-37, an Immunomodulatory Peptide with Promising Cell-Penetrating Properties

    Directory of Open Access Journals (Sweden)

    Marc Ferdinand Lensink


    Full Text Available Cationic antimicrobial peptides are major components of innate immunity and help control the initial steps of the infectious process. They are expressed not only by immunocytes, but also by epithelial cells. They share an amphipathic secondary structure with a polar cationic site, which explains their tropism for prokaryote membranes and their hydrophobic site contributing to the destructuration of these membranes. LL-37 is the only cationic antimicrobial peptide derived from human cathelicidin. LL-37 can also cross the plasma membrane of eukaryotic cells, probably through special domains of this membrane called lipid rafts. This transfer could be beneficial in the context of vaccination: the activation of intracellular toll-like receptors by a complex formed between CpG oligonucleotides and LL-37 could conceivably play a major role in the building of a cellular immunity involving NK cells.

  7. Fullerene C60 Penetration into Leukemic Cells and Its Photoinduced Cytotoxic Effects (United States)

    Franskevych, D.; Palyvoda, K.; Petukhov, D.; Prylutska, S.; Grynyuk, I.; Schuetze, C.; Drobot, L.; Matyshevska, O.; Ritter, U.


    Fullerene C60 as a representative of carbon nanocompounds is suggested to be promising agent for application in photodynamic therapy due to its unique physicochemical properties. The goal of this study was to estimate the accumulation of fullerene C60 in leukemic cells and to investigate its phototoxic effect on parental and resistant to cisplatin leukemic cells. Stable homogeneous water colloid solution of pristine C60 with average 50-nm diameter of nanoparticles was used in experiments. Fluorescent labeled C60 was synthesized by covalent conjugation of C60 with rhodamine B isothiocyanate. The results of confocal microscopy showed that leukemic Jurkat cells could effectively uptake fullerene C60 from the medium. Light-emitting diode lamp (100 mW cm-2, λ = 420-700 nm) was used for excitation of accumulated C60. A time-dependent decrease of viability was detected when leukemic Jurkat cells were exposed to combined treatment with C60 and visible light. The cytotoxic effect of photoexcited C60 was comparable with that induced by H2O2, as both agents caused 50% decrease of cell viability at 24 h at concentrations about 50 μM. Using immunoblot analysis, protein phosphotyrosine levels in cells were estimated. Combined action of C60 and visible light was followed by decrease of cellular proteins phosphorylation on tyrosine residues though less intensive as compared with that induced by H2O2 or protein tyrosine kinase inhibitor staurosporine. All tested agents reduced phosphorylation of 55, 70, and 90 kDa proteins while total suppression of 26 kDa protein phosphorylation was specific only for photoexcited C60.

  8. Potent inhibition of late stages of hepadnavirus replication by a modified cell penetrating peptide

    DEFF Research Database (Denmark)

    Abdul, Fabien; Ndeboko, Bénédicte; Buronfosse, Thierry;


    by confocal laser scanning microscopy indicating severe structural changes of preS/S. Sucrose gradient analysis of supernatants from Deca-(Arg)8-treated cells showed unaffected naked viral nucleocapsids release, which was concomitant with a complete arrest of virion and surface protein-containing subviral...... particle secretion. This is the first report showing that a CPP is able to drastically block hepadnaviral release from infected cells by altering late stages of viral morphogenesis via interference with enveloped particle formation, without affecting naked nucleocapsid egress, thus giving a view inside...

  9. Two functional motifs define the interaction, internalization and toxicity of the cell-penetrating antifungal peptide PAF26 on fungal cells.

    Directory of Open Access Journals (Sweden)

    Alberto Muñoz

    Full Text Available The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW and PAF96 (RKKAAA, were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i its interaction with the cell envelope; (ii its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the

  10. [Cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy]. (United States)

    Tan, Jiao; Wang, Ya-Ping; Wang, Hui-Xin; Liang, Jian-Ming; Zhang, Meng; Sun, Xun; Huang, Yong-Zhuo


    To develop a cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy, we prepared the AVPI-LMWP/pTRAIL self-assembled complexes containing a therapeutic combination of peptide drug AVPI and DNA drug TRAIL. The chimeric apoptotic peptide AVPI-LMWP was synthesized using the standard solid-phase synthesis. The cationic AVPI-LMWP could condense pTRAIL by electrostatic interaction. The physical-chemical properties of the AVPI-LMWP/pTRAIL complexes were characterized. The cellular uptake efficiency and the inhibitory activity of the AVPI-LMWP/pTRAIL complexes on tumor cell were also performed. The results showed that the AVPI-LMWP/pTRAIL complexes were successfully prepared by co-incubation. With the increase of mass ratio (AVPI-LMWP/DNA), the particle size was decreased and the zeta potential had few change. Agarose gel electrophoresis showed that AVPI-LMWP could fully bind and condense pTRAIL at a mass ratio above 15:1. Cellular uptake efficiency was improved along with the increased ratio of W(AVPI-LMWP)/WpTRAIL. The in vitro cytotoxicity experiments demonstrated that the AVPI-LMWP/pTRAIL (W:W = 20:1) complexes was significantly more effective than the pTRAIL, AVPI-LMWP alone or LMWP/pTRAIL complexes on inhibition of HeLa cell growth. Our studies indicated that the AVPI-LMWP/pTRAIL co-delivery system could deliver plasmid into HeLa cell and induce tumor cell apoptosis efficiently, which showed its potential in cancer therapy using combination of apoptoic peptide and gene drugs.

  11. Antitumor activity of tripterine via cell-penetrating peptide-coated nanostructured lipid carriers in a prostate cancer model

    Directory of Open Access Journals (Sweden)

    Yuan L


    Full Text Available Ling Yuan,1 Congyan Liu,2 Yan Chen,2 Zhenhai Zhang,2 Lei Zhou,1 Ding Qu2 1Department of Pharmaceutics, School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, 2Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu, People's Republic of China Background: The purpose of this study was to evaluate the antitumor effect of cell-penetrating peptide-coated tripterine-loaded nanostructured lipid carriers (CT-NLC on prostate tumor cells in vitro and in vivo. Methods: CT-NLC were developed to improve the hydrophilicity of tripterine. The antiproliferative effects of CT-NLC, tripterine-loaded nanostructured lipid carriers (T-NLC, and free tripterine in a human prostatic carcinoma cell line (PC-3 and a mouse prostate carcinoma cell line (RM-1 were evaluated using an MTT assay. The advantage of CT-NLC over T-NLC and free tripterine with regard to antitumor activity in vivo was evaluated in a prostate tumor-bearing mouse model. The induced tumor necrosis factor-alpha and interleukin-6 cytokine content was investigated by enzyme-linked immunosorbent assay to determine the effect of CT-NLC, T-NLC, and free tripterine on immune responses. Histologic and TUNEL assays were carried out to investigate the mechanisms of tumor necrosis and apoptosis. Results: CT-NLC, T-NLC, and free tripterine showed high antiproliferative activity in a dose-dependent manner, with an IC50 of 0.60, 0.81, and 1.02 µg/mL in the PC-3 cell line and 0.41, 0.54, and 0.89 µg/mL in the RM-1 cell line after 36 hours. In vivo, the tumor inhibition rates for cyclophosphamide, high-dose (4 mg/kg and low-dose (2 mg/kg tripterine, high-dose (4 mg/kg and low-dose (2 mg/kg T-NLC, high-dose (4 mg/kg and low-dose (2 mg/kg CT-NLC were 76.51%, 37.07%, 29.53%, 63.56%, 48.25%, 72.68%, and 54.50%, respectively, showing a dose-dependent pattern. The induced tumor necrosis factor-alpha and interleukin-6 cytokine content

  12. Enhanced cellular delivery of cell-penetrating peptide-peptide nucleic acid conjugates by photochemical internalization

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E


    )) or tetraphenylporphyrin tetrasulfonic acid (TPPS). Cellular uptake of the PNA conjugates were evaluated by using a sensitive cellular method with HeLa pLuc705 cells based on the splicing correction of luciferase gene by targeting antisense oligonucleotides to a cryptic splice site of the mutated luciferase gene....... The cellular efficacy of CPP conjugates were evaluated by measuring luciferase activity as a result of splicing correction and was also confirmed by RT-PCR analysis of luciferase pre-mRNA....

  13. Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells. (United States)

    Crosera, Matteo; Prodi, Andrea; Mauro, Marcella; Pelin, Marco; Florio, Chiara; Bellomo, Francesca; Adami, Gianpiero; Apostoli, Pietro; De Palma, Giuseppe; Bovenzi, Massimo; Campanini, Marco; Filon, Francesca Larese


    Titanium dioxide nanoparticles (TiO2NPs) suspensions (concentration 1.0 g/L) in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue(®) and propidium iodide, PI, uptake assays) was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact and damaged skin. Titanium was found in the epidermal layer after 24 h of exposure (0.47 ± 0.33 μg/cm(2)) while in the dermal layer, the concentration was below the limit of detection. Damaged skin, in its whole, has shown a similar concentration (0.53 ± 0.26 μg/cm(2)). Cytotoxicity studies on HaCaT cells demonstrated that TiO2NPs induced cytotoxic effects only at very high concentrations, reducing cell viability after seven days of exposure with EC50s of 8.8 × 10(-4) M (MTT assay), 3.8 × 10(-5) M (AlamarBlue(®) assay), and 7.6 × 10(-4) M (PI uptake, index of a necrotic cell death). Our study demonstrated that TiO2NPs cannot permeate intact and damaged skin and can be found only in the stratum corneum and epidermis. Moreover, the low cytotoxic effect observed on human HaCaT keratinocytes suggests that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.

  14. Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells

    Directory of Open Access Journals (Sweden)

    Matteo Crosera


    Full Text Available Titanium dioxide nanoparticles (TiO2NPs suspensions (concentration 1.0 g/L in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue® and propidium iodide, PI, uptake assays was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact and damaged skin. Titanium was found in the epidermal layer after 24 h of exposure (0.47 ± 0.33 μg/cm2 while in the dermal layer, the concentration was below the limit of detection. Damaged skin, in its whole, has shown a similar concentration (0.53 ± 0.26 μg/cm2. Cytotoxicity studies on HaCaT cells demonstrated that TiO2NPs induced cytotoxic effects only at very high concentrations, reducing cell viability after seven days of exposure with EC50s of 8.8 × 10−4 M (MTT assay, 3.8 × 10−5 M (AlamarBlue® assay, and 7.6 × 10−4 M (PI uptake, index of a necrotic cell death. Our study demonstrated that TiO2NPs cannot permeate intact and damaged skin and can be found only in the stratum corneum and epidermis. Moreover, the low cytotoxic effect observed on human HaCaT keratinocytes suggests that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.

  15. Study on the Penetrability of PEP-1-P27mt for Cell Membranes in Vitro

    Institute of Scientific and Technical Information of China (English)

    YAN Shirong; ZHU Minglei; QIU Fangcheng; WANG Lilin; QU Shen


    Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, prokaryotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS transformed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentrations in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.

  16. Comparison of Franz cells and microdialysis for assessing salicylic acid penetration through human skin. (United States)

    Leveque, N; Makki, S; Hadgraft, J; Humbert, Ph


    The purpose of the present study was to compare Franz cells (FC) and microdialysis (MD) for monitoring the skin absorption of salicylic acid (SA). The influence of pH on SA flux was also assessed by these two techniques. Excised abdominal human skin was used in the experiments. SA was dissolved in phosphate buffer solutions of pH 2, 5 and 7 (2 mg/ml). SA concentrations in the receptor FC solutions and in MD samples were assessed by high performance liquid chromatography (HPLC). The results demonstrate that the flux of SA decreased with increased pH. The profiles permeation determined by Franz cells and microdialysis were similar. However, whatever the pH, the SA flux was higher with microdialysis than with Franz cells. The results showed that SA percutaneous permeation conformed to the pH partition hypothesis. The flux of SA was different when it was determined by the two techniques. The collect of SA, by these two techniques is different. The results of the two techniques are compared and discussed.

  17. State of the Art in the Studies on Crotamine, a Cell Penetrating Peptide from South American Rattlesnake

    Directory of Open Access Journals (Sweden)

    Irina Kerkis


    Full Text Available Animal venoms comprise a naturally selected cocktail of bioactive peptides/proteins and other molecules, each of which playing a defined role thanks to the highly specific interactions with diverse molecular targets found in the prey. Research focused on isolation, structural, and functional characterizations of novel natural biologics (bioactive peptides/proteins from natural sources has a long way to go through from the basic science to clinical applications. Herein, we overview the structural and functional characteristics of the myoneurotoxin crotamine, firstly isolated from the South American rattlesnake venom. Crotamine is the first venom peptide classified as a natural cell penetrating and antimicrobial peptide (CPP and AMP with a more pronounced antifungal activity. In contrast to other known natural CPPs and AMPs, crotamine demonstrates a wide spectrum of biological activities with potential biotechnological and therapeutic values. More recent studies have demonstrated the selective in vitro anticancer activity of crotamine. In vivo, using a murine melanoma model, it was shown that crotamine delays tumor implantation, inhibits tumor cells proliferation, and also increases the survival of mice engrafted with subcutaneous melanoma. The structural and functional properties and also the possible biotechnological applications of minimized molecules derived from crotamine are also discussed.

  18. Full membrane spanning self-assembled monolayers as model systems for UHV-based studies of cell-penetrating peptides

    Energy Technology Data Exchange (ETDEWEB)

    Franz, Johannes [Max Planck Institute for Polymer Research, Mainz (Germany); Graham, Daniel J. [Univ. of Washington, Seattle, WA (United States). NESAC/BIO; Schmüser, Lars [Max Planck Institute for Polymer Research, Mainz (Germany); Baio, Joe E. [Oregon State Univ., Corvallis, OR (United States); Lelle, Marco [Max Planck Institute for Polymer Research, Mainz (Germany); Peneva, Kalina [Max Planck Institute for Polymer Research, Mainz (Germany); Müllen, Klaus [Max Planck Institute for Polymer Research, Mainz (Germany); Castner, David G. [Univ. of Washington, Seattle, WA (United States). NESAC/BIO; Bonn, Mischa [Max Planck Institute for Polymer Research, Mainz (Germany); Weidner, Tobias [Max Planck Institute for Polymer Research, Mainz (Germany)


    Biophysical studies of the interaction of peptides with model membranes provide a simple yet effective approach to understand the transport of peptides and peptide based drug carriers across the cell membrane. Therein, the authors discuss the use of self-assembled monolayers fabricated from the full membrane-spanning thiol (FMST) 3-((14-((4'-((5-methyl-1-phenyl-35-(phytanyl)oxy-6,9,12,15,18,21,24,27,30,33,37-undecaoxa-2,3-dithiahenpentacontan-51-yl)oxy)-[1,1'-biphenyl]-4-yl)oxy)tetradecyl)oxy)-2-(phytanyl)oxy glycerol for ultrahigh vacuum (UHV) based experiments. UHV-based methods such as electron spectroscopy and mass spectrometry can provide important information about how peptides bind and interact with membranes, especially with the hydrophobic core of a lipid bilayer. Moreover, near-edge x-ray absorption fine structure spectra and x-ray photoelectron spectroscopy (XPS) data showed that FMST forms UHV-stable and ordered films on gold. XPS and time of flight secondary ion mass spectrometry depth profiles indicated that a proline-rich amphipathic cell-penetrating peptide, known as sweet arrow peptide is located at the outer perimeter of the model membrane.

  19. Solid formulation of cell-penetrating peptide nanocomplexes with siRNA and their stability in simulated gastric conditions. (United States)

    Ezzat, Kariem; Zaghloul, Eman M; El Andaloussi, Samir; Lehto, Taavi; El-Sayed, Ramy; Magdy, Tarek; Smith, C I Edvard; Langel, Ulo


    Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention. Here, we show that a newly developed CPP, PepFect 14 (PF14), forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect is obtained at low siRNA doses with a unique kinetic profile. Furthermore, the solid dispersion technique is utilized to formulate PF14/siRNA nanocomplexes into solid formulations that are as active as the freshly prepared nanocomplexes in solution. Importantly, the nanocomplexes are stable and active in mediating RNAi response after incubation with simulated gastric fluid (SGF) that is highly acidic. These results demonstrate the activity of PF14 in delivering and protecting siRNA in different pharmaceutical forms and biological environments.

  20. Synergistic Enhancement of Antitumor Efficacy by PEGylated Multi-walled Carbon Nanotubes Modified with Cell-Penetrating Peptide TAT (United States)

    Hu, Shanshan; Wang, Tong; Pei, Xibo; Cai, He; Chen, Junyu; Zhang, Xin; Wan, Qianbing; Wang, Jian


    In the present study, a cell-penetrating peptide, the transactivating transcriptional factor (TAT) domain from HIV, was linked to PEGylated multi-walled carbon nanotubes (MWCNTs) to develop a highly effective antitumor drug delivery system. FITC was conjugated on MWCNTs-polyethylene glycol (PEG) and MWCNTs-PEG-TAT to provide fluorescence signal for tracing the cellular uptake of the nanocarrier. After loaded with an anticancer agent, doxorubicin (DOX) via π - π stacking interaction, the physicochemical characteristics, release profile and biological evaluation of the obtained nano-sized drug carrier were investigated. The DOX loaded MWCNTs-PEG and MWCNTs-PEG-TAT drug carriers both displayed appropriate particle size, excellent stability, high drug loading, and pH-dependent drug release profile. Nevertheless, compared with DOX-MWCNTs-PEG, DOX-MWCNTs-PEG-TAT showed improved cell internalization, intracellular distribution and potentiated anticancer efficacy due to the TAT-mediated membrane translocation, endosomal escape and nuclear targeting. Furthermore, the therapeutic efficacy of DOX was not compromised after being conjugated with MWCNTs-PEG-TAT and the proposed nanocarrier was also confirmed to have a good biocompatibility. In conclusion, our results suggested that the unique combination of TAT and MWCNTs as a multifunctional drug delivery system might be a powerful tool for improved anticancer drug development.

  1. pH-Responsive Triblock Copolymeric Micelles Decorated with a Cell-Penetrating Peptide Provide Efficient Doxorubicin Delivery (United States)

    Ng, Khen Eng; Amin, Mohd Cairul Iqbal Mohd; Katas, Haliza; Amjad, Muhammad Wahab; Butt, Adeel Masood; Kesharwani, Prashant; Iyer, Arun K.


    This study developed novel triblock pH-responsive polymeric micelles (PMs) using cholic acid-polyethyleneimine-poly- l-arginine (CA-PEI-pArg) copolymers. PEI provided pH sensitivity, while the hydrophilic cell-penetrating pArg peptide promoted cellular PM internalization. The copolymers self-assembled into PMs in aqueous solution at above the critical micelle concentration (2.98 × 10-7 M) and encapsulated doxorubicin in the core region, with a 34.2% ( w/ w) entrapment efficiency. PMs showed pH-dependent swelling, increasing in size by almost sevenfold from pH 7.4 to 5.0. Doxorubicin release was pH-dependent, with about 65% released at pH 5.0, and 32% at pH 7.4. Cellular uptake, assessed by confocal microscopy and flow cytometry, was enhanced by using doxorubicin-loaded CA-PEI-pArg PMs, as compared to free doxorubicin and DOX-loaded CA-PEI PMs. Moreover, 24-h incubation of these PMs with a human breast cancer cell line produced greater cytotoxicity than free doxorubicin. These results indicate that pH-responsive CA-PEI-pArg micelles could provide a versatile delivery system for targeted cancer therapy using hydrophobic drugs.

  2. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria. (United States)

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang


    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  3. A cell-penetrating antibody fragment against HIV-1 Rev has high antiviral activity: characterization of the paratope. (United States)

    Zhuang, Xiaolei; Stahl, Stephen J; Watts, Norman R; DiMattia, Michael A; Steven, Alasdair C; Wingfield, Paul T


    The HIV-1 protein Rev oligomerizes on viral transcripts and directs their nuclear export. Previously, a Fab against Rev generated by phage display was used to crystallize and solve the structure of the Rev oligomerization domain. Here we have investigated the capability of this Fab to block Rev oligomerization and inhibit HIV-1 replication. The Fab itself did not have antiviral activity, but when a Tat-derived cell-penetrating peptide was appended, the resulting molecule (FabRev1-Tat) was strongly inhibitory of three different CCR5-tropic HIV-1 isolates (IC50 = 0.09-0.44 μg/ml), as assessed by suppression of reverse transcriptase activity in infected peripheral blood mononuclear cells, and had low cell toxicity (TC50 > 100 μg/ml). FabRev1-Tat was taken up by both peripheral blood mononuclear and HEK293T cells, appearing in both the cytoplasm and nucleus, as shown by immunofluorescence confocal laser scanning microscopy. Computational alanine scanning was used to identify key residues in the complementarity-determining regions to guide mutagenesis experiments. Residues in the light chain CDR3 (LCDR3) were assessed to be important. Residues in LCDR3 were mutated, and LCDR3-Tyr(92) was found to be critical for binding to Rev, as judged by surface plasmon resonance and electron microscopy. Peptides corresponding to all six CDR regions were synthesized and tested for Rev binding. None of the linear peptides had significant affinity for Rev, but four of the amide-cyclic forms did. Especially cyclic-LCDR3 (LGGYPAASYRTA) had high affinity for Rev and was able to effectively depolymerize Rev filaments, as shown by both surface plasmon resonance and electron microscopy.

  4. Titanium Dioxide Nanoparticle Penetration into the Skin and Effects on HaCaT Cells


    Matteo Crosera; Andrea Prodi; Marcella Mauro; Marco Pelin; Chiara Florio; Francesca Bellomo; Gianpiero Adami; Pietro Apostoli; Giuseppe Palma; Massimo Bovenzi; Marco Campanini; Francesca Larese Filon


    Titanium dioxide nanoparticles (TiO2NPs) suspensions (concentration 1.0 g/L) in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue® and propidium iodide, PI, uptake assays) was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact an...

  5. Early endosomal escape of a cyclic cell-penetrating peptide allows effective cytosolic cargo delivery. (United States)

    Qian, Ziqing; LaRochelle, Jonathan R; Jiang, Bisheng; Lian, Wenlong; Hard, Ryan L; Selner, Nicholas G; Luechapanichkul, Rinrada; Barrios, Amy M; Pei, Dehua


    Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4-12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.

  6. The spacer arm length in cell-penetrating peptides influences chitosan/siRNA nanoparticle delivery for pulmonary inflammation treatment (United States)

    Jeong, Eun Ju; Choi, Moonhwan; Lee, Jangwook; Rhim, Taiyoun; Lee, Kuen Yong


    Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of R9Gn-chitosan/siRNA nanoparticles were investigated in vitro. Increasing the spacing arm length did not significantly affect the complex formation between R9Gn-chitosan and siRNA. However, R9G10-chitosan was much more effective in delivering genes both in vitro and in vivo compared with non-modified chitosan (without the peptide) and R9-chitosan (without the spacer arm). Chitosan derivatives modified with oligoarginine containing a spacer arm can be considered as potential delivery vehicles for various genes.Although chitosan and its derivatives have been frequently utilized as delivery vehicles for small interfering RNA (siRNA), it is challenging to improve the gene silencing efficiency of chitosan-based nanoparticles. In this study, we hypothesized that controlling the spacer arm length between a cell-penetrating peptide (CPP) and a nanoparticle could be critical to enhancing the cellular uptake as well as the gene silencing efficiency of conventional chitosan/siRNA nanoparticles. A peptide consisting of nine arginine units (R9) was used as a CPP, and the spacer arm length was controlled by varying the number of glycine units between the peptide (R9Gn) and the nanoparticle (n = 0, 4, and 10). Various physicochemical characteristics of

  7. Thrombin-activatable fibrinolysis inhibitor activity in healthy and diseased dogs

    DEFF Research Database (Denmark)

    Jessen, Lisbeth Rem; Wiinberg, Bo; Kjelgaard-Hansen, Mads


    Background: In people, increased thrombin-activatable fibrinolysis inhibitor (TAFI) antigen has been associated with increased risk of thrombosis, and decreased TAFI may contribute to bleeding diathesis. TAFI activity in dogs has been described in experimental models, but not in dogs...... with spontaneous disease. Objective: The aim of this study was to compare TAFI activity in healthy dogs with TAFI activity in dogs with spontaneous disease. Methods: Plasma samples from 20 clinically healthy Beagles and from 35 dogs with various diseases were analyzed using a commercial chromogenic assay...... that measured TAFI activity relative to activity in standardized pooled human plasma. Results: Median TAFI activity for the 20 Beagles was 46.1% (range 32.2-70.8%) compared with 62.6% (29.1-250%) for the 35 diseased dogs, and 14/35 (40%) had TAFI activities >the upper limit for controls. The highest individual...

  8. Far-red light activatable, multifunctional prodrug for fluorescence optical imaging and combinational treatment. (United States)

    Bio, Moses; Rajaputra, Pallavi; Nkepang, Gregory; You, Youngjae


    We recently developed "photo-unclick chemistry", a novel chemical tool involving the cleavage of aminoacrylate by singlet oxygen, and demonstrated its application to visible light-activatable prodrugs. In this study, we prepared an advanced multifunctional prodrug, Pc-(L-CA4)2, composed of the fluorescent photosensitizer phthalocyanine (Pc), an SO-labile aminoacrylate linker (L), and a cytotoxic drug combretastatin A-4 (CA4). Pc-(L-CA4)2 had reduced dark toxicity compared with CA4. However, once illuminated, it showed improved toxicity similar to CA4 and displayed bystander effects in vitro. We monitored the time-dependent distribution of Pc-(L-CA4)2 using optical imaging with live mice. We also effectively ablated tumors by the illumination with far-red light to the mice, presumably through the combined effects of photodynamic therapy (PDT) and released chemotherapy drug, without any sign of acute systemic toxicity.

  9. siRNA-cell-penetrating peptides complexes as a combinatorial therapy against chronic myeloid leukemia using BV173 cell line as model. (United States)

    Freire, João Miguel; Rego de Figueiredo, Inês; Valle, Javier; Veiga, Ana Salomé; Andreu, David; Enguita, Francisco J; Castanho, Miguel A R B


    Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a single gene mutation, a reciprocal translocation that originates the Bcr-Abl gene with constitutive tyrosine kinase activity. As a monogenic disease, it is an optimum target for RNA silencing therapy. We developed a siRNA-based therapeutic approach in which the siRNA is delivered by pepM or pepR, two cell-penetrating peptides (CPPs) derived from the dengue virus capsid protein. These peptides have a dual role: siRNA delivery into cells and direct action as bioportides, i.e. intracellularly bioactive CPPs, targetting cancer-related signaling processes. Both pepM and pepR penetrate the positive Bcr-Abl(+) Cell Line (BV173). Five in silico designed anti-Bcr-Abl siRNA were selected for in vitro analysis after thorough screening. The Bcr-Abl downregulation kinetics (48h to 168h) was followed by quantitative PCR. The bioportide action of the peptide vectors was evaluated by genome-wide microarray analysis and further validated by testing BV173 cell cycle and cell proliferation monitoring different genes involved in housekeeping/cell stress (RPL13A, HPRT1), cell proliferation (ki67), cell apoptosis (Caspase 3 and Caspase 9) and cell cycle steps (CDK2, CCDN2, CDKN1A). Assays with a commercial transfection agent were carried out for comparison purposes. Maximal Bcr-Abl gene knockdown was observed for one of the siRNA when delivered by pepM at 120h. Both pepM and pepR showed downregulation effects on proliferative CML-related signaling pathways having direct impact on BV173 cell cycle and proliferation, thus reinforcing the siRNA effect by acting as anticancer molecules. With this work we show the therapeutic potential of a CPP shuttle that combines intrinsic anticancer properties with the ability to deliver functional siRNA into CML cell models. By such combination, the pepM-siRNA conjugates lowered Bcr-Abl gene expression levels more extensively than conventional siRNA delivery technologies and

  10. Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent,diethylenetriamine pentaacetic acid gadolinium

    Institute of Scientific and Technical Information of China (English)

    GUO You-min; LIU Min; YANG Jun-le; GUO Xiao-juan; WANG Si-cen; DUAN Xiao-yi; WANG Peng


    Background The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance (MR) contrast agent. Cell penetrating peptide (CPP) is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging in vitro. Methods Fluorescein-5-isothiocyanate (FITC) and Gd-DTPA respectively labeled with CPP (FITC-CPP, Gd-DTPA-CPP) were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T1WI signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide reduction assay (MTT). Results The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry (FITC-CPP MW=2163.34, Gd-DTPA-CPP MW=2285.99), were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T1 weighted imaging (T1WI) signal, and that the T1WI signal intensity was increasing in a time-dependent manner (r=0.972, P=0.001), while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different (P=0.225). By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant (F=0.006, P=1.000) effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group. Conclusions The newly constructed CPP based on polyarginine can translocate cells by carrying FITC

  11. A novel cell penetrating peptide carrier for the delivery of nematocidal proteins drug (United States)

    Kim, Jea Hyun

    Nematodes have recently become a primary source of harmful diseases to the environment that inflict harsh damages to pine trees and marine species. However, nematodes cannot be killed by normal pesticides or chemicals due to their thick outer protective layer mainly composed of collagen and cuticles. Thus, a novel approach to trigger intracellular delivery of chemicals through the layers of nematodes is required. In this study, the selection of the novel CPP was carefully progressed through protein database and serial digested fragmentation, internalization of each amino sequence was analyzed through flow cytometry and confocal microscope. As one of the most effective CPP material, JH 1.6 was compared with other major CPPs and its cellular toxicity was investigated. Furthermore, JH 1.6 was attached to various RNA, DNA, and proteins and internalization efficiency was evaluated for mammalian cells. To examine its effects on nematodes in vivo, JH 1.6 was conjugated with nematocidal protein - botulinum neurotoxin (BnT) and treated in C.elegans as a model animal. The results showed that JH 1.6 had high relative internalization rate and low cellular toxicity compared to other major CPP such as TAT and GV1001 peptides.

  12. Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin. (United States)

    Liu, Di; Guo, Hua; Zheng, Wenyun; Zhang, Na; Wang, Tianwen; Wang, Ping; Ma, Xingyuan


    Heat-labile enterotoxin (LT) is a protein toxin produced by enterotoxigenic Escherichia coli (ETEC). As a bacterial toxin, LT holotoxin can enter intestinal epithelial cells and cause diarrhea. In addition, LT is also a powerful mucosal adjuvant capable of enhancing the strong immune responses to co-administered antigens. However, the LT immunological mechanism is still not clear in some aspects, especially with the respect to how the LTA subunit functions alone. Here, we discovered that the A2 domain of LTA could carry a fluorescent protein into cells, whose function is similar to a cell-penetrating peptide. The transmembrane-transporting ability of the A2 domain is non-specific in its cell-penetrating function, which was shown through testing with different cell types. Moreover, the LTA2 fusion protein penetrated a fluorescently labeled cell membrane that identified LTA2 internalization through membrane transport pathways, and showed it finally localized in the endoplasmic reticulum. Furthermore, low-temperature stress and pharmacological agent treatments showed that the LTA2 internalization route is a temperature-dependent process involving the clathrin-mediated endocytosis and the macropinocytosis pathways. These results could explain the internalization of the LTA subunit alone without the LTB pentamer, contributing to a better understanding of LTA working as a mucosal adjuvant; they also suggest that the A2 domain could be used as a novel transport vehicle for research and treatment of disease.

  13. Efficiency of cell-penetrating peptides on the nasal and intestinal absorption of therapeutic peptides and proteins. (United States)

    Khafagy, El-Sayed; Morishita, Mariko; Kamei, Noriyasu; Eda, Yoshimi; Ikeno, Yohei; Takayama, Kozo


    The purpose of our study was to investigate the potential of cell-penetrating peptides; penetratin as novel delivery vector, on the systemic absorption of therapeutic peptides and proteins across different mucosal administration sites. The absorption-enhancing feasibility of l- and d-penetratin (0.5mM) was used for glucagon-like peptide-1 (GLP-1), and exendin-4 as novel antidiabetic therapy, in addition to interferon-beta (IFN-beta) as protein biotherapeutic model from nasal and intestinal route of administration was evaluated as first time in rats. Nasal route is the most feasible for the delivery of therapeutic peptides coadministered with penetratin whereas the intestinal route appears to be more restricted. The absolute bioavailability (BA (%)) values depend on the physichochemical characters of drugs, stereoisomer character of penetratin, and site of administration. Penetratin significantly increased the nasal more than intestinal absorption of GLP-1 and exendin-4, as the BA for nasal and intestinal administration of GLP-1 was 15.9% and 5%, and for exendin-4 were 7.7% and 1.8%, respectively. Moreover, the BA of IFN-beta coadministered with penetratin was 11.1% and 0.17% for nasal and intestinal administration, respectively. From these findings, penetratin is a promising carrier for transmucosal delivery of therapeutic peptides and macromolecules as an alternative to conventional parenteral routes.

  14. Hemocompatible poly(NIPAm-MBA-AMPS) colloidal nanoparticles as carriers of anti-inflammatory cell penetrating peptides. (United States)

    Bartlett, Rush L; Medow, Matthew R; Panitch, Alyssa; Seal, Brandon


    Anionic copolymer systems containing sulfated monomers have great potential for delivery of cationic therapeutics, but N-isopropylacrylamide (NIPAm) 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) copolymer nanoparticles have seen limited characterization to date with regard to physical properties relevant to loading and release of therapeutics. Characterization of polymeric nanoparticles incorporating AMPS showed an increased size and decreased thermodynamic swelling ratios of AMPS containing particles as compared to NIPAm nanoparticles lacking AMPS. Particles with increasing AMPS addition showed an increased propensity for uniformity, intraparticle colloidal stability, and drug loading capacity. Peptide encapsulated in particles was shielded from peptide degradation in serum. Particles were shown not impede blood coagulation or to cause hemolysis. This study has demonstrated that AMPS incorporation into traditional NIPAm nanoparticles presents a tunable parameter for changing particle LCST, size, swelling ratio, ζ potential, and cationic peptide loading potential. This one-pot synthesis results in a thermosensitive anionic nanoparticle system that is a potentially useful platform to deliver cationic cell penetrating peptides.

  15. Tumor penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tambet eTeesalu


    Full Text Available Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC, contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is

  16. Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers. (United States)

    Magzoub, Mazin; Pramanik, Aladdin; Gräslund, Astrid


    Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine prion protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (DeltapH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of DeltapH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of DeltapH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: DeltapH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.

  17. Binding of oligoarginine to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide. (United States)

    Gonçalves, Elisabete; Kitas, Eric; Seelig, Joachim


    Cell-penetrating peptides (CPPs) comprise a group of arginine-rich oligopeptides that are able to deliver exogenous cargo into cells. A first step in the internalization of CPPs is their binding to the cell surface, a reaction likely to involve membrane phospholipids and/or heparan sulfate proteoglycans (HSPGs). The present work characterizes the interaction of R(9), one of the most efficient CPPs, with either heparan sulfate (HS) or lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Isothermal titration calorimetry shows that R(9) binds to HS with high affinity. Assuming that HS has n independent and equivalent binding sites for R(9), we find an association constant of 3.1 x 10(6) M(-1) at 28 degrees C. At this temperature, the reaction enthalpy is DeltaH(degrees)pep = - 5.5 kcal/mol and approximately 7 R(9) molecules bind per HS chain, which is equivalent to approximately 0.95 cationic/anionic charge ratio. Delta decreases in magnitude upon an increase in temperature, and the reaction becomes entropy-driven at higher temperatures (>or=37 degrees C). The positive heat-capacity change entailed by this reaction (DeltaC(degrees)P = +167 cal mol(-1) K(-1)) indicates the loss of polar residues on R(9)-HS binding, suggesting that hydrophobic forces play no major role on binding. Calorimetric analysis of the interaction of R(9) with POPC/POPG (75:25) vesicles reveals an association constant of 8.2 x 10(4) M(-1) at 28 degrees C. Using a surface partition equilibrium model to correct for electrostatic effects, we find an intrinsic partition constant of approximately 900 M(-1), a value that is also confirmed by electrophoretic mobility measurements. This corresponds to an electrostatic contribution of approximately 33% to the total free energy of binding. Deuterium nuclear magnetic resonance (NMR) shows no change in the headgroup conformation of POPC and POPG, suggesting

  18. Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Etienne van Bracht

    Full Text Available Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

  19. Region-Dependent Role of Cell-Penetrating Peptides in Insulin Absorption Across the Rat Small Intestinal Membrane. (United States)

    Khafagy, El-Sayed; Iwamae, Ruisha; Kamei, Noriyasu; Takeda-Morishita, Mariko


    We have reported that the cell-penetrating peptide (CPP) penetratin acts as a potential absorption enhancer in oral insulin delivery systems and that this action occurs through noncovalent intermolecular interactions. However, the region-dependent role of CPPs in intestinal insulin absorption has not been clarified. To identify the intestinal region where CPPs have the most effect in increasing insulin absorption, the region-dependent action of penetratin was investigated using in situ closed intestinal loops in rats. The order of the insulin area under the insulin concentration-time curve (AUC) increase effect by L-penetratin was ileum > jejunum > duodenum > colon. By contrast, the AUC order after coadministration of insulin with D-penetratin was colon > duodenum ≥ jejunum and ileum. We also compared the effects of the L- and D-forms of penetratin, R8, and PenetraMax on ileal insulin absorption. Along with the CPPs used in this study, L- and D-PenetraMax produced the largest insulin AUCs. An absorption study using ilea pretreated with CPPs showed that PenetraMax had no irreversible effect on the intestinal epithelial membrane. The degradation of insulin in the presence of CPPs was assessed in rat intestinal enzymatic fluid. The half-life (t 1/2) of insulin increased from 14.5 to 23.7 and 184.7 min in the presence of L- and D-PenetraMax, respectively. These enzymatic degradation-resistant effects might contribute partly to the increased ileal absorption of insulin induced by D-PenetraMax. In conclusion, this study demonstrated that the ability of the L- and D-forms of penetratin to increase intestinal insulin absorption was maximal in the ileum and the colon, respectively, and that D-PenetraMax is a powerful but transient enhancer of oral insulin absorption.


    Directory of Open Access Journals (Sweden)

    Zhadyra Sagykyzy Shagyrova


    Full Text Available Abstract: Nano-sized carriers can help to reduce toxicity and improve clinical efficacy of drugs. Virus-like particles (VLPs are biocompatible and biodegradable self-assembling nanoparticles, which show great promise as carriers for substances for targeted delivery and controlled release. Either chemical conjugation of physical incorporation without formation of covalent bonds is possible to load substances of interest into VLPs.Objectives: To produce VLPs from recombinant viral capsid protein (HBcAg and test feasibility of methods of formation of chemical and physical conjugates of VLPs with substances of pharmacological interest.Methods: Virus-like particles composed from recombinant hepatitis B core antigen (HBcAg were produced by recombinant expression in E.coli and purified by successive centrifugation through sucrose gradients. Peptide transportan 10 was synthesized and used for carbodiimide (EDC-mediated conjugation to VLPs. Doxorubicin (DOX was loaded into the nucleic acid-containing VLPs to form physical conjugate.Results: VLPs with chemically attached moieties of cell-penetrating peptide transportan 10 were produced. The conjugate was examined in SDS-PAGE to confirm presence of conjugation products. Conjugation efficiency (molar ration peptide/protein in the conjugate reaches 0.5:1 (i.e. 50% of protein chains have one attached peptide moiety. The nucleic acid-containing VLPs can be loaded with the DOX forming stable non-covalent physical conjugate.Conclusion: Recombinantly expressed VLPs allow easy attaching of small molecules making them a convenient platform to develop drug carriers.

  1. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells. (United States)

    Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung


    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

  2. Enlarging the scope of cell penetrating prenylated peptides to include farnesylated “CAAX” box sequences and diverse cell types (United States)

    Ochocki, Joshua D.; Igbavboa, Urule; Wood, W. Gibson; Wattenberg, Elizabeth V.; Distefano, Mark D.


    Protein prenylation is a post-translational modification that is present in a large number of proteins; it has been proposed to be responsible for membrane association and protein-protein interactions which contribute to its role in signal transduction pathways. Research has been aimed at inhibiting prenylation with farnesyltransferase inhibitors (FTIs) based on the finding that the farnesylated protein Ras is implicated in 30% of human cancers. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation as it occurs in living cells. Here we describe the preparation of a series of farnesylated peptides that contain sequences recognized by protein farnesyltransferase. Using a combination of flow cytometry and confocal microscopy, we show that these peptides enter a variety of different cell types. A related peptide where the farnesyl group has been replaced by a disulfide-linked decyl group is also shown to be able to efficiently enter cells. These results highlight the applicability of these peptides as a platform for further study of protein prenylation and subsequent processing in live cells. PMID:20584014

  3. Hierarchy of Specific Lipid-Peptide Interactions Produces the Activity of Cell-penetrating and Cell-permeating Peptides (United States)

    Davis, Matthew; Parente, Daniel; Gordon, Vernita; Mishra, Abhijit; Schmidt, Nathan; Yang, Lihua; Coridan, Robert; Som, Abhigyan; Tew, Gregory; Wong, Gerard


    Protein transduction domains can cross cell membranes with high efficiency, even when carrying a variety of cargos, and thus has strong biotechnological potential. The molecular mechanism of entry, however, is not well understood. We use small-angle x-ray scattering (SAXS) and confocal microscopy to systematically study the interaction of the TAT and ANTP PTD with model membranes of variable composition. Their membrane transduction activity requires the presence of both PE and PS lipids in the membrane. Antimicrobial peptides (AMP's) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogs of AMP's, such as the family of phenylene ethynylene antimicrobial oligomers (AMO's), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. PE lipid greatly enhances permeating activity of AMO in these membranes, showing the importance of specific lipid composition for the activity of cell-permeating peptides. Since bacterial cell membranes are richer in PE lipids than are eukaryotic cell membranes, this may indicate a mechanism for antimicrobial specificity.

  4. The effect of "on/off" molecular switching on the photophysical and photochemical properties of axially calixarene substituted activatable silicon(iv)phthalocyanine photosensitizers. (United States)

    Güngör, Ömer; Altınbaş Özpınar, Gül; Durmuş, Mahmut; Ahsen, Vefa


    Silicon(iv) phthalocyanines ( and ) bearing two calixarene groups as axial ligands were synthesized. Surprisingly, both phthalocyanines were obtained as two different isomers ( and ) depending on the distance between calixarene benzene groups and the phthalocyanine ring. DFT and TD-DFT computations were performed to model plausible structures of these isomers and to simulate electronic absorption spectra. These isomers converted into each other depending on the polarity of the used solvent, temperature and light irradiation. The photophysical and photochemical properties of each isomer were investigated in dimethylsulfoxide (DMSO) for the determination of photodynamic therapy (PDT) activities of these compounds. The more blue-shifted isomers ( and ) showed higher fluorescence quantum yields and singlet oxygen generation compared to more red-shifted counterparts ( and ). This behavior is extremely important for developing activatable photosensitizers for cancer treatment by PDT. Although these photosensitizers produce lower singlet oxygen in normal cells, they produce higher singlet oxygen (six times higher for ) in cancer cells since these photosensitizers converted to more blue-shifted isomers by using light irradiation.

  5. Curb challenges of the "Trojan Horse" approach: smart strategies in achieving effective yet safe cell-penetrating peptide-based drug delivery. (United States)

    Huang, Yongzhuo; Jiang, Yifan; Wang, Huiyuan; Wang, Jianxin; Shin, Meong Cheol; Byun, Youngro; He, Huining; Liang, Yanqin; Yang, Victor C


    Cell-penetrating peptide (CPP)-mediated intracellular drug delivery system, often specifically termed as "the Trojan horse approach", has become the "holy grail" in achieving effective delivery of macromolecular compounds such as proteins, DNA, siRNAs, and drug carriers. It is characterized by the unique cell- (or receptor-), temperature-, and payload-independent mechanisms, therefore offering potent means to improve poor cellular uptake of a variety of macromolecular drugs. Nevertheless, this "Trojan horse" approach also acts like a double-edged sword, causing serious safety and toxicity concerns to normal tissues or organs for in vivo application, due to lack of target selectivity of the powerful cell penetrating activity. To overcome this problem of potent yet non-selective penetration vs. targeting delivery, a number of "smart" strategies have been developed in recent years, including controllable CPP-based drug delivery systems based on various stimuli-responsive mechanisms. This review article provides a fundamental understanding of these smart systems, as well as a discussion of their real-time in vivo applicability.

  6. Differential neuroprotective potential of CRMP2 peptide aptamers conjugated to cationic, hydrophobic, and amphipathic cell penetrating peptides

    Directory of Open Access Journals (Sweden)

    Aubin eMoutal


    Full Text Available The microtubule-associated axonal specification collapsin response mediator protein 2 (CRMP2 is a novel target for neuroprotection. A CRMP2 peptide (TAT-CBD3 conjugated to the HIV transactivator of transcription (TAT protein’s cationic cell penetrating peptide motif (CPP protected neurons in the face of toxic levels of Ca2+ influx leaked in via N-methyl-D-aspartate receptor (NMDAR hyperactivation. Here we tested whether replacing the hydrophilic TAT motif with alternative cationic (nona-arginine (R9, hydrophobic (membrane transport sequence (MTS of k-fibroblast growth factor or amphipathic (model amphipathic peptide (MAP CPPs could be superior to the neuroprotection bestowed by TAT-CBD3. In giant plasma membrane vesicles (GPMVs derived from cortical neurons, the peptides translocated across plasma membranes with similar efficiencies. Cortical neurons, acutely treated with peptides prior to a toxic glutamate challenge, demonstrated enhanced efflux of R9-CBD3 compared to others. R9-CBD3 inhibited N-methyl-D-aspartate (NMDA-evoked Ca2+ influx to a similar extent as TAT-CBD3 while MTS-CBD3 was ineffective which correlated with the ability of R9- and TAT-CBD3, but not MTS-CBD3, to block NMDAR interaction with CRMP2. Unrestricted Ca2+ influx through NMDARs leading to delayed calcium dysregulation and neuronal cell death was blocked by all peptides but MAP-CBD3. When applied acutely for 10 minutes, R9-CBD3 was more effective than TAT-CBD3 at neuroprotection while MTS- and MAP-CBD3 were ineffective. In contrast, long-term (> 24 hours treatment with MTS-CBD3 conferred neuroprotection where TAT-CBD3 failed. Neither peptide altered surface trafficking of NMDARs. Neuroprotection conferred by MTS-CBD3 peptide is likely due to its increased uptake coupled with decreased efflux when compared to TAT-CBD3. Overall, our results demonstrate that altering CPPs can bestow differential neuroprotective potential onto the CBD3 cargo.

  7. Selective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells. (United States)

    Yeh, Tzyy-Harn; Chen, Yun-Ru; Chen, Szu-Ying; Shen, Wei-Chiang; Ann, David K; Zaro, Jennica L; Shen, Li-Jiuan


    Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5-7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP HBHAc was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with p

  8. The current distribution in an electrochemical cell. Part V. The determination of the depth of the current line penetration between the edges of the electrodes and the side walls of the cell

    Directory of Open Access Journals (Sweden)



    Full Text Available A method for the calculation of the depth of the current line penetration between the edges of the electrodes and the side walls of the cell in a cell with plane parallel electrode arrangement is proposed. The method is verified by the calculation of the polarization curves for the cells in which the electrode edges do not touch the side walls of the cell. The agreement between the calculated and the measured values was fair.

  9. DNA-histone complexes as ligands amplify cell penetration and nuclear targeting of anti-DNA antibodies via energy-independent mechanisms. (United States)

    Zannikou, Markella; Bellou, Sofia; Eliades, Petros; Hatzioannou, Aikaterini; Mantzaris, Michael D; Carayanniotis, George; Avrameas, Stratis; Lymberi, Peggy


    We have generated three monoclonal cell-penetrating antibodies (CPAbs) from a non-immunized lupus-prone (NZB × NZW)F1 mouse that exhibited high anti-DNA serum titres. These CPAbs are polyreactive because they bind to DNA and other cellular components, and localize mainly in the nucleus of HeLa cells, albeit with a distinct nuclear labelling profile. Herein, we have examined whether DNA-histone complexes (DHC) binding to CPAbs, before cell entry, could modify the cell penetration of CPAbs or their nuclear staining properties. By applying confocal microscopy and image analysis, we found that extracellular binding of purified CPAbs to DHC significantly enhanced their subsequent cell-entry, both in terms of percentages of positively labelled cells and fluorescence intensity (internalized CPAb amount), whereas there was a variable effect on their nuclear staining profile. Internalization of CPAbs, either alone or bound to DHC, remained unaltered after the addition of endocytosis-specific inhibitors at 37° or assay performance at 4°, suggesting the involvement of energy-independent mechanisms in the internalization process. These findings assign to CPAbs a more complex pathogenetic role in systemic lupus erythematosus where both CPAbs and nuclear components are abundant.

  10. Structure analysis and conformational transitions of the cell penetrating peptide transportan 10 in the membrane-bound state.

    Directory of Open Access Journals (Sweden)

    Susanne Fanghänel

    Full Text Available Structure analysis of the cell-penetrating peptide transportan 10 (TP10 revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan was found to exhibit prominent characteristics of (i amphiphilic α-helices, (ii intrinsically disordered peptides, as well as (iii β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state (19F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L- or D-enantiomer of 3-(trifluoromethyl-bicyclopent-[1.1.1]-1-ylglycine (CF3-Bpg as a reporter group for (19F-NMR. Using the L-epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼ 55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D-CF3-Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D-CF3-Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the

  11. Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI

    Directory of Open Access Journals (Sweden)

    Kristensen Torsten


    Full Text Available Abstract Background TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI, and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. Results The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. Conclusion The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.

  12. Resistance of Escherichia coli to nourseothricin (streptothricin): reduced penetrability of the cell wall as an additional, possibly unspecific mechanism. (United States)

    Seltmann, G


    The resistance of E. coli strains to the antibiotic nourseothricin is known to be caused by an acetyltransferase acetylating the beta-lysine chain of the antibiotic. In addition, most of the resistant strains exhibit reduced penetrability of the outer membrane, presumably caused by a reduced amount of available negative charges. This was shown using crystal violet, Congo red, or the hydrophobic antibiotic novobiocin as indicators.

  13. Identification of a cell-penetrating peptide domain from human beta-defensin 3 and characterization of its anti-inflammatory activity

    Directory of Open Access Journals (Sweden)

    Lee JY


    Full Text Available Jue Yeon Lee,1,* Jin Sook Suh,2,* Jung Min Kim,1 Jeong Hwa Kim,1 Hyun Jung Park,1 Yoon Jeong Park,1,2 Chong Pyoung Chung1 1Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Chungcheongbuk-do, Republic of Korea; 2Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Human beta-defensins (hBDs are crucial factors of intrinsic immunity that function in the immunologic response to a variety of invading enveloped viruses, bacteria, and fungi. hBDs can cause membrane depolarization and cell lysis due to their highly cationic nature. These molecules participate in antimicrobial defenses and the control of adaptive and innate immunity in every mammalian species and are produced by various cell types. The C-terminal 15-mer peptide within hBD3, designated as hBD3-3, was selected for study due to its cell- and skin-penetrating activity, which can induce anti-inflammatory activity in lipopolysaccharide-treated RAW 264.7 macrophages. hBD3-3 penetrated both the outer membrane of the cells and mouse skin within a short treatment period. Two other peptide fragments showed poorer penetration activity compared to hBD3-3. hBD3-3 inhibited the lipopolysaccharide-induced production of inducible nitric oxide synthase, nitric oxide, and secretory cytokines, such as interleukin-6 and tumor necrosis factor in a concentration-dependent manner. Moreover, hBD3-3 reduced the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. Further investigation also revealed that hBD3-3 downregulated nuclear factor kappa B-dependent inflammation by directly suppressing the degradation of phosphorylated-IκBα and by downregulating active nuclear factor kappa B p65. Our findings indicate that hBD3-3 may be conjugated with drugs of interest to ensure their proper translocation to

  14. In vivo near-infrared fluorescence imaging of FAP-expressing tumors with activatable FAP-targeted, single-chain Fv-immunoliposomes. (United States)

    Rüger, Ronny; Tansi, Felista L; Rabenhold, Markus; Steiniger, Frank; Kontermann, Roland E; Fahr, Alfred; Hilger, Ingrid


    Molecular and cellular changes that precede the invasive growth of solid tumors include the release of proteolytic enzymes and peptides in the tumor stroma, the recruitment of phagocytic and lymphoid infiltrates and alteration of the extracellular matrix. The reactive tumor stroma consists of a large number of myofibroblasts, characterized by high expression of fibroblast activation protein alpha (FAP). FAP, a type-II transmembrane sialoglycoprotein is an attractive target in diagnosis and therapy of several pathologic disorders especially cancer. In the underlying work, a fluorescence-activatable liposome (fluorescence-quenched during circulation and fluorescence activation upon cellular uptake), bearing specific single-chain Fv fragments directed against FAP (scFv'FAP) was developed, and its potential for use in fluorescence diagnostic imaging of FAP-expressing tumor cells was evaluated by whole body fluorescence imaging. The liposomes termed anti-FAP-IL were prepared via post-insertion of ligand-phospholipid-conjugates into preformed DY-676-COOH-containing liposomes. The anti-FAP-IL revealed a homogeneous size distribution and showed specific interaction and binding with FAP-expressing cells in vitro. The high level of fluorescence quenching of the near-infrared fluorescent dye sequestered in the aqueous interior of the liposomes enables fluorescence imaging exclusively upon uptake and degradation by cells, which results in fluorescence activation. Only FAP-expressing cells were able to take up and activate fluorescence of anti-FAP-IL in vitro. Furthermore, anti-FAP-IL accumulated selectively in FAP-expressing xenograft models in vivo, as demonstrated by blocking experiments using free scFv'FAP. The local tumor fluorescence intensities were in agreement with the intrinsic degree of FAP-expression in different xenograft models. Thus, anti-FAP-IL can serve as a suitable in vivo diagnostic tool for pathological disorders accompanied by high FAP-expression.

  15. Multispectral photoacoustic imaging of tumours in mice injected with an enzyme-activatable photoacoustic probe (United States)

    Hirasawa, Takeshi; Iwatate, Ryu J.; Kamiya, Mako; Okawa, Shinpei; Urano, Yasuteru; Ishihara, Miya


    Photoacoustic (PA) imaging offers depth-resolved images of optical absorbers with the spatial resolution of ultrasound imaging. To enhance tumour contrast, tumour-specific probes are used as contrast agents. We synthesised a colourless PA probe that is activated in the presence of γ-glutamyltranspeptidase, a cancer-associated enzyme, to show its original colour and fluorescence. We have acquired high specificity fluorescence images of small tumours, using a fluorescent probe based on similar enzymatic reactions. Here, we developed a PA imaging technique to detect the PA probe. In PA imaging, depending on the concentration and excitation wavelength of the probe, the intensities of the probe signals may be lower than those of the background signals produced by intrinsic optical absorbers such as haemoglobin. For probe imaging in the presence of strong background signals, multispectral photoacoustic (MS-PA) imaging was evaluated. In MS-PA imaging, the spectral fitting method, which distinguishes the probe signals from background signals using reference spectra, has been widely used. To compensate for the decrease of fluence due to optical attenuation in biological tissue, we used a simplified compensation method that calculates fluence inside biological tissues by the Monte-Carlo model using published data on optical properties of biological tissues. The validity of the method was confirmed using tissue-mimicking phantoms. Finally, MS-PA imaging of a mouse subcutaneous tumour injected with the activatable probe was demonstrated. In conclusion, our MS-PA imaging technique afforded successful detection of the activated probe in the tumour, and time-increase of PA signals were successfully observed.

  16. Combined HLA matched limbal stem cells allograft with amniotic membrane transplantation as a prophylactic surgical procedure to prevent corneal graft rejection after penetrating keratoplasty: case report

    Directory of Open Access Journals (Sweden)

    Paolo Capozzi


    Full Text Available Purpose. To determine if the use of combined HLA matched limbal stem cells allograft with amniotic membrane transplantation (AMT is a safe and effective prophylactic surgical procedure to prevent corneal graft after penetrating keratoplasty (PK. Methods. We report the case of a 17 years old patient with a history of congenital glaucoma, trabeculectomy and multiple corneal graft rejections, presenting total limbal cell deficiency. To reduce the possibility of graft rejection in the left eye after a new PK, a two step procedure was performed. At first the patient underwent a combined HLA matched limbal stem cells allograft (LAT and AMT and then, 10 months later, a new PK. Results. During 12 months of follow-up, the corneal graft remained stable and smooth, with no sign of graft rejection. Conclusions. In our patient, the prophylactic use of LAT from HLA-matched donors and AMT before PK, may result in a better prognosis of corneal graft survival.

  17. Fusion of cell-penetrating peptides to thermally responsive biopolymer improves tumor accumulation of p21 peptide in a mouse model of pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Walker LR


    Full Text Available Leslie R Walker,1 Jung Su Ryu,1 Eddie Perkins,2 Lacey R McNally,3 Drazen Raucher1 1Department of Biochemistry, 2Department of Neurosurgery, University of Mississippi Medical Center, Jackson, MS, USA; 3Division of Hematology and Oncology, University of Louisville, Louisville, KY, USAAbstract: Current therapies for the treatment of pancreatic cancer are limited. The limitations of this type of treatment are abundant. The majority of chemotherapeutic agents used in clinics are highly toxic to both tumor cells and normal tissues due to the lack of specificity. Resistance can develop due to overexposure of these agents. To address these issues, these agents must be made more exclusive toward the tumor site. We have developed a macromolecular carrier based on the sequence of the biopolymer elastin-like polypeptide (ELP that is able to aggregate upon reaching the externally heated tumor environment. This carrier is specific to the tumor as it only aggregates at the heated tumor site. ELP is soluble below its transition temperature but will aggregate when the temperature is raised above its transition temperature. ELP was modified by p21, a cell cycle inhibitory peptide, and the addition of Bac, a cell-penetrating peptide with nuclear localization capabilities. In this study, p21-ELP-Bac and its control, ELP-p21, were used in cell proliferation studies using the pancreatic cancer cell lines Panc-1, MiaPaca-2, and S2013. ELP-p21 had little effect on proliferation, while the half maximal inhibitory concentration of p21-ELP-Bac was ~30 µM. As translocation across the plasma membrane is a limiting step for delivery of macromolecules, these polypeptides were utilized in a pancreatic xenograft model to study the plasma clearance, biodistribution, tumor accumulation, and tumor reduction capabilities of the polypeptide with and without a cell-penetrating peptide.Keywords: elastin-like polypeptide, peptide, targeted drug delivery, macromolecule

  18. Activatable albumin-photosensitizer nanoassemblies for triple-modal imaging and thermal-modulated photodynamic therapy of cancer. (United States)

    Hu, Dehong; Sheng, Zonghai; Gao, Guanhui; Siu, Fungming; Liu, Chengbo; Wan, Qian; Gong, Ping; Zheng, Hairong; Ma, Yifan; Cai, Lintao


    Photodynamic therapy (PDT) is a noninvasive and effective approach for cancer treatment. The main bottlenecks of clinical PDT are poor selectivity of photosensitizer and inadequate oxygen supply resulting in serious side effects and low therapeutic efficiency. Herein, a thermal-modulated reactive oxygen species (ROS) strategy using activatable human serum albumin-chlorin e6 nanoassemblies (HSA-Ce6 NAs) for promoting PDT against cancer is developed. Through intermolecular disulfide bond crosslinking and hydrophobic interaction, Ce6 photosensitizer is effectively loaded into the HSA NAs, and the obtained HSA-Ce6 NAs exhibit excellent reduction response, as well as enhanced tumor accumulation and retention. By the precision control of the overall body temperature instead of local tumor temperature increasing from 37 °C to 43 °C, the photosensitization reaction rate of HSA-Ce6 NAs increases 20%, and the oxygen saturation of tumor tissue raise 52%, significantly enhancing the generation of ROS for promoting PDT. Meanwhile, the intrinsic fluorescence and photoacoustic properties, and the chelating characteristic of porphyrin ring can endow the HSA-Ce6 NAs with fluorescence, photoacoustic and magnetic resonance triple-modal imaging functions. Upon irradiation of low-energy near-infrared laser, the tumors are completely suppressed without tumor recurrence and therapy-induced side effects. The robust thermal-modulated ROS strategy combined with albumin-based activatable nanophotosensitizer is highly potential for multi-modal imaging-guided PDT and clinical translation.

  19. The potent antimicrobial properties of cell penetrating peptide-conjugated silver nanoparticles with excellent selectivity for Gram-positive bacteria over erythrocytes (United States)

    Liu, Lihong; Yang, Jun; Xie, Jianping; Luo, Zhentao; Jiang, Jiang; Yang, Yi Yan; Liu, Shaomin


    Silver nanoparticles are of great interest for use as antimicrobial agents. Studies aimed at producing potent nano-silver biocides have focused on manipulation of particle size, shape, composition and surface charge. Here, we report the cell penetrating peptide catalyzed formation of antimicrobial silver nanoparticles in N,N-dimethylformamide. The novel nano-composite demonstrated a distinctly enhanced biocidal effect toward bacteria (Gram-positive Bacillus subtilis, Gram-negative Escherichia coli) and pathogenic yeast (Candida albicans), as compared to triangular and extremely small silver nanoparticles. In addition, a satisfactory biocompatibility was verified by a haemolysis test. Our results provide a paradigm in developing strategies that can maximize the silver nanoparticle application potentials while minimizing the toxic effects.Silver nanoparticles are of great interest for use as antimicrobial agents. Studies aimed at producing potent nano-silver biocides have focused on manipulation of particle size, shape, composition and surface charge. Here, we report the cell penetrating peptide catalyzed formation of antimicrobial silver nanoparticles in N,N-dimethylformamide. The novel nano-composite demonstrated a distinctly enhanced biocidal effect toward bacteria (Gram-positive Bacillus subtilis, Gram-negative Escherichia coli) and pathogenic yeast (Candida albicans), as compared to triangular and extremely small silver nanoparticles. In addition, a satisfactory biocompatibility was verified by a haemolysis test. Our results provide a paradigm in developing strategies that can maximize the silver nanoparticle application potentials while minimizing the toxic effects. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr34254a

  20. Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification (United States)

    Illien, Françoise; Rodriguez, Nicolas; Amoura, Mehdi; Joliot, Alain; Pallerla, Manjula; Cribier, Sophie; Burlina, Fabienne; Sagan, Sandrine


    The mechanism of cell-penetrating peptides entry into cells is unclear, preventing the development of more efficient vectors for biotechnological or therapeutic purposes. Here, we developed a protocol relying on fluorometry to distinguish endocytosis from direct membrane translocation, using Penetratin, TAT and R9. The quantities of internalized CPPs measured by fluorometry in cell lysates converge with those obtained by our previously reported mass spectrometry quantification method. By contrast, flow cytometry quantification faces several limitations due to fluorescence quenching processes that depend on the cell line and occur at peptide/cell ratio >6.108 for CF-Penetratin. The analysis of cellular internalization of a doubly labeled fluorescent and biotinylated Penetratin analogue by the two independent techniques, fluorometry and mass spectrometry, gave consistent results at the quantitative and qualitative levels. Both techniques revealed the use of two alternative translocation and endocytosis pathways, whose relative efficacy depends on cell-surface sugars and peptide concentration. We confirmed that Penetratin translocates at low concentration and uses endocytosis at high μM concentrations. We further demonstrate that the hydrophobic/hydrophilic nature of the N-terminal extremity impacts on the internalization efficiency of CPPs. We expect these results and the associated protocols to help unraveling the translocation pathway to the cytosol of cells.

  1. An efficient PEGylated liposomal nanocarrier containing cell-penetrating peptide and pH-sensitive hydrazone bond for enhancing tumor-targeted drug delivery

    Directory of Open Access Journals (Sweden)

    Ding Y


    Full Text Available Yuan Ding,1,* Dan Sun,1,* Gui-Ling Wang,1 Hong-Ge Yang,1 Hai-Feng Xu,1 Jian-Hua Chen,2 Ying Xie,1,3 Zhi-Qiang Wang4 1Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing, 2School of Medicine, Jianghan University, Wuhan, 3State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, People’s Republic of China; 4Department of Chemistry and Biochemistry, Kent State University Geauga, Burton, OH, USA *These authors contributed equally to this work Abstract: Cell-penetrating peptides (CPPs as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into

  2. Hydrothermal synthesis of nitrogen-doped carbon dots with real-time live-cell imaging and blood–brain barrier penetration capabilities (United States)

    Lu, Shousi; Guo, Shanshan; Xu, Pingxiang; Li, Xiaorong; Zhao, Yuming; Gu, Wei; Xue, Ming


    Nitrogen-doped carbon dots (N-CDs) were synthesized using a one-pot hydrothermal treatment with citric acid in the presence of polyethylenimine. Transmission electron microscopy analysis revealed that the N-CDs were monodispersed and quasi-spherical with an average size of ~2.6 nm. Under ultraviolet irradiation the N-CDs emitted a strong blue luminescence with a quantum yield as high as 51%. Moreover, the N-CDs exhibited a negligible cytotoxicity and could be applied as efficient nanoprobes for real-time imaging of live cells. In addition, the ability of the N-CDs to cross the blood–brain barrier (BBB) in a concentration-dependent manner was demonstrated using an in vitro BBB model. Therefore, these PEI-passivated N-CDs with real-time live-cell imaging and BBB-penetration capabilities hold promise for traceable drug delivery to the brain. PMID:27932880

  3. Development of cell-penetrating peptide-based drug leads to inhibit MDMX:p53 and MDM2:p53 interactions. (United States)

    Philippe, Grégoire; Huang, Yen-Hua; Cheneval, Olivier; Lawrence, Nicole; Zhang, Zhen; Fairlie, David P; Craik, David J; de Araujo, Aline Dantas; Henriques, Sónia Troeira


    The transcription factor p53 has a tumor suppressor role in leading damaged cells to apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2 and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53 interactions with nanomolar potency in vitro. With the aim of improving its cellular inhibitory activity, we produced a series of constrained pDI analogs featuring lactam staples that stabilize the bioactive helical conformation and fused them with a cell-penetrating peptide to increase cytosol delivery. We compared pDI and its analogs on their inhibitory potency, toxicity, and ability to enter cancer cells. Overall, the results show that these analogs keep their nanomolar affinity for MDM2 and MDMX and are highly active against cancer cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 853-863, 2016.

  4. Cell-penetrating superoxide dismutase attenuates oxidative stress-induced senescence by regulating the p53-p21Cip1 pathway and restores osteoblastic differentiation in human dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Park YJ


    Full Text Available Yoon Jung Choi,1,* Jue Yeon Lee,2,* Chong Pyoung Chung,2 Yoon Jeong Park,1,21Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea; 2Research Institute, Nano Intelligent Biomedical Engineering, Seoul, Republic of Korea*These authors contributed equally to this workBackground: Human dental pulp stem cells (DPSCs have potential applications in tissue regeneration because of their convenient cell harvesting procedures and multipotent capacity. However, the tissue regenerative potential of DPSCs is known to be negatively regulated by aging in long-term culture and under oxidative stress. With an aim of reducing cellular senescence and oxidative stress in DPSCs, an intracellular delivery system for superoxide dismutase 1 (SOD1 was developed. We conjugated SOD1 with a cell-penetrating peptide known as low-molecular weight protamine (LMWP, and investigated the effect of LMWP-SOD1 conjugates on hydrogen peroxide-induced cellular senescence and osteoblastic differentiation.Results: LMWP-SOD1 significantly attenuated enlarged and flattened cell morphology and increased senescence-associated β-galactosidase activity. Under the same conditions, LMWP-SOD1 abolished activation of the cell cycle regulator proteins, p53 and p21Cip1, induced by hydrogen peroxide. In addition, LMWP-SOD1 reversed the inhibition of osteoblastic differentiation and downregulation of osteogenic gene markers induced by hydrogen peroxide. However, LMWP-SOD1 could not reverse the decrease in odontogenesis caused by hydrogen peroxide.Conclusion: Overall, cell-penetrating LMWP-SOD1 conjugates are effective for attenuation of cellular senescence and reversal of osteoblastic differentiation of DPSCs caused by oxidative stress inhibition. This result suggests potential application in the field of antiaging and tissue engineering to overcome the limitations of senescent stem cells.Keywords: superoxide

  5. Intestinal Cell Tight Junctions Limit Invasion of Candida albicans through Active Penetration and Endocytosis in the Early Stages of the Interaction of the Fungus with the Intestinal Barrier.

    Directory of Open Access Journals (Sweden)

    Marianne Goyer

    Full Text Available C. albicans is a commensal yeast of the mucous membranes in healthy humans that can also cause disseminated candidiasis, mainly originating from the digestive tract, in vulnerable patients. It is necessary to understand the cellular and molecular mechanisms of the interaction of C. albicans with enterocytes to better understand the basis of commensalism and pathogenicity of the yeast and to improve the management of disseminated candidiasis. In this study, we investigated the kinetics of tight junction (TJ formation in parallel with the invasion of C. albicans into the Caco-2 intestinal cell line. Using invasiveness assays on Caco-2 cells displaying pharmacologically altered TJ (i.e. differentiated epithelial cells treated with EGTA or patulin, we were able to demonstrate that TJ protect enterocytes against invasion of C. albicans. Moreover, treatment with a pharmacological inhibitor of endocytosis decreased invasion of the fungus into Caco-2 cells displaying altered TJ, suggesting that facilitating access of the yeast to the basolateral side of intestinal cells promotes endocytosis of C. albicans in its hyphal form. These data were supported by SEM observations of differentiated Caco-2 cells displaying altered TJ, which highlighted membrane protrusions engulfing C. albicans hyphae. We furthermore demonstrated that Als3, a hypha-specific C. albicans invasin, facilitates internalization of the fungus by active penetration and induced endocytosis by differentiated Caco-2 cells displaying altered TJ. However, our observations failed to demonstrate binding of Als3 to E-cadherin as the trigger mechanism of endocytosis of C. albicans into differentiated Caco-2 cells displaying altered TJ.

  6. Quantification of Cell-Penetrating Peptide Associated with Polymeric Nanoparticles Using Isobaric-Tagging and MALDI-TOF MS/MS (United States)

    Chiu, Jasper Z. S.; Tucker, Ian G.; McDowell, Arlene


    High sensitivity quantification of the putative cell-penetrating peptide di-arginine-histidine (RRH) associated with poly (ethyl-cyanoacrylate) (PECA) nanoparticles was achieved without analyte separation, using a novel application of isobaric-tagging and high matrix-assisted laser desorption/ionization coupled to time-of-flight (MALDI-TOF) mass spectrometry. Isobaric-tagging reaction equilibrium was reached after 5 min, with 90% or greater RRH peptide successfully isobaric-tagged after 60 min. The accuracy was greater than 90%, which indicates good reliability of using isobaric-tagged RRH as an internal standard for RRH quantification. The sample intra- and inter-spot coefficients of variations were less than 11%, which indicate good repeatability. The majority of RRH peptides in the nanoparticle formulation were physically associated with the nanoparticles (46.6%), whereas only a small fraction remained unassociated (13.7%). The unrecovered RRH peptide (~40%) was assumed to be covalently associated with PECA nanoparticles.

  7. Penetration Testing of the OPRA Regolith Penetrator (United States)

    El Shafie, A.; Kegege, O.; Barrows, S.; Roe, L.; Ulrich, R.


    Our work focuses on the mechanical design and penetration forces for the Optical Probe for Regolith Analysis. This is a spike-shaped probe delivered to a planet, asteroid, or cometary body by a lander to provide IR spectroscopy of the subsurface.

  8. Antibody-mediated activation of a defective beta-D-galactosidase: dimeric form of the activatable mutant enzyme. (United States)

    Conway de Macario, E; Ellis, J; Guzman, R; Rotman, B


    Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC, and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody,

  9. Polypyrrole-based nanotheranostics for activatable fluorescence imaging and chemo/photothermal dual therapy of triple-negative breast cancer (United States)

    Park, Dongjin; Ahn, Kyung-Ohk; Jeong, Kyung-Chae; Choi, Yongdoo


    Here, we fabricated polypyrrole nanoparticles (PPys) (termed HA10-PPy, HA20-PPy, and HA40-PPy) doped with different average molecular weight hyaluronic acids (HAs) (10, 20, and 40 kDa, respectively), and evaluated the effect of molecular weight of doped HA on photothermal induction, fluorescence quenching, and drug loading efficiencies. Doxorubicin-loaded HA-doped PPys (DOX@HA-PPys) could be used for imaging and therapy of triple-negative breast cancer (TNBC). Fluorescence turn-on, stimuli-responsive drug release, and photo-induced heating of DOX@HA-PPys enabled not only activatable fluorescence imaging but also subsequent chemo/photothermal dual therapy for TNBC. In particular, we illustrated the potential usefulness of the photothermal effect of the nanoparticles for overcoming chemoresistance in TNBC.

  10. Functional study of p38 mitogen-activated protein kinase based on cell-penetrating peptide delivery system

    Institute of Scientific and Technical Information of China (English)

    Liping Yang; Yongming Yao; Zhiyong Sheng; Xiaomei Zhu; Yong Jiang


    Objective p38 Mitogen-activated protein kinase (MAPK) is a crossing center of various pathways. In this study, protein transduction system based on human immunodeficiency virus (HIV)-1 transactivator of transcription (TAT), which is an efficient delivery peptide of the foreign proteins into cells, was employed to study p38 MAPK functions in eukaryotic cells. Methods p38 And its dominant negative form, p38AF, were constructed into pET-His-TAT vector correctly to verify that the recombinant plasmids were well-founded through restriction enzyme digestion and DNA sequencing. The two proteins, His-TAT-p38 and His-TAT-p38AF, were expressed and purified in Escherichia coli by SDS-PAGE. Then they were incubated with ECV304 cells respectively and readily transduced into cells in a time-dependent and dose-dependent manner. The cells were stimulated by sorbitol. Activating transcription factor (ATF) 2 phosphorylation level was checked using Western blot to assess the activity of endogenous p38. Results Compared with controls, it was found that His-TAT-p38 increased the level ofATF2 phosphorylation in sorbitol-stimulated ECV304 cells, while His-TAT-p38AF inhibited it, indicating p38 MAPK protein delivery system based on TAT was constructed successfully. TAT-p38 and its dominant negative form possessed high biological activity after transduction into ECV304 cells by TAT protein delivery system. The results showed that p38AF fused with TAT could inhibit the transduction of endogenous p38 signal pathway in part, and other pathway might regulate p38 phosphorylation. Conclusions Our study provides a novel pathway to inhibit p38 signal pathway and establish a new method to study p38 function.

  11. The antimicrobial domains of wheat puroindolines are cell-penetrating peptides with possible intracellular mechanisms of action.

    Directory of Open Access Journals (Sweden)

    Rebecca L Alfred

    Full Text Available The puroindoline proteins (PINA and PINB of wheat display lipid-binding properties which affect the grain texture, a critical parameter for wheat quality. Interestingly, the same proteins also display antibacterial and antifungal properties, attributed mainly to their Tryptophan-rich domain (TRD. Synthetic peptides based on this domain also display selectivity towards bacterial and fungal cells and do not cause haemolysis of mammalian cells. However, the mechanisms of these activities are unclear, thus limiting our understanding of the in vivo roles of PINs and development of novel applications. This study investigated the mechanisms of antimicrobial activities of synthetic peptides based on the TRD of the PINA and PINB proteins. Calcein dye leakage tests and transmission electron microscopy showed that the peptides PuroA, Pina-M and Pina-W→F selectively permeabilised the large unilamellar vesicles (LUVs made with negatively charged phospholipids mimicking bacterial membranes, but were ineffective against LUVs made with zwitterionic phospholipids mimicking eukaryotic membranes. Propidium iodide fluorescence tests of yeast (Saccharomyces cerevisiae cells showed the peptides were able to cause loss of membrane integrity, PuroA and Pina-M being more efficient. Scanning electron micrographs of PINA-based peptide treated yeast cells showed the formation of pits or pores in cell membranes and release of cellular contents. Gel retardation assays indicated the peptides were able to bind to DNA in vitro, and the induction of filamental growth of E. coli cells indicated in vivo inhibition of DNA synthesis. Together, the results strongly suggest that the PIN-based peptides exert their antimicrobial effects by pore formation in the cell membrane, likely by a carpet-like mechanism, followed by intracellular mechanisms of activity.

  12. Fusion of a Short HA2-Derived Peptide Sequence to Cell-Penetrating Peptides Improves Cytosolic Uptake, but Enhances Cytotoxic Activity

    Directory of Open Access Journals (Sweden)

    Igor Kitanovic


    Full Text Available Cell-penetrating peptides (CPP have become a widely used tool for efficient cargo delivery into cells. However, one limiting fact is their uptake by endocytosis causing the enclosure of the CPP-cargo construct within endosomes. One often used method to enhance the outflow into the cytosol is the fusion of endosome-disruptive peptide or protein sequences to CPP. But, until now, no studies exist investigating the effects of the fusion peptide to the cellular distribution, structural arrangements and cytotoxic behaviour of the CPP. In this study, we attached a short modified sequence of hemagglutinin subunit HA2 to different CPP and analysed the biologic activity of the new designed peptides. Interestingly, we observed an increased cytosolic distribution but also highly toxic activities in the micromolar range against several cell lines. Structural analysis revealed that attachment of the fusion peptide had profound implications on the whole conformation of the peptide, which might be responsible for membrane interaction and endosome disruption.

  13. Hair follicle targeting, penetration enhancement and Langerhans cell activation make cyanoacrylate skin surface stripping a promising delivery technique for transcutaneous immunization with large molecules and particle-based vaccines. (United States)

    Vogt, Annika; Hadam, Sabrina; Deckert, Iliane; Schmidt, Julia; Stroux, Andrea; Afraz, Zahra; Rancan, Fiorenza; Lademann, Jürgen; Combadiere, Behazine; Blume-Peytavi, Ulrike


    Transcutaneous immunization (TCI) requires targeting of a maximum number of skin antigen-presenting cells as non-invasive as possible on small skin areas. In two clinical trials, we introduced cyanoacrylate skin surface stripping (CSSS) as a safe method for TCI. Here, using ex vivo human skin, we demonstrate that one CSSS procedure removed only 30% of stratum corneum, but significantly increased the penetration of 200 nm polystyrene particles deep into vellus and intermediate hair follicles from where they could not been retrieved by conventional tape stripping. Two subsequent CSSS had no striking additional effect. CSSS increased particle penetration in superficial stratum corneum and induced Langerhans cell activation. Formulation in amphiphilic ointment or massage did not substantially influences the interfollicular penetration profiles. Hair follicle (HF) targeting by CSSS could become a highly effective tool for TCI when combined with carrier-based delivery and is gaining new attention as our understanding on the HF immune system increases.

  14. Parallel Synthesis of Cell-Penetrating Peptide Conjugates of PMO Toward Exon Skipping Enhancement in Duchenne Muscular Dystrophy

    NARCIS (Netherlands)

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A.; Williams, Donna L.; Deuss, Peter; Gait, Michael J.


    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELecti

  15. The equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions. (United States)

    Osterrieder, N; Neubauer, A; Brandmuller, C; Braun, B; Kaaden, O R; Baines, J D


    Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted. To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed. Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11. A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gene inserted into the EHV-1 strain RacL11 gM gene (open reading frame 52) was purified, and cells infected with L11deltagM did not contain detectable gM. L11deltagM exhibited approximately 100-fold lower titers and a more than 2-fold reduction in plaque size relative to wild-type EHV-1 when grown and titrated on noncomplementing cells. Viral titers were reduced only 10-fold when L11deltagM was grown on the complementing cell line TCgM and titrated on noncomplementing cells. L11deltagM also exhibited slower penetration kinetics compared with those of the parental EHV-1 RacL11. It is concluded that EHV-1 gM plays important roles in the penetration of virus into the target cell and in spread of EHV-1 from cell to cell.

  16. Transfection of infectious RNA and DNA/RNA layered vectors of semliki forest virus by the cell-penetrating peptide based reagent PepFect6.

    Directory of Open Access Journals (Sweden)

    Kalle Pärn

    Full Text Available Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.

  17. A single-cell technique for the measurement of membrane potential, membrane conductance, and the efflux of rapidly penetrating solutes in Amphiuma erythrocytes. (United States)

    Stoner, L C; Kregenow, F M


    We describe a single-cell technique for measuring membrane potential, membrane resistance, and the efflux of rapidly penetrating solutes such as Cl and H2O. Erythrocytes from Amphiuma means were aspirated into a Sylgard (Dow Corning Corp.)-coated capillary. The aspirated cell separated a solution within the capillary from a solution in the bath. Each of these two solutions was contiguous with approximately 5% of the total membrane surface. Microelectrodes placed concentrically within the capillary permit the measurement of intracellular voltage, specific membrane resistance, and the electrical seal between the two solutions. The intracellular voltage averaged -17.7 mV (pH 7.6) and changed as either intra- or extracellular chloride was varied. The average specific membrane resistance measured by passing current across the exposed membrane surface was 110 ohm-cm2. 36Cl and tritiated H2O fluxes (0.84 +/- 0.05 x 10(-6) M . cm-2 . min-1 and 6.4 +/- 1.5 x 10(-3) M . cm-2 . min-1, respectively) were determined by noting the rate at which isotope leaves the cell and crosses the membrane exposed to the bath. Our measured values for the flux of 36Cl and tritiated H2O approximate reported values for free-floating cells. 36Cl efflux, in addition, is inhibited by 4-acetamido-4'-isothiocyano-stilbene 2,2'-disulfonic acid (SITS) and furosemide, known inhibitors of the anion exchange mechanism responsible for the rapid anion fluxes of red blood cells. One can also demonstrate directly that > 89% of 36Cl efflux is "electrically silent" by analyzing the flux in the presence of an imposed transcellular voltage.

  18. Metasploit penetration testing cookbook

    CERN Document Server

    Agarwal, Monika


    This book follows a Cookbook style with recipes explaining the steps for penetration testing with WLAN, VOIP, and even cloud computing. There is plenty of code and commands used to make your learning curve easy and quick.This book targets both professional penetration testers as well as new users of Metasploit, who wish to gain expertise over the framework and learn an additional skill of penetration testing, not limited to a particular OS. The book requires basic knowledge of scanning, exploitation, and the Ruby language.

  19. Cell-penetrating peptides, novel synthetic nucleic acids, and regulation of gene function : Reconnaissance for designing functional conjugates


    Guterstam, Peter


    Our genome operates by sending instructions, conveyed by mRNA, for the manufacture of proteins from chromosomal DNA in the nucleus of the cell to the protein synthesizing machinery in the cytoplasm. Alternative splicing is a natural process in which a single gene can encode multiple related proteins. During RNA splicing, introns are selectively removed resulting in alternatively spliced gene products. Alternatively spliced protein products can have very different biological effects, such that...

  20. Sucrose esters increase drug penetration, but do not inhibit p-glycoprotein in caco-2 intestinal epithelial cells. (United States)

    Kiss, Lóránd; Hellinger, Éva; Pilbat, Ana-Maria; Kittel, Ágnes; Török, Zsolt; Füredi, András; Szakács, Gergely; Veszelka, Szilvia; Sipos, Péter; Ózsvári, Béla; Puskás, László G; Vastag, Monika; Szabó-Révész, Piroska; Deli, Mária A


    Sucrose fatty acid esters are increasingly used as excipients in pharmaceutical products, but few data are available on their toxicity profile, mode of action, and efficacy on intestinal epithelial models. Three water-soluble sucrose esters, palmitate (P-1695), myristate (M-1695), laurate (D-1216), and two reference absorption enhancers, Tween 80 and Cremophor RH40, were tested on Caco-2 cells. Caco-2 monolayers formed a good barrier as reflected by high transepithelial resistance and positive immunostaining for junctional proteins claudin-1, ZO-1, and β-catenin. Sucrose esters in nontoxic concentrations significantly reduced resistance and impedance, and increased permeability for atenolol, fluorescein, vinblastine, and rhodamine 123 in Caco-2 monolayers. No visible opening of the tight junctions was induced by sucrose esters assessed by immunohistochemistry and electron microscopy, but some alterations were seen in the structure of filamentous actin microfilaments. Sucrose esters fluidized the plasma membrane and enhanced the accumulation of efflux transporter ligands rhodamine 123 and calcein AM in epithelial cells, but did not inhibit the P-glycoprotein (P-gp)-mediated calcein AM accumulation in MES-SA/Dx5 cell line. These data indicate that in addition to their dissolution-increasing properties sucrose esters can enhance drug permeability through both the transcellular and paracellular routes without inhibiting P-gp.

  1. A mouse model for inducible overexpression of Prdm14 results in rapid-onset and highly penetrant T-cell acute lymphoblastic leukemia (T-ALL

    Directory of Open Access Journals (Sweden)

    Brandi L. Carofino


    PRDM14 functions in embryonic stem cell (ESC maintenance to promote the expression of pluripotency-associated genes while suppressing differentiation genes. Expression of PRDM14 is tightly regulated and typically limited to ESCs and primordial germ cells; however, aberrant expression is associated with tumor initiation in a wide variety of human cancers, including breast cancer and leukemia. Here, we describe the generation of a Cre-recombinase-inducible mouse model for the spatial and temporal control of Prdm14 misexpression [ROSA26 floxed-stop Prdm14 (R26PR]. When R26PR is mated to either of two Cre lines, Mx1-cre or MMTV-cre, mice develop early-onset T-cell acute lymphoblastic leukemia (T-ALL with median overall survival of 41 and 64 days for R26PR;Mx1-cre and R26PR;MMTV-cre, respectively. T-ALL is characterized by the accumulation of immature single-positive CD8 cells and their widespread infiltration. Leukemia is preceded by a dramatic expansion of cells resembling hematopoietic stem cells and lymphoid-committed progenitors prior to disease onset, accompanied by a blockage in B-cell differentiation at the early pro-B stage. Rapid-onset PRDM14-induced T-ALL requires factors that are present in stem and progenitor cells: R26PR;dLck-cre animals, which express Prdm14 starting at the double-positive stage of thymocyte development, do not develop disease. PRDM14-induced leukemic cells contain high levels of activated NOTCH1 and downstream NOTCH1 targets, including MYC and HES1, and are sensitive to pharmacological inhibition of NOTCH1 with the γ-secretase inhibitor DAPT. Greater than 50% of human T-ALLs harbor activating mutations in NOTCH1; thus, our model carries clinically relevant molecular aberrations. The penetrance, short latency and involvement of the NOTCH1 pathway will make this hematopoietic R26PR mouse model ideal for future studies on disease initiation, relapse and novel therapeutic drug combinations. Furthermore, breeding R26PR to additional Cre

  2. Hydrothermal synthesis of nitrogen-doped carbon dots with real-time live-cell imaging and blood–brain barrier penetration capabilities

    Directory of Open Access Journals (Sweden)

    Lu SS


    Full Text Available Shousi Lu,1,2 Shanshan Guo,1 Pingxiang Xu,1 Xiaorong Li,1 Yuming Zhao,1 Wei Gu,3 Ming Xue1 1Department of Pharmacology, Beijing Laboratory for Biomedical Detection Technology and Instrument, School of Basic Medical Sciences, Capital Medical University, 2China Rehabilitation Research Center, 3Department of Chemistry and Biology, School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing, China Abstract: Nitrogen-doped carbon dots (N-CDs were synthesized using a one-pot hydrothermal treatment with citric acid in the presence of polyethylenimine. Transmission electron microscopy analysis revealed that the N-CDs were monodispersed and quasi-spherical with an average size of ~2.6 nm. Under ultraviolet irradiation the N-CDs emitted a strong blue luminescence with a quantum yield as high as 51%. Moreover, the N-CDs exhibited a negligible cytotoxicity and could be applied as efficient nanoprobes for real-time imaging of live cells. In addition, the ability of the N-CDs to cross the blood–brain barrier (BBB in a concentration-dependent manner was demonstrated using an in vitro BBB model. Therefore, these PEI-passivated N-CDs with real-time live-cell imaging and BBB-penetration capabilities hold promise for traceable drug delivery to the brain. Keywords: hydrothermal synthesis, nitrogen-doped carbon dots, bioimaging

  3. Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood-Brain Barrier. (United States)

    Bera, Swapna; Kar, Rajiv K; Mondal, Susanta; Pahan, Kalipada; Bhunia, Anirban


    Cell-penetrating peptides (CPPs) have shown promise in nonpermeable therapeutic drug delivery, because of their ability to transport a variety of cargo molecules across the cell membranes and their noncytotoxicity. Drosophila antennapedia homeodomain-derived CPP penetratin (RQIKIWFQNRRMKWKK), being rich in positively charged residues, has been increasingly used as a potential drug carrier for various purposes. Penetratin can breach the tight endothelial network known as the blood-brain barrier (BBB), permitting treatment of several neurodegenerative maladies, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. However, a detailed structural understanding of penetratin and its mechanism of action is lacking. This study defines structural features of the penetratin-derived peptide, DK17 (DRQIKIWFQNRRMKWKK), in several model membranes and describes a membrane-induced conformational transition of the DK17 peptide in these environments. A series of biophysical experiments, including high-resolution nuclear magnetic resonance spectroscopy, provides the three-dimensional structure of DK17 in different membranes mimicking the BBB or total brain lipid extract. Molecular dynamics simulations support the experimental results showing preferential binding of DK17 to particular lipids at atomic resolution. The peptide conserves the structure of the subdomain spanning residues Ile6-Arg11, despite considerable conformational variation in different membrane models. In vivo data suggest that the wild type, not a mutated sequence, enters the central nervous system. Together, these data highlight important structural and functional attributes of DK17 that could be utilized in drug delivery for neurodegenerative disorders.

  4. Photoacoustic Imaging: Semiconducting Oligomer Nanoparticles as an Activatable Photoacoustic Probe with Amplified Brightness for In Vivo Imaging of pH (Adv. Mater. 19/2016). (United States)

    Miao, Qingqing; Lyu, Yan; Ding, Dan; Pu, Kanyi


    Despite the great potential of photoacoustic imaging in the life sciences, the development of smart activatable photoacoustic probes remains elusive. On page 3662, K. Pu and co-workers report a facile nanoengineering approach based on semiconducting oligomer nano-particles to develop ratiometric photoacoustic probes with amplified brightness and enhanced sensing capability for accurate photoacoustic mapping of pH in the tumors of living mice.

  5. Penetration testing with Perl

    CERN Document Server

    Berdeaux, Douglas


    If you are an expert Perl programmer interested in penetration testing or information security, this guide is designed for you. However, it will also be helpful for you even if you have little or no Linux shell experience.

  6. Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA. (United States)

    Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F


    Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

  7. Prolongation of life in rats with malignant glioma by intranasal siRNA/drug codelivery to the brain with cell-penetrating peptide-modified micelles. (United States)

    Kanazawa, Takanori; Morisaki, Kazuki; Suzuki, Shohei; Takashima, Yuuki


    New therapeutic strategies are required to develop candidate drugs and ensure efficient delivery of these drugs to the brain and the central nervous system (CNS). Small interfering RNA (siRNA)-based therapies have been investigated as potential novel approaches for the treatment of brain disorders. Previously, we showed that Tat, a cell-penetrating peptide derived from HIV-Tat, and the modified block copolymers (MPEG-PCL-Tat) can form stable complexes with siRNA or can be loaded with an anticancer drug and efficiently deliver the drugs to the brain tissue via intranasal delivery. In this study, to develop a novel, efficient, and safe therapeutic strategy for managing brain disorders, we used MPEG-PCL-Tat micelles with a nose-to-brain delivery system to investigate its therapeutic effects on a rat model of malignant glioma using siRNA with a Raf-1 (siRaf-1)/camptothecin (CPT) codelivery system. MPEG-PCL-Tat and CPT-loaded MPEG-PCL-Tat can form a stable complex with siRNA with a particle size from 60 to 200 nm and a positive charge at N/P ratios up to 5. Additionally, MPEG-PCL-Tat/siRaf-1 and CPT-loaded MPEG-PCL-Tat/siRaf-1 have fostered cell death in rat glioma cells after the high cellular uptake of siRaf-1/drug by the MPEG-PCL-Tat carrier. Furthermore, compared to the unloaded MPEG-PCL-Tat/siRaf-1 complex, a CPT-loaded MPEG-PCL-Tat/siRaf-1 complex achieved the high therapeutic effect because of the additive effects of CPT and siRaf-1. These results indicate that drug/siRNA codelivery using MPEG-PCL-Tat nanomicelles with nose-to-brain delivery is an excellent therapeutic approach for brain and CNS diseases.

  8. Novel pH-sensitive charge-reversal cell penetrating peptide conjugated PEG-PLA micelles for docetaxel delivery: in vitro study. (United States)

    Ouahab, Ammar; Cheraga, Nihad; Onoja, Vitus; Shen, Yan; Tu, Jiasheng


    In order to create a pH-sensitive charge-reversal system for cell penetrating peptides (CPP) to prevent non-specific internalization of the drug; and concomitantly enhance the physical stability and tumor targetability of poly(ethylene glycol)-poly(d,l-lactide) (PEG-PLA) micelles, two sets of novel PEG-PLA micelles were developed. Cell penetrating decapeptide arginine-glycine (RG)5 and a pH-sensitive masking decapeptide histidine-glutamic acid (HE)5 were conjugated at the PEG free end to produce pH sensitive with peptides outside micelles (PHPO), while the pH sensitive with peptides inside micelles (PHPI) are the micelles obtained with the two peptides conjugated to the free end of the PLA block. The polymers were successfully synthesized and characterized by (1)H NMR and GPC. The mixed micelles were prepared and characterized for their loading efficiency, particle size and zeta potential. The surface charge of PHPO was greatly affected by the pH of the solution and (RG)5:(HE)5 ratio at the surface. The pH value of the solution at which the surface charge of PHPO reversed could be manipulated by the feed ratio of (RG)5-PEG-PLA (RGO) and (HE)5-PEG-PLA (HEO), hence, HEO:RGO molar ratio of 45:55 was selected for tumor targeting. Docetaxel (DTX) was sufficiently solubilized by DTX-PHPO with a loading efficiency of 90.18 ± 1.65%. At pH 7.4, DTX loaded mPEG-PLA (DTX-PM) (41.2 ± 0.3 nm), DTX-PHPO (195.3 ± 1.9 nm) and DTX-PHPI (190.9 ± 4.5 nm) showed sustained DTX release of less than 55% within 48 h. However, at pH 6.8 DTX-PHPI released 87.29 ± 0.24%, while DTX-PHPO released 70.49 ± 0.39% of the initial DTX amount within 48 h. Moreover, the physical stability of DTX-PHPO was increased due to the electrostatic interaction of the two peptides. The cellular uptake of DTX-PHPO in SGC-7901 cells and the cell killing effect tested on MCF-7 cells were enhanced by 2 folds at pH 6.8 compared to pH 7.4. Hence, DTX-PHPO is highly pH-sensitive in mildly acidic pH and exhibited

  9. Delivery of siRNA to the brain using a combination of nose-to-brain delivery and cell-penetrating peptide-modified nano-micelles. (United States)

    Kanazawa, T; Akiyama, F; Kakizaki, S; Takashima, Y; Seta, Y


    The potential for RNA-based agents to serve as effective therapeutics for central nerve systems (CNS) disorders has been successfully demonstrated in vitro. However, the blood-brain barrier limits the distribution of systemically administered therapeutics to the CNS, posing a major challenge for drug development aimed at combatting CNS disorders. Therefore, the development of effective strategies to enhance siRNA delivery to the brain is of great interest in clinical and pharmaceutical fields. To improve the efficiency of small interfering RNA (siRNA) delivery to the brain, we developed a nose-to-brain delivery system combined with cell-penetrating peptide (CPP) modified nano-micelles comprising polyethylene glycol-polycaprolactone (PEG-PCL) copolymers conjugated with the CPP, Tat (MPEG-PCL-Tat). In this study, we describe intranasal brain delivery of siRNA or dextran (Mw: 10,000 Da) as a model siRNA, by using MPEG-PCL-Tat. Intranasal delivery of dextran with MPEG-PCL-Tat improved brain delivery compared to intravenous delivery of dextran either with or without MPEG-PCL-Tat. We also studied the intranasal transfer of MPEG-PCL-Tat to the brain via the olfactory and trigeminal nerves, the putative pathways to the brain from the nasal cavity. We found that MPEG-PCL-Tat accelerated transport along the olfactory and trigeminal nerve pathway because of its high permeation across the nasal mucosa.

  10. Visualization and Quantitative Assessment of the Brain Distribution of Insulin through Nose-to-Brain Delivery Based on the Cell-Penetrating Peptide Noncovalent Strategy. (United States)

    Kamei, Noriyasu; Shingaki, Tomotaka; Kanayama, Yousuke; Tanaka, Misa; Zochi, Riyo; Hasegawa, Koki; Watanabe, Yasuyoshi; Takeda-Morishita, Mariko


    Our recent work suggested that intranasal coadministration with the cell-penetrating peptide (CPP) penetratin increased the brain distribution of the peptide drug insulin. The present study aimed to distinctly certify the ability of penetratin to facilitate the nose-to-brain delivery of insulin by quantitatively evaluating the distribution characteristics in brain using radioactive (64)Cu-NODAGA-insulin. Autoradiography and analysis using a gamma counter of brain areas demonstrated that the accumulation of radioactivity was greatest in the olfactory bulb, the anterior part of the brain closest to the administration site, at 15 min after intranasal administration of (64)Cu-NODAGA-insulin with l- or d-penetratin. The brain accumulation of (64)Cu-NODAGA-insulin with penetratin was confirmed by ELISA using unlabeled insulin in which intact insulin was delivered to the brain after intranasal coadministration with l- or d-penetratin. By contrast, quantification of cerebrospinal fluid (CSF) samples showed increased insulin concentration in only the anterior portion of the CSF at 15 min after intranasal coadministration with l-penetratin. This study gives the first concrete proof that penetratin can accelerate the direct transport of insulin from the nasal cavity to the brain parenchyma. Further optimization of intranasal administration with CPP may increase the efficacy of delivery of biopharmaceuticals to the brain while reducing the risk of systemic drug exposure.

  11. Cell-penetrating peptide and antibiotic combination therapy: a potential alternative to combat drug resistance in methicillin-resistant Staphylococcus aureus. (United States)

    Randhawa, Harmandeep Kaur; Gautam, Ankur; Sharma, Minakshi; Bhatia, Rakesh; Varshney, Grish C; Raghava, Gajendra Pal Singh; Nandanwar, Hemraj


    The diverse pattern of resistance by methicillin-resistant Staphylococcus aureus (MRSA) is the major obstacle in the treatment of its infections. The key reason of resistance is the poor membrane permeability of drug molecules. Over the last decade, cell-penetrating peptides (CPPs) have emerged as efficient drug delivery vehicles and have been exploited to improve the intracellular delivery of numerous therapeutic molecules in preclinical studies. Therefore, to overcome the drug resistance, we have investigated for the first time the effects of two CPPs (P3 and P8) in combination with four antibiotics (viz. oxacillin, erythromycin, norfloxacin, and vancomycin) against MRSA strains. We found that both CPPs internalized into the MRSA efficiently at very low concentration (combinations of CPPs (≤10 μM) and antibiotics showed high toxicity against MRSA as compared to antibiotics alone. The significant finding is that P3 and P8 could lower the MICs against oxacillin, norfloxacin, and vancomycin to susceptible levels (generally antibiotics. In summary, CPPs assist to restore the effectiveness of antibiotics at much lower concentration, eliminate the antibiotic toxicity, and represent the CPP-antibiotic combination therapy as a potential novel weapon to combat MRSA infections.

  12. Inhibiting α-synuclein oligomerization by stable cell-penetrating β-synuclein fragments recovers phenotype of Parkinson's disease model flies.

    Directory of Open Access Journals (Sweden)

    Ronit Shaltiel-Karyo

    Full Text Available The intracellular oligomerization of α-synuclein is associated with Parkinson's disease and appears to be an important target for disease-modifying treatment. Yet, to date, there is no specific inhibitor for this aggregation process. Using unbiased systematic peptide array analysis, we identified molecular interaction domains within the β-synuclein polypeptide that specifically binds α-synuclein. Adding such peptide fragments to α-synuclein significantly reduced both amyloid fibrils and soluble oligomer formation in vitro. A retro-inverso analogue of the best peptide inhibitor was designed to develop the identified molecular recognition module into a drug candidate. While this peptide shows indistinguishable activity as compared to the native peptide, it is stable in mouse serum and penetrates α-synuclein over-expressing cells. The interaction interface between the D-amino acid peptide and α-synuclein was mapped by Nuclear Magnetic Resonance spectroscopy. Finally, administering the retro-inverso peptide to a Drosophila model expressing mutant A53T α-synuclein in the nervous system, resulted in a significant recovery of the behavioral abnormalities of the treated flies and in a significant reduction in α-synuclein accumulation in the brains of the flies. The engineered retro-inverso peptide can serve as a lead for developing a novel class of therapeutic agents to treat Parkinson's disease.

  13. PEGylation of the peptide Bac7(1-35) reduces renal clearance while retaining antibacterial activity and bacterial cell penetration capacity. (United States)

    Benincasa, Monica; Zahariev, Sotir; Pelillo, Chiara; Milan, Annalisa; Gennaro, Renato; Scocchi, Marco


    The proline-rich antibacterial peptide Bac7(1-35) protects mice against Salmonella typhimurium infection, despite its rapid clearance. To overcome this problem the peptide was linked to a polyethylene glycol (PEG) molecule either via a cleavable ester bond or via a non-hydrolysable amide bond. Both the PEGylated conjugates retained most of the in vitro activity against S. typhimurium. In addition, the ester bond was cleaved in human serum or plasma, releasing a carboxymethyl derivative of Bac7(1-35) which accounts for a higher activity of this peptide with relative to the other, non-hydrolysable form. Both PEGylated peptides maintained the capacity of the unconjugated form to kill bacteria without permeabilizing the bacterial membranes, by penetrating into cells. They exploited the same transporter as unmodified Bac7(1-35), suggesting it has the capacity to internalize quite sizeable cargo if this is linked to Bac7 fragment. PEGylation allows the peptide to have a wide distribution in mice, and a slow renal clearance, indicating that this strategy would improve the bioavailability of Bac7, and in principle of other antimicrobial peptides. This can be an equally important issue to reducing cytotoxicity for therapeutic use of these antibacterials.

  14. A Coevolutionary Arms Race between Hosts and Viruses Drives Polymorphism and Polygenicity of NK Cell Receptors

    NARCIS (Netherlands)

    Carrillo-Bustamante, Paola; Kesmir, C.; de Boer, Rob J


    Natural killer cell receptors (NKRs) monitor the expression of major histocompatibility class I (MHC-I) and stress molecules to detect unhealthy tissue, such as infected or tumor cells. The NKR gene family shows a remarkable genetic diversity, containing several genes encoding receptors with activat

  15. Physics of Armour penetration

    Directory of Open Access Journals (Sweden)

    Sampooran Singh


    Full Text Available When a “Shaped-Charge” projectile is spin-stabilized during its flight, its penetration is greatly reduced, hence such a projectile is fin-stabilized and consequently the range is less and accuracy is poor. Attempts are being made to combine the penetrating power of a bazooka, and range and accuracy of rifled weapon. Just as the introduction of fire-arms brought about the extinction of the armour knights because a bullet could easily penetrate the kind of armour that the knights could put on; similarly “Shaped-Charge” projectiles having the range and accuracy of a rifled weapon will seriously affect the tactical use of amour in the battle-field.

  16. Enhancing Anticancer Effect of Gefitinib across the Blood-Brain Barrier Model Using Liposomes Modified with One α-Helical Cell-Penetrating Peptide or Glutathione and Tween 80. (United States)

    Lin, Kuan-Hung; Hong, Shu-Ting; Wang, Hsiang-Tsui; Lo, Yu-Li; Lin, Anya Maan-Yuh; Yang, James Chih-Hsin


    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib, have been demonstrated to effectively treat the patients of extracranial non-small cell lung cancer (NSCLC). However, these patients often develop brain metastasis (BM) during their disease course. The major obstacle to treat BM is the limited penetration of anticancer drugs across the blood-brain barrier (BBB). In the present study, we utilized gefitinib-loaded liposomes with different modifications to improve gefitinib delivery across the in vitro BBB model of bEnd.3 cells. Gefitinib was encapsulated in small unilamellar liposomes modified with glutathione (GSH) and Tween 80 (SUV-G+T; one ligand plus one surfactant) or RF (SUV-RF; one α-helical cell-penetrating peptide). GSH, Tween 80, and RF were tested by the sulforhodamine B (SRB) assay to find their non-cytotoxic concentrations on bEnd.3 cells. The enhancement on gefitinib across the BBB was evaluated by cytotoxicity assay on human lung adenocarcinoma PC9 cells under the bEnd.3 cells grown on the transwell inserts. Our findings showed that gefitinib incorporated in SUV-G+T or SUV-RF across the bEnd.3 cells significantly reduced the viability of PC9 cells more than that of free gefitinib. Furthermore, SUV-RF showed no cytotoxicity on bEnd.3 cells and did not affect the transendothelial electrical resistance (TEER) and transendothelial permeability of sodium fluorescein across the BBB model. Moreover, flow cytometry and confocal laser scanning microscopy were employed to evaluate the endocytosis pathways of SUV-RF. The results indicated that the uptake into bEnd.3 cells was mainly through adsorptive-mediated mechanism via electrostatic interaction and partially through clathrin-mediated endocytosis. In conclusion, cell penetrating peptide-conjugated SUV-RF shed light on improving drug transport across the BBB via modulating the transcytosis pathway(s).

  17. Ground penetrating radar

    CERN Document Server

    Daniels, David J


    Ground-penetrating radar has come to public attention in recent criminal investigations, but has actually been a developing and maturing remote sensing field for some time. In the light of recent expansion of the technique to a wide range of applications, the need for an up-to-date reference has become pressing. This fully revised and expanded edition of the best-selling Surface-Penetrating Radar (IEE, 1996) presents, for the non-specialist user or engineer, all the key elements of this technique, which span several disciplines including electromagnetics, geophysics and signal processing. The

  18. Session: Hard Rock Penetration

    Energy Technology Data Exchange (ETDEWEB)

    Tennyson, George P. Jr.; Dunn, James C.; Drumheller, Douglas S.; Glowka, David A.; Lysne, Peter


    This session at the Geothermal Energy Program Review X: Geothermal Energy and the Utility Market consisted of five presentations: ''Hard Rock Penetration - Summary'' by George P. Tennyson, Jr.; ''Overview - Hard Rock Penetration'' by James C. Dunn; ''An Overview of Acoustic Telemetry'' by Douglas S. Drumheller; ''Lost Circulation Technology Development Status'' by David A. Glowka; ''Downhole Memory-Logging Tools'' by Peter Lysne.

  19. Penetration through the Skin Barrier

    DEFF Research Database (Denmark)

    Nielsen, Jesper Bo; Benfeldt, Eva; Holmgaard, Rikke


    The skin is a strong and flexible organ with barrier properties essential for maintaining homeostasis and thereby human life. Characterizing this barrier is the ability to prevent some chemicals from crossing the barrier while allowing others, including medicinal products, to pass at varying rates......-through diffusion cells) as well as in vivo methods (microdialysis and microperfusion). Then follows a discussion with examples of how different characteristics of the skin (age, site and integrity) and of the penetrants (size, solubility, ionization, logPow and vehicles) affect the kinetics of percutaneous...

  20. Chiral, J-Aggregate-Forming Dyes for Alternative Signal Modulation Mechanisms in Self-Immolative Enzyme-Activatable Optical Probes. (United States)

    Sloniec-Myszk, Jagoda; Resch-Genger, Ute; Hennig, Andreas


    Enzyme-activatable optical probes are important for future advances in cancer imaging, but may easily suffer from low signal-to-background ratios unless not optimized. To address this shortcoming, numerous mechanisms to modulate the fluorescence signal have been explored. We report herein newly synthesized probes based on self-immolative linkers containing chiral J-aggregate-forming dyes. Signal modulation by formation of chiral J-aggregates is yet unexplored in optical enzyme probe design. The comprehensive characterization of the probes by absorption, CD, fluorescence, and time-resolved fluorescence spectroscopy revealed dye-dye interactions not observed for the free dyes in solution as well as dye-protein interactions with the enzyme. This suggested that J-aggregate formation is challenging to achieve with current probe design and that interactions of the dyes with the enzyme may interfere with achieving high signal-to-background ratios. The detailed understanding of the interactions provided herein provides valuable guidelines for the future design of similar probes.

  1. Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

    Directory of Open Access Journals (Sweden)

    Park Sung


    Full Text Available Abstract Background Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli have been unsuccessful. Results Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM of the precursor proteins by two orders of magnitude. Conclusion A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.

  2. The cell-penetrating peptide domain from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) has anti-inflammatory activity in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jue-Yeon; Seo, Yoo-Na; Park, Hyun-Jung [Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Park, Yoon-Jeong, E-mail: [Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Dental Regenerative Biotechnology, Dental Research Institute and School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Chung, Chong-Pyoung, E-mail: [Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Periodontology, College of Dentistry, Seoul National University, Seoul (Korea, Republic of)


    Highlights: Black-Right-Pointing-Pointer HBP sequence identified from HB-EGF has cell penetration activity. Black-Right-Pointing-Pointer HBP inhibits the NF-{kappa}B dependent inflammatory responses. Black-Right-Pointing-Pointer HBP directly blocks phosphorylation and degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer HBP inhibits nuclear translocation of NF-{kappa}B p65 subunit. -- Abstract: A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cytokines (TNF-{alpha} and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-{alpha} and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-{kappa}B-dependent inflammatory responses by directly blocking the phosphorylation and degradation of I{kappa}B{alpha} and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-{kappa}B. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.

  3. Penetration of erythromycin through Staphylococcus epidermidis biofilm

    Institute of Scientific and Technical Information of China (English)

    LIN Mao-hu; HE Lei; GAO Jie; LIU Yun-xi; SUO Ji-jiang; XING Yu-bin; JIA Ning


    Background The catheter related infection caused by Staphylococcus epiderrnidis biofilm is increasing and difficult to treat by antimicrobial chemotherapy.The properties of biofilms that give rise to antibiotic resistance are only partially understood.This study aimed to elucidate the penetration of erythromycin through Staphylococcus epidermidis biofilm.Methods The penetration ratio of erythromycin through Staphylococcus epidermidis biofilms of 1457,1457-msrA,and wild isolate S68 was detected by biofilm penetration model at different time points according to the standard regression curve.The RNNDNA ratio and the cell density within the biofilms were observed by confocal laser microscope and transmission electromicroscope,respectively.Results The penetration ratios of erythromycin through the biofilms of 1457,1457-msrA,and S68 after cultivation for 36 hours were 0.93,0.55 and 0.4,respectively.The erythromycin penetration ratio through 1457 biofilm (0.58 after 8 hours)was higher than that through the other two (0.499 and 0.31 after 24 hours).Lower growth rate of the cells in biofilm was shown,with reduction of RNA/DNA proportion observed by confocal laser microscope through acridine orange stain.Compared with the control group observed by transmission electrmicroscope,the cell density of biofilm air face was lower than that of agar face,with more cell debris.Conclusions Erythromycin could penetrate to the Staphylococcus epidermidis biofilm,but could not kill the cells thoroughly.The lower growth rate of the cells within biofilm could help decreasing the erythromycin susceptibility.

  4. Comparative density functional theory and density functional tight binding study of arginine and arginine-rich cell penetrating peptide TAT adsorption on anatase TiO2. (United States)

    Li, Wenxuan; Kotsis, Konstantinos; Manzhos, Sergei


    We present a comparative density functional theory (DFT) and density functional tight binding (DFTB) study of geometries and electronic structures of arginine (Arg), arginine adsorbed on the anatase (101) surface of titania in several adsorption configurations, and of an arginine-rich cell penetrating peptide TAT and its adsorption on the surface of TiO2. Two DFTB parameterizations are considered, tiorg-0-1/mio-1-1 and matsci-0-3. While there is good agreement in the structures and relative energies of Arg and peptide conformers between DFT and DFTB, both adsorption geometries and energies are noticeably different for Arg adsorbed on TiO2. The tiorg-0-1/mio-1-1 parameterization performs better than matsci-0-3. We relate this difference to the difference in electronic structures resulting from the two methods (DFT and DFTB) and specifically to the band alignment between the molecule and the oxide. We show that the band alignment of TAT and TiO2 modeled with DFTB is qualitatively correct but that with DFT using the PBE functional is not. This is specific to the modeling of large molecules where the HOMO is close to the conduction band of the oxide. We therefore report a case where the approximate DFT-based method - DFTB (with which the correct band structure can be effectively obtained) - performs better than the DFT itself with a functional approximation feasible for the modeling of large bio-inorganic interfaces, i.e. GGA (as opposed to hybrid functionals which are impractical at such a scale). Our results highlight the utility of the DFTB method for the modeling of bioinorganic interfaces not only from the CPU cost perspective but also from the accuracy point of view.

  5. An unusual MAP kinase is required for efficient penetration of the plant surface by Ustilago maydis. (United States)

    Brachmann, Andreas; Schirawski, Jan; Müller, Philip; Kahmann, Regine


    In Ustilago maydis, pathogenic development is controlled by a heterodimer of the two homeodomain proteins bW and bE. We have identified by RNA fingerprinting a b-regulated gene, kpp6, which encodes an unusual MAP kinase. Kpp6 is similar to a number of other fungal MAP kinases involved in mating and pathogenicity, but contains an additional N-terminal domain unrelated to other proteins. Transcription of the kpp6 gene yields two transcripts differing in length, but encoding proteins of identical mass. One transcript is upregulated by the bW/bE heterodimer, while the other is induced after pheromone stimulation. kpp6 deletion mutants are attenuated in pathogenicity. kpp6(T355A,Y357F) mutants carrying a non-activatable allele of kpp6 are more severely compromised in pathogenesis. These strains can still form appressoria, but are defective in the subsequent penetration of the plant cuticle. Kpp6 is expressed during all stages of the sexual life cycle except mature spores. We speculate that Kpp6 may respond to a plant signal and regulate the genes necessary for efficient penetration of plant tissue.

  6. Cellular penetration and nuclear importation properties of {sup 111}In-labeled and {sup 123}I-labeled HIV-1 tat peptide immunoconjugates in BT-474 human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cornelissen, Bart [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Hu, Meiduo [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); McLarty, Kristin [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Costantini, Dan [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada); Reilly, Raymond M. [Division of Nuclear Medicine, University Health Network, Toronto, ON, M5S 3M2 (Canada) and Department of Medical Imaging, University of Toronto, Toronto, ON, M5S 3M2 (Canada) and Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON, M5S 3M2 (Canada) and Toronto General Research Institute, University Health Network, Toronto, ON, M5S 3M2 (Canada)]. E-mail:


    Introduction: Our objective was to compare the cell penetration and nuclear importation properties of {sup 111}In-labeled and {sup 123}I-labeled immunoconjugates (ICs) composed of 16-mer peptides (GRKKRRQRRRPPQGYG) derived from HIV-1 transactivator of transcription (tat) protein and anti-mouse IgG (mIgG) in BT-474 breast cancer (BC) cells. Methods: [{sup 111}In]tat ICs were constructed by site-specific conjugation of tat peptides to NaIO{sub 4} {sup -}-oxidized carbohydrates in the Fc domain of diethylenetriaminepentaacetic-acid-modified anti-mIgG antibodies. Immunoreactivity against mIgG was assessed in a competition assay. The kinetics of the accumulation of [{sup 111}In]anti-mIgG-tat IC and [{sup 123}I]anti-mIgG-tat ICs in BT-474 cells and the elimination of radioactivity from cells, cytoplasm or nuclei were determined. The effects of excess tat peptides or NH{sub 4}Cl (an inhibitor of endosomal acidification) on cellular uptake and nuclear importation of [{sup 111}In]anti-mIgG-tat were measured. Results: [{sup 111}In]anti-mIgG-tat was >97% radiochemically pure and exhibited preserved immunoreactivity with mIgG epitopes. [{sup 123}I]Anti-mIgG-tat penetrated BT-474 cells more rapidly than [{sup 111}In]anti-mIgG-tat ICs and achieved a 1.5-fold to a 2-fold higher uptake in cells and nuclei. Cell penetration and nuclear uptake of [{sup 111}In]anti-mIgG-tat were inhibited by excess tat peptides and NH{sub 4}Cl. Elimination of radioactivity from BT-474 cells and nuclei was more rapid and complete for {sup 123}I-labeled than for {sup 111}In-labeled anti-mIgG-tat ICs. Conclusion: Tat peptides derived from HIV-1 tat protein promoted the penetration and nuclear uptake of radioactivity following the incubation of {sup 111}In-labeled and {sup 123}I-labeled anti-mIgG antibodies with BT-474 human BC cells. {sup 111}In-labeled tat ICs are feasible for inserting radionuclides into cancer cells with potential for targeting intracellular and, particularly, nuclear epitopes for

  7. Python penetration testing essentials

    CERN Document Server



    If you are a Python programmer or a security researcher who has basic knowledge of Python programming and want to learn about penetration testing with the help of Python, this book is ideal for you. Even if you are new to the field of ethical hacking, this book can help you find the vulnerabilities in your system so that you are ready to tackle any kind of attack or intrusion.

  8. Market penetration of biodiesel

    Directory of Open Access Journals (Sweden)

    Kenneth R. Szulczyk, Bruce A. McCarl


    Full Text Available This research examines in detail the technology and economics of substituting biodiesel for diesel #2. This endeavor examines three areas. First, the benefits of biodiesel are examined, and the technical problems of large-scale implementation. Second, the biodiesel production possibilities are examined for soybean oil, corn oil, tallow, and yellow grease, which are the largest sources of feedstocks for the United States. Examining in detail the production possibilities allows to identity the extent of technological change, production costs, byproducts, and greenhouse gas (GHG emissions. Finally, a U.S. agricultural model, FASOMGHG was used to predict market penetration of biodiesel, given technological progress, variety of technologies and feedstocks, market interactions, energy prices, and carbon dioxide equivalent prices. FASOMGHG has several interesting results. First, diesel fuel prices have an expansionary impact on the biodiesel industry. The higher the diesel fuel prices, the more biodiesel is produced. However, given the most favorable circumstances, the maximum biodiesel market penetration is 9% in 2030 with a wholesale diesel price of $4 per gallon. Second, the two dominant sources of biodiesel are from corn and soybeans. Sources like tallow and yellow grease are more limited, because they are byproducts of other industries. Third, GHG prices have an expansionary impact on the biodiesel prices, because biodiesel is quite GHG efficient. Finally, U.S. government subsidies on biofuels have an expansionary impact on biodiesel production, and increase market penetration at least an additional 3%.

  9. Market penetration of biodiesel

    Energy Technology Data Exchange (ETDEWEB)

    Szulczyk, Kenneth R. [Department of Economics, Orbita 3, Suleyman Demirel University, Almaty, 050043 (Kazakhstan); McCarl, Bruce A. [Department of Agricultural Economics, 2124 TAMU, Texas A& amp; M University, College Station, Texas, 77843 (United States)


    This research examines in detail the technology and economics of substituting biodiesel for diesel number 2. This endeavor examines three areas. First, the benefits of biodiesel are examined, and the technical problems of large-scale implementation. Second, the biodiesel production possibilities are examined for soybean oil, corn oil, tallow, and yellow grease, which are the largest sources of feedstocks for the United States. Examining in detail the production possibilities allows to identity the extent of technological change, production costs, byproducts, and greenhouse gas (GHG) emissions. Finally, a U.S. agricultural model, FASOMGHG was used to predict market penetration of biodiesel, given technological progress, variety of technologies and feedstocks, market interactions, energy prices, and carbon dioxide equivalent prices. FASOMGHG has several interesting results. First, diesel fuel prices have an expansionary impact on the biodiesel industry. The higher the diesel fuel prices, the more biodiesel is produced. However, given the most favorable circumstances, the maximum biodiesel market penetration is 9% in 2030 with a wholesale diesel price of $4 per gallon. Second, the two dominant sources of biodiesel are from corn and soybeans. Sources like tallow and yellow grease are more limited, because they are byproducts of other industries. Third, GHG prices have an expansionary impact on the biodiesel prices, because biodiesel is quite GHG efficient. Finally, U.S. government subsidies on biofuels have an expansionary impact on biodiesel production, and increase market penetration at least an additional 3%.

  10. An Overview of Penetration Testing



    Penetration testing is a series of activities undertaken to identify and exploit security vulnerabilities. It helps confirm the effectiveness or ineffectiveness of the security measures that have been implemented. This paper provides an overview of penetration testing. It discusses the benefits, the strategies and the methodology of conducting penetration testing. The methodology of penetration testing includes three phases: test preparation, test and test analysis. The test phase involves ...

  11. Microbial Penetration through Nutrient-Saturated Berea Sandstone. (United States)

    Jenneman, G E; McInerney, M J; Knapp, R M


    Penetration times and penetration rates for a motile Bacillus strain growing in nutrient-saturated Berea sandstone cores were determined. The rate of penetration was essentially independent of permeabilities above 100 mdarcys and rapidly declined for permeabilities below 100 mdarcys. It was found that these penetration rates could be grouped into two statistically distinct classes consisting of rates for permeabilities above 100 mdarcys and rates for those below 100 mdarcys. Instantaneous penetration rates were found to be zero order with respect to core length for cores with permeabilities above 100 mdarcys and first order with respect to core length for cores with permeabilities below 100 mdarcys. The maximum observed penetration rate was 0.47 cm . h, and the slowest was 0.06 cm . h; however, these rates may be underestimates of the true penetration rate, since the observed rates included the time required for growth in the flask as well as the core. The relationship of penetration time to the square of the length of the core suggested that cells penetrated high-permeability cores as a band and low-permeability cores in a diffuse fashion. The motile Enterobacter aerogenes strain penetrated Berea sandstone cores three to eight times faster than did the nonmotile Klebsiella pneumoniae strain when cores of comparable length and permeability were used. A penetration mechanism based entirely on motility predicted penetration times that were in agreement with the observed penetration times for motile strains. The fact that nonmotile strains penetrated the cores suggested that filamentous or unrestricted growth, or both, may also be important.

  12. An Overview of Penetration Testing

    Directory of Open Access Journals (Sweden)

    Aileen G. Bacudio


    Full Text Available Penetration testing is a series of activities undertaken to identify and exploit security vulnerabilities. It helps confirm the effectiveness or ineffectiveness of the security measures that have been implemented. This paper provides an overview of penetration testing. It discusses the benefits, the strategies and the methodology of conducting penetration testing. The methodology of penetration testing includes three phases: test preparation, test and test analysis. The test phase involves the following steps: information gathering, vulnerability analysis, and vulnerability exploit. This paper further illustrates how to apply this methodology to conduct penetration testing on two example web applications.

  13. Cell-penetrating Peptide YARA-mediated Transduction of Enhanced Green Fluorescent Protein into Human Vascular Smooth Muscle Cells%YARA介导增强型绿色荧光蛋白穿透人血管平滑肌细胞

    Institute of Scientific and Technical Information of China (English)

    陈思思; 王家宁; 黄永章; 郭凌郧; 孔霞


    Objective To investigate the penetrating ability of fusion protein YARA-EGFP with human vascular smooth muscle cells (HVSMC). Methods The prokaryotic expression plastnids YARA-EGFP was constructed and transformed into E. coli BL21 (DE3) to express fusion protein YARA-EGFP. The fusion protein YARA-EGFP was purified with Ni2 + -resin affinity chromatography and transduced into HVSMC. Results YARA-EGFP fusion protein could transduce into HVSMC and distribute in cytoplasm and nucleus after 6 h incubation. Conclusion The successful expression and purification of YARA-EGFP fusion protein can transduce into human vascular smooth muscle cells. This study provides a basis for the research on transduction of antiproliferative proteins such as p27 and p21 mediated by the cell-penetrating peptide, YARA,in protein therapy for the diabetic vascular diseases.%目的:研究细胞穿透肽YARA介导大分子蛋白穿透人血管平滑肌细施(human vascular smooth musle cells,HVSMC)细胞膜的能力.方法:用基因工程的方法制备并纯化YARA-EGFP融合蛋白,将其和培养的人平滑肌细胞共同孵育,在荧光显微镜下直接观察YARA介导目的蛋白EGFP转导入人平滑肌细胞的能力.结果:荧光显微镜下观察到,荧光蛋白穿透细胞膜进入并分布在入血管平滑肌细胞内.结论:YARA能有效携带目的蛋白进入人血管平滑肌细胞,这为将来用细胞穿透肽YARA介导有生物活性的大分子抗血管平滑肌细胞增殖,进行糖尿病血管病变的蛋白治疗奠定了基础.

  14. Enhanced chlorhexidine skin penetration with eucalyptus oil

    Directory of Open Access Journals (Sweden)

    Worthington Tony


    Full Text Available Abstract Background Chlorhexidine digluconate (CHG is a widely used skin antiseptic, however it poorly penetrates the skin, limiting its efficacy against microorganisms residing beneath the surface layers of skin. The aim of the current study was to improve the delivery of chlorhexidine digluconate (CHG when used as a skin antiseptic. Method Chlorhexidine was applied to the surface of donor skin and its penetration and retention under different conditions was evaluated. Skin penetration studies were performed on full-thickness donor human skin using a Franz diffusion cell system. Skin was exposed to 2% (w/v CHG in various concentrations of eucalyptus oil (EO and 70% (v/v isopropyl alcohol (IPA. The concentration of CHG (μg/mg of skin was determined to a skin depth of 1500 μm by high performance liquid chromatography (HPLC. Results The 2% (w/v CHG penetration into the lower layers of skin was significantly enhanced in the presence of EO. Ten percent (v/v EO in combination with 2% (w/v CHG in 70% (v/v IPA significantly increased the amount of CHG which penetrated into the skin within 2 min. Conclusion The delivery of CHG into the epidermis and dermis can be enhanced by combination with EO, which in turn may improve biocide contact with additional microorganisms present in the skin, thereby enhancing antisepsis.

  15. Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

    Energy Technology Data Exchange (ETDEWEB)

    Cuneo, Kyle C. [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Mito, Jeffrey K.; Javid, Melodi P. [Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States); Ferrer, Jorge M. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Kim, Yongbaek [Department of Clinical Pathology, College of Veterinary Medicine, Seoul National University, Seoul (Korea, Republic of); Lee, W. David [The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Bawendi, Moungi G. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Brigman, Brian E. [Department of Orthopedic Surgery, Duke University School of Medicine, Durham, North Carolina (United States); Kirsch, David G., E-mail: [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States)


    Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

  16. Penetration Testing: A Review

    Directory of Open Access Journals (Sweden)

    Kumar Shravan


    Full Text Available : Network Security is becoming more and more crucial as the volume of data being exchanged on the Internet increases. When people use Internet, they have certain expectations. They expect confidentiality, data integrity and authentication (CIA Standards. In 1980’s most organizations had only a physical boundary that needed protection of the assets. Today, due to the changes in the way resources are made available Organizations are forced to verify that their assets are protected from both the external and internal threats that our working environment has enabled. Due to the increasing dependency of our society on networked information system the overall security of these systems should be measured and improved. The most accurate method to evaluate organization’s information security stance is to observe how it stands up against an attack. Network administrators have often tried their best by improving their network security, however with rapid surface of new exploits; the best way of ensuring that the system is secure is to attempt penetration testing. This would be the most effective way to find exploits and to proof whether a system is vulnerable. Penetration test ing often allows the security analyst to find new vulnerabilities.

  17. Penetration Dynamics of Earth Penetration Warhead into Composite Target Media

    Directory of Open Access Journals (Sweden)

    P. K. Roy


    Full Text Available Attempts have been made to develop a suitable computer code that can find solutions to the axi-symmetric penetration of an Earth Penetrating Warhead yielding complete space-time histories of the resistive force offered by the target medium. The consequent warhead deceleration and velocity reduction, the resulting axial compressive stress developed in warhead casing as the penetration process progresses into the composite target media consisting of hard concrete of specified thickness followed by earth soil have been discussed.

  18. Cancer theranostics with near-infrared light-activatable multimodal nanoparticles. (United States)

    Melancon, Marites P; Zhou, Min; Li, Chun


    Nanomaterials that interact with light provide a unique opportunity for applications in biophotonic nanomedicine. Image-guided therapies could be designed based on multifunctional nanoparticles (NPs). Such NPs have a strong and tunable surface plasmon resonance absorption in the near-infrared region and can be detected using multiple imaging modalities (magnetic resonance imaging, nuclear imaging, and photoacoustic imaging). These novel nanostructures, once introduced, are expected to home in on solid tumors either via a passive targeting mechanism (i.e., the enhanced permeability and retention effect) or via an active targeting mechanism facilitated by ligands bound to their surfaces. Once the NPs reach their target tissue, their activity can then be turned on using an external stimulus. For example, photothermal conducting NPs primarily act by converting light energy into heat. As a result, the temperature in the treatment volume is elevated above the thermal damage threshold, which kills the cells. This process, termed photothermal ablation therapy (PTA), is effective, but it is also unlikely to kill all tumor cells when used alone. In addition to PTA, photothermal conducting NPs can also efficiently trigger the release of drugs and activate RNA interference. A multimodal approach, which permits simultaneous PTA therapy, chemotherapy, and therapeutic RNA interference, has the potential to completely eradicate residual diseased cells. In this Account, we provide an up-to-date review of the synthesis and characterization, functionalization, and in vitro and in vivo evaluation of NIR lightactivatable multifunctional nanostructures used for imaging and therapy. We emphasize research on hollow gold nanospheres, magnetic core-shell gold nanoshells, and semiconductor copper monosulfide NPs. We discuss three types of novel drug delivery systems in which hollow gold nanospheres are used to mediate controlled drug release.

  19. Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins. (United States)

    Kobayashi, Tomonori; Komatsu, Toru; Kamiya, Mako; Campos, Cláudia; González-Gaitán, Marcos; Terai, Takuya; Hanaoka, Kenjiro; Nagano, Tetsuo; Urano, Yasuteru


    Optical highlighters are photoactivatable fluorescent molecules that exhibit pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. Here, we present a novel design strategy for a new class of caged BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophores, based on the use of photoremovable protecting groups (PRPGs) with high reduction potentials that serve as both a photosensitive unit and a fluorescence quencher via photoinduced electron transfer (PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated, with activation ratios exceeding 600-fold in aqueous solutions. We then combined this photoactivatable fluorophore with a SNAP (mutant of O(6)-alkylguanine DNA alkyltransferase) ligand to obtain a small-molecule-based optical highlighter for visualization of protein dynamics, using the well-established SNAP tag technology. As proof of concept, we demonstrate spatiotemporal imaging of the fusion protein of epidermal growth factor receptor (EGFR) with SNAP tag in living cells. We also demonstrate highlighting of cells of interest in live zebrafish embryos, using the fusion protein of histone 2A with SNAP tag.

  20. Localizing Ground-Penetrating Radar (United States)


    ing Ground-Penetrating Radar (LGPR) uses very high frequency (VHF) radar reflections of underground features to generate base- line maps and then...Innovative ground- penetrating radar that maps underground geological features provides autonomous vehicles with real-time localization. Localizing...NOV 2014 2. REPORT TYPE 3. DATES COVERED 00-00-2014 to 00-00-2014 4. TITLE AND SUBTITLE Localizing Ground-Penetrating Radar 5a. CONTRACT NUMBER

  1. Penetration Tester's Open Source Toolkit

    CERN Document Server

    Faircloth, Jeremy


    Great commercial penetration testing tools can be very expensive and sometimes hard to use or of questionable accuracy. This book helps solve both of these problems. The open source, no-cost penetration testing tools presented do a great job and can be modified by the user for each situation. Many tools, even ones that cost thousands of dollars, do not come with any type of instruction on how and in which situations the penetration tester can best use them. Penetration Tester's Open Source Toolkit, Third Edition, expands upon existing instructions so that a professional can get the most accura

  2. Physical Penetration Testing: A Whole New Story in Penetration Testing

    NARCIS (Netherlands)

    Dimkov, Trajce; Pieters, Wolter


    Physical penetration testing plays an important role in assuring a company that the security policies are properly enforced and that the security awareness of the employees is on the appropriate level. In physical penetration tests the tester physically enters restricted locations and directly inter

  3. Membrane damage as first and DNA as the secondary target for anti-candidal activity of antimicrobial peptide P7 derived from cell-penetrating peptide ppTG20 against Candida albicans. (United States)

    Li, Lirong; Song, Fengxia; Sun, Jin; Tian, Xu; Xia, Shufang; Le, Guowei


    P7, a peptide analogue derived from cell-penetrating peptide ppTG20, possesses antibacterial and antitumor activities without significant hemolytic activity. In this study, we investigated the antifungal effect of P7 and its anti-Candida acting mode in Candida albicans. P7 displayed antifungal activity against the reference C. albicans (MIC = 4 μM), Aspergilla niger (MIC = 32 μM), Aspergillus flavus (MIC = 8 μM), and Trichopyton rubrum (MIC = 16 μM). The effect of P7 on the C. albicans cell membrane was examined by investigating the calcein leakage from fungal membrane models made of egg yolk l-phosphatidylcholine/ergosterol (10 : 1, w/w) liposomes. P7 showed potent leakage effects against fungal liposomes similar to Melittin-treated cells. C. albicans protoplast regeneration assay demonstrated that P7 interacted with the C. albicans plasma membrane. Flow cytometry of the plasma membrane potential and integrity of C. albicans showed that P7 caused 60.9 ± 1.8% depolarization of the membrane potential of intact C. albicans cells and caused 58.1 ± 3.2% C. albicans cell membrane damage. Confocal laser scanning microscopy demonstrated that part of FITC-P7 accumulated in the cytoplasm. DNA retardation analysis was also performed, which showed that P7 interacted with C. albicans genomic DNA after penetrating the cell membrane, completely inhibiting the migration of genomic DNA above the weight ratio (peptide : DNA) of 6. Our results indicated that the plasma membrane was the primary target, and DNA was the secondary intracellular target of the mode of action of P7 against C. albicans. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  4. Failure Engineered Heavy Metal Penetrators (United States)


    ARMY RESEARCH LABORATORY Failure Engineered Heavy Metal Penetrators, Phase I, SBIR ARL-CR-5· R. Cavalieri, W. Tiarn, and D. Nicholson prepared...REPORT DATE S. REPORT TYPE AND DATES COVERED December 1992 Final Report-1/1/92 - 7/31/92 4. TITLE AND SUBTITLE FAILURE ENGINEERED HEAVY METAL PENETRATORS

  5. Top Sounder Ice Penetration (United States)

    Porter, D. L.; Goemmer, S. A.; Sweeney, J. H.


    Ice draft measurements are made as part of normal operations for all US Navy submarines operating in the Arctic Ocean. The submarine ice draft data are unique in providing high resolution measurements over long transects of the ice covered ocean. The data has been used to document a multidecadal drop in ice thickness, and for validating and improving numerical sea-ice models. A submarine upward-looking sonar draft measurement is made by a sonar transducer mounted in the sail or deck of the submarine. An acoustic beam is transmitted upward through the water column, reflecting off the bottom of the sea ice and returning to the transducer. Ice thickness is estimated as the difference between the ship's depth (measured by pressure) and the acoustic range to the bottom of the ice estimated from the travel time of the sonar pulse. Digital recording systems can provide the return off the water-ice interface as well as returns that have penetrated the ice. Typically, only the first return from the ice hull is analyzed. Information regarding ice flow interstitial layers provides ice age information and may possibly be derived with the entire return signal. The approach being investigated is similar to that used in measuring bottom sediment layers and will involve measuring the echo level from the first interface, solving the reflection loss from that transmission, and employing reflection loss versus impedance mismatch to ascertain ice structure information.

  6. Electromagnetic Field Penetration Studies (United States)

    Deshpande, M.D.


    A numerical method is presented to determine electromagnetic shielding effectiveness of rectangular enclosure with apertures on its wall used for input and output connections, control panels, visual-access windows, ventilation panels, etc. Expressing EM fields in terms of cavity Green's function inside the enclosure and the free space Green's function outside the enclosure, integral equations with aperture tangential electric fields as unknown variables are obtained by enforcing the continuity of tangential electric and magnetic fields across the apertures. Using the Method of Moments, the integral equations are solved for unknown aperture fields. From these aperture fields, the EM field inside a rectangular enclosure due to external electromagnetic sources are determined. Numerical results on electric field shielding of a rectangular cavity with a thin rectangular slot obtained using the present method are compared with the results obtained using simple transmission line technique for code validation. The present technique is applied to determine field penetration inside a Boeing-757 by approximating its passenger cabin as a rectangular cavity filled with a homogeneous medium and its passenger windows by rectangular apertures. Preliminary results for, two windows, one on each side of fuselage were considered. Numerical results for Boeing-757 at frequencies 26 MHz, 171-175 MHz, and 428-432 MHz are presented.

  7. An Earth Penetrating Modeling Assessment

    Energy Technology Data Exchange (ETDEWEB)

    Stokes, E; Yarrington, P; Glenn, L


    Documentation of a study to assess the capability of computer codes to predict lateral loads on earth penetrating projectiles under conditions of non-normal impact. Calculations simulated a set of small scale penetration tests into concrete targets with oblique faces at angles of 15 and 30 degrees to the line-of-flight. Predictive codes used by the various calculational teams cover a wide range of modeling approaches from approximate techniques, such as cavity expansion, to numerical methods, such as finite element codes. The modeling assessment was performed under the auspices of the Phenomenology Integrated Product Team (PIPT) for the Robust Nuclear Earth Penetrator Program (RNEP). Funding for the penetration experiments and modeling was provided by multiple earth penetrator programs.

  8. Stereoselective synthesis of light-activatable perfluorophenylazide-conjugated carbohydrates for glycoarray fabrication and evaluation of structural effects on protein binding by SPR imaging. (United States)

    Deng, Lingquan; Norberg, Oscar; Uppalapati, Suji; Yan, Mingdi; Ramström, Olof


    A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors proved especially efficient and provided the final compounds in up to 70% total yield with high anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The results indicate better performance and equal efficiency of S- and O-linked structures with intermediate linker length.

  9. Different patterns of nuclear and mitochondrial penetration by the G3 PAMAM dendrimer and its biotin–pyridoxal bioconjugate BC-PAMAM in normal and cancer cells in vitro

    Directory of Open Access Journals (Sweden)

    Uram Ł


    Full Text Available Łukasz Uram,1 Magdalena Szuster,1 Aleksandra Filipowicz,2 Krzysztof Gargasz,3 Stanisław Wołowiec,3 Elżbieta Wałajtys-Rode4 1Bioorganic Chemistry Laboratory, Faculty of Chemistry, Rzeszow University of Technology, 2Cosmetology Department, University of Information Technology and Management in Rzeszow, 3Institute of Nursery and Health Sciences, Faculty of Medicine, University of Rzeszow, Rzeszow, 4Department of Drug Technology and Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland Abstract: The intracellular localization and colocalization of a fluorescently labeled G3 amine-terminated cationic polyamidoamine (PAMAM dendrimer and its biotin–pyridoxal (BC-PAMAM bioconjugate were investigated in a concentration-dependent manner in normal human fibroblast (BJ and squamous epithelial carcinoma (SCC-15 cell lines. After 24 hours treatment, both cell lines revealed different patterns of intracellular dendrimer accumulation depending on their cytotoxic effects. Cancer cells exhibited much higher (20-fold tolerance for native PAMAM treatment than fibroblasts, whereas BC-PAMAM was significantly toxic only for fibroblasts at 50 µM concentration. Fibroblasts accumulated the native and bioconjugated dendrimers in a concentration-dependent manner at nontoxic range of concentration, with significantly lower bioconjugate loading. After reaching the cytotoxicity level, fluorescein isothiocyanate-PAMAM accumulation remains at high, comparable level. In cancer cells, native PAMAM loading at higher, but not cytotoxic concentrations, was kept at constant level with a sharp increase at toxic concentration. Mander’s coefficient calculated for fibroblasts and cancer cells confirmed more efficient native PAMAM penetration as compared to BC-PAMAM. Significant differences in nuclear dendrimer penetration were observed for both cell lines. In cancer cells, PAMAM signals amounted to ~25%–35% of the total nuclei area at all

  10. Physical Penetration Testing: A Whole New Story in Penetration Testing



    Physical penetration testing plays an important role in assuring a company that the security policies are properly enforced and that the security awareness of the employees is on the appropriate level. In physical penetration tests the tester physically enters restricted locations and directly interacts with the employees to convince them to break a policy or provide credentials. The physical access and the direct interaction with the employees complicate the execution of the tests and have e...

  11. Percutaneous penetration through slightly damaged skin

    DEFF Research Database (Denmark)

    Nielsen, Jesper B


    Guidelines for experimental studies of percutaneous penetration prescribe optimal barrier integrity of the skin. The barrier integrity of the skin exposed in occupational or household situations is, however, not always ideal, and skin problems are among the most dominant reasons for absence from...... work. We have therefore evaluated an experimental model for percutaneous penetration through slightly damaged skin. The influence of a slight damage to the skin was evaluated using five pesticides covering a wide range of solubilities. We used an experimental model with static diffusion cells mounted...... with human skin. A slight damage to the barrier integrity was induced by pre-treatment of the skin with sodium lauryl sulphate (SLS) before pesticide exposure. The experimental model with 3 h pre-treatment with SLS (0.1% or 0.3%) assured a significant but controlled damage to the barrier integrity, a damage...

  12. Cement penetration after patella venting. (United States)

    Jones, Christopher W; Lam, Li-On; Butler, Adam; Wood, David J; Walsh, William R


    There is a high rate of patellofemoral complications following total knee arthroplasty. Optimization of the cement-bone interface by venting and suction of the tibial plateau has been shown to improve cement penetration. Our study was designed to investigate if venting the patella prior to cementing improved cement penetration. Ten paired cadaver patellae were allocated prior to resurfacing to be vented or non-vented. Bone mineral density (BMD) was measured by DEXA scanning. In vented specimens, a 1.6 mm Kirschner wire was used to breach the anterior cortex at the center. Specimens were resurfaced with standard Profix instrumentation and Versabond bone cement (Smith and Nephew PLC, UK). Cement penetration was assessed from Faxitron and sectioned images by a digital image software package (ImageJ V1.38, NIH, USA). Wilcoxon rank sum test was used to assess the difference in cement penetration between groups. The relationship between BMD and cement penetration was analyzed by Pearson correlation coefficient. There was a strong negative correlation between peak BMD and cement penetration when analyzed independent of experimental grouping (r(2)=-0.812, p=0.004). Wilcoxon rank sum testing demonstrated no significant difference (rank sum statistic W=27, p=0.579) in cement penetration between vented (10.53%+/-4.66; mean+/-std dev) and non-vented patellae (11.51%+/-6.23; mean+/-std dev). Venting the patella using a Kirschner wire does not have a significant effect on the amount of cement penetration achieved in vitro using Profix instrumentation and Versabond cement.

  13. Novel approach for characterizing the specific shunt resistance caused by the penetration of the front contact through the p-n junction in solar cell

    Institute of Scientific and Technical Information of China (English)

    Zhang Lucheng; Shen Hui


    on process was completely compatible with the industrial silicon fabrication sequence, which was of great convenience. The measurement results give informations on the solar cell structure, material ingredients, and process parameters.

  14. Ultrastructural study of adherence to and penetration of cultured cells by two invasive Escherichia coli strains isolated from infants with enteritis.



    The adherence of invasive Escherichia coli strains 444-3 and 469-3 to human erythrocytes and to cultured HeLa and HEp-2 cells has been examined by electron microscopy. Bacteria elaborating type 1 fimbriae, glycocalyces , and nonfimbrial mannose-resistant hemagglutinins specific for human erythrocytes were identified in cultures of both strains, and each of these different bacterial surface components appeared to be involved in attachment of 444-3 and 469-3 to cultured epithelial cells or huma...

  15. Liposome surface charge influence on skin penetration behaviour. (United States)

    Gillet, A; Compère, P; Lecomte, F; Hubert, P; Ducat, E; Evrard, B; Piel, G


    Vesicular systems have shown their ability to increase dermal and transdermal drug delivery. Their mechanism of drug transport into and through the skin has been investigated but remains a much debated question. Several researchers have outlined that drug penetration can be influenced by modifying the surface charge of liposomes. In the present work we study the influence of particle surface charge on skin penetration. The final purpose is the development of a carrier system which is able to enhance the skin delivery of two model drugs, betamethasone and betamethasone dipropionate. Liposomes were characterised by their size, morphology, zeta potential, encapsulation efficiency and stability. Ex vivo diffusion studies using Franz diffusion cells were performed. Confocal microscopy was performed to visualise the penetration of fluorescently labelled liposomes into the skin. This study showed the potential of negatively charged liposomes to enhance the skin penetration of betamethasone and betamethasone dipropionate.

  16. Case Report: Penetrating Cardiac Injury

    Directory of Open Access Journals (Sweden)

    Adem Grbolar


    Full Text Available Summary: Penetrating cardiac injurys caused by gunshots and penetrating tools have high mortality rates. The way of injury, how the cardiac area is effected and the presence of cardiac tamponadecauses mortality in different rates. However the better treatment quality of hospitals, increasingoperative techniques, and internel care unit quality has not been change during the years. Searching the literature, we want to present a 42 years old male patient whowas injured by knife and had a 1 cm skin wound on chest with cardiac tamponade. After sternotomy a 7 cm laseration was observed in heart. Cardioraphy was performed.

  17. Stylet penetration activities by aphids

    NARCIS (Netherlands)

    Tjallingii, W.F.


    The composition of stylet penetration behaviour in aphids and its possible role in food-plant selection is the subject of these studies. Evidence is presented that the labium is devoid of external chemoreceptors (Chapter 1). In addition to other morphological data (Wensler & Filshie, 1969; Wensler,

  18. Penetration Physics of Armor Glass (United States)


    Penetration Response of Borosilicate Glass during Short Rod Impact”, Proc. 23rd Int. Symp. Ballistics, 2, 1251-1258, Graficas Couche, Madrid, Spain (”, Proc. 23rd Int. Symp. Ballistics, 2, 1049-1056, Graficas Couche, Madrid, Spain (2007). 8D. R. Curran, “Comparison of Mesomechanical and

  19. Simulation of laser penetration efficiency (United States)

    Semak, V. V.; Miller, T. F.


    The results of numerical simulation of laser beam interaction with a hypothetical metallic material with properties similar to a steel alloy are reported. The numerical simulation was performed using a physical model that includes detailed consideration of surface evaporation, evaporative cooling of the surface and evaporation recoil induced melt ejection. The laser beam ‘penetration’ is considered in terms of melting through the sample or drilling through the sample due to both evaporation and recoil ejection of material. As a demonstration of the predictive capabilities of the model, the average velocity of penetration through a material with steel-like properties is numerically predicted for various laser interaction parameters such as, laser beam radius, laser pulse duration (including CW regime), laser pulse energy and pulse repetition. In particular, the average penetration velocities through a sample due to melting are compared for pulsed and CW lasers of the same power. For the sake of another demonstration of penetration simulation, the temporal dynamics of the position of melt front relative to the sample surface irradiated by a laser beam was computed for different laser pulse repetition rates and constant average laser power. An illustration of the penetration efficiency (W parameter) defined as the amount of energy per unit volume delivered into a target in order to achieve either melting of drilling through a target wall is shown in a wide range of laser pulse parameters covering regimes corresponding to domination of melting through and drilling through.

  20. FAA Fluorescent Penetrant Laboratory Inspections

    Energy Technology Data Exchange (ETDEWEB)



    The Federal Aviation Administration Airworthiness Assurance NDI Validation Center currently assesses the capability of various non-destructive inspection (NDI) methods used for analyzing aircraft components. The focus of one such exercise is to evaluate the sensitivity of fluorescent liquid penetrant inspection. A baseline procedure using the water-washable fluorescent penetrant method defines a foundation for comparing the brightness of low cycle fatigue cracks in titanium test panels. The analysis of deviations in the baseline procedure will determine an acceptable range of operation for the steps in the inspection process. The data also gives insight into the depth of each crack and which step(s) of the inspection process most affect penetrant sensitivities. A set of six low cycle fatigue cracks produced in 6.35-mm thick Ti-6Al-4V specimens was used to conduct the experiments to produce sensitivity data. The results will document the consistency of the crack readings and compare previous experiments to find the best parameters for water-washable penetrant.

  1. The Penetration of Shaped Charges

    Institute of Scientific and Technical Information of China (English)

    Wu Mingde


    @@ In order to get the best perforating effect, many facts have to be considered. Perforating job is often design by computer. A perforating engineer has to decide the perforating technology and procedure, the gun and charge system, the shot density and phasing, the penetration and hole size, etc.

  2. Association between the Thr325Ile polymorphism of the thrombin-activatable fibrinolysis inhibitor and stroke in the Ludwigshafen Risk and Cardiovascular Health Study. (United States)

    Kozian, Detlef H; Lorenz, Martin; März, Winfried; Cousin, Emmanuelle; Mace, Sandrine; Deleuze, Jean-Francois


    The thrombin-activatable fibrinolysis inhibitor (TAFI) is a key mediator in the regulation of endogenous fibrinolysis, down-regulating clot lysis by degrading the C-terminal lysine residues from fibrin, which are important for binding and activating plasminogen. Elevated TAFI antigen levels have been suggested to be associated with promoter variants and the Ala147Thr polymorphism; increased TAFI stability and antifibrinolytic potential instead have been associated with the Thr325Ile polymorphism. We investigated the influence of these two polymorphisms on cardiovascular and thrombotic events in patients of the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. The LURIC study is a prospective cohort study comprising more than 3,300 patients aimed at identifying biochemical and genetic markers for metabolic and cardiovascular diseases. We demonstrate that the Ile/Ile genotype at position 325 of TAFI associates with the incidence of stroke and the age at onset of first stroke in patients of the LURIC cohort. Both the incidence of stroke and the risk of a premature event are higher in TAFI Ile325Ile patients with predisposing risk factors for thrombotic events such as diabetes mellitus, myocardial infarction or hypertension, alone or in combination. In contrast, no significant association was identified for the TAFI Ala147Thr polymorphism. The robust association of the TAFI Thr325Ile polymorphism with the incidence and the age at onset of first stroke strongly suggests a key role for TAFI in the pathogenetic mechanism of stroke.

  3. The combined transduction of copper, zinc-superoxide dismutase and catalase mediated by cell-penetrating peptide, PEP-1, to protect myocardium from ischemia-reperfusion injury

    Directory of Open Access Journals (Sweden)

    Guo Ling-Yun


    Full Text Available Abstract Background Our previous studies indicate that either PEP-1-superoxide dismutase 1 (SOD1 or PEP-1-catalase (CAT fusion proteins protects myocardium from ischemia-reperfusion-induced injury in rats. The aim of this study is to explore whether combined use of PEP-1-SOD1 and PEP-1-CAT enhances their protective effects. Methods SOD1, PEP-1-SOD1, CAT or PEP-1-CAT fusion proteins were prepared and purified by genetic engineering. In vitro and in vivo effects of these proteins on cell apoptosis and the protection of myocardium after ischemia-reperfusion injury were measured. Embryo cardiac myocyte H9c2 cells were used for the in vitro studies. In vitro cellular injury was determined by the expression of lactate dehydrogenase (LDH. Cell apoptosis was quantitatively assessed with Annexin V and PI double staining by Flow cytometry. In vivo, rat left anterior descending coronary artery (LAD was ligated for one hour followed by two hours of reperfusion. Hemodynamics was then measured. Myocardial infarct size was evaluated by TTC staining. Serum levels of myocardial markers, creatine kinase-MB (CK-MB and cTnT were quantified by ELISA. Bcl-2 and Bax expression in left ventricle myocardium were analyzed by western blot. Results In vitro, PEP-1-SOD1 or PEP-1-CAT inhibited LDH release and apoptosis rate of H9c2 cells. Combined transduction of PEP-1-SOD1 and PEP-1-CAT, however, further reduced the LDH level and apoptosis rate. In vivo, combined usage of PEP-1-SOD1 and PEP-1-CAT produced a greater effect than individual proteins on the reduction of CK-MB, cTnT, apoptosis rate, lipoxidation end product malondialdehyde, and the infarct size of myocardium. Functionally, the combination of these two proteins further increased left ventricle systolic pressure, but decreased left ventricle end-diastolic pressure. Conclusion This study provided a basis for the treatment or prevention of myocardial ischemia-reperfusion injury with the combined usage of PEP-1-SOD1

  4. Energy spectra of He + ions penetrating thick biological targets (United States)

    Xia, Yueyuan; Tan, Chunyu; Mu, Yuguang; Wang, Ruijin; Zhang, Jianhua; Liu, Xiangdong; Liu, Jitian; Yu, Zhengliang


    Energy spectra of 500 keV-1MeV He + ion penetrating 50 μ m- 100 μ m thick seed coat of maize, fruit peel of grape and of tomato, are measured. The results indicate that these thick biological targets, as seen by the penetrating ions, are inhomogeneous, and there are open paths, along which the incident ions can penetrate the targets easily. While most of the incident ions are stopped in the targets, some of the penetrating ions only lose a small fraction of their initial incident energy. The penetration energy spectra show a pure electronic stopping feature. Transmission electron microscope (TEM) micrographs taken from these samples with thickness of 30 μ m indicate that 150 keV electron beam from the TEM can penetrate the thick samples to give very good images with clear contrast. The electronic structures of β-1,4 glucosan molecular chains, which is deemed as the most important constituent of the cell walls of seed coats and peels of fruits, are calculated to show the possible open-path directions which exist in biological samples.

  5. LeciPlex, invasomes, and liposomes: A skin penetration study. (United States)

    Shah, Sanket M; Ashtikar, Mukul; Jain, Ankitkumar S; Makhija, Dinesh T; Nikam, Yuvraj; Gude, Rajiv P; Steiniger, Frank; Jagtap, Aarti A; Nagarsenker, Mangal S; Fahr, Alfred


    The present study compares three vesicular systems, cationic LeciPlex, invasomes, and conventional liposomes for their ability to deliver drugs deep into the skin. Skin penetration ability of the three vesicular systems was studied for two drugs namely idebenone (antioxidant/anticancer) and azelaic acid (antiacne). All systems showed sizes in nanometer range with small polydispersity indices. Vesicular systems were characterized by CryoTEM studies to understand the differences in morphology of the vesicular systems. Ex vivo human skin penetration studies suggested a pattern in penetration of drugs in different layers of the skin: LeciPlex showed higher penetration for idebenone whereas invasomes showed higher penetration of azelaic acid. Ex vivo study using a fluorescent dye (DiI) was performed to understand the differences in the penetration behavior of the three vesicular systems on excised human skin. In vitro cytotoxicity studies on B16F10 melanoma cell lines revealed, when loaded with idebenone, LeciPlex formulations had the superior activity followed by invasomes and liposomes. In vitro antimicrobial study of azelaic acid loaded systems on Propionibacterium acne revealed high antimicrobial activity for DDAB leciplex followed by almost equal activity for invasomes and CTAB LeciPlex followed by liposomes. Whereas antiacne efficacy study in rats for azelaic acid loaded systems, invasomes exhibited the best antiacne efficacy followed by liposomes and LeciPlex.

  6. Akon - A Penetrator for Europa (United States)

    Jones, Geraint


    Jupiter's moon Europa is one of the most intriguing objects in our Solar System. This 2000km-wide body has a geologically young solid water ice crust that is believed to cover a global ocean of liquid water. The presence of this ocean, together with a source of heating through tidal forces, make Europa a conceivable location for extraterrestrial life. The science case for exploring all aspects of this icy world is compelling. NASA has selected the Europa Mission (formerly Europa Clipper) to study Europa in detail in the 2020s through multiple flybys, and ESA's JUICE mission will perform two flybys of the body in the 2030s. The US agency has extended to the European Space Agency an invitation to provide a contribution to their mission. European scientists interested in Europa science and exploration are currently organizing themselves, in the framework of a coordinated Europa M5 Inititative to study concurrently the main options for this ESA contribution, from a simple addition of individual instruments to the NASA spacecraft, to a lander to investigate Europa's surface in situ. A high speed lander - a penetrator - is by far the most promising technology to achieve this latter option within the anticipated mass constraints, and studies of such a hard lander, many funded by ESA, are now at an advanced level. An international team to formally propose an Europa penetrator to ESA in response to the anticipated ESA M5 call is growing. The working title of this proposal is Akon (Άκων), named after the highly accurate javelin gifted to Europa by Zeus in ancient Greek mythology. We present plans for the Akon penetrator, which would impact Europa's surface at several hundred metres per second, and travel up to several metres into the moon's subsurface. To achieve this, the penetrator would be delivered to the surface by a dedicated descent module, to be destroyed on impact following release of the penetrator above the surface. It is planned that the instruments to be

  7. Multi-targeting exploration of new 2-aminothiazolyl quinolones: Synthesis, antimicrobial evaluation, interaction with DNA, combination with topoisomerase IV and penetrability into cells. (United States)

    Cheng, Yu; Avula, Srinivasa Rao; Gao, Wei-Wei; Addla, Dinesh; Tangadanchu, Vijai Kumar Reddy; Zhang, Ling; Lin, Jian-Mei; Zhou, Cheng-He


    A series of new potentially multi-targeting antimicrobial 2-aminothiazolyl quinolones were designed, synthesized and characterized by (1)H NMR, (13)C NMR, IR, MS and HRMS spectra. Bioactive assay manifested that some of the prepared compounds showed moderate to good antibacterial and antifungal activities. Noticeably, compound 10f could effectively inhibit the growth of B. typhi and MRSA with MIC values of 1 and 8 μg/mL, respectively. Experimental results revealed that compound 10f was membrane-active and had the ability to rapidly kill the tested strains and effectively prevent the development of bacterial resistance. Moreover, this compound also exhibited low toxicity against L929 cells. Molecular docking indicated that compound 10f could bind with topoisomerase IV-DNA complexes through hydrogen bonds and hydrophobic interactions. Quantum chemical studies were also performed on 10f to understand the structural features essential for activity. The preliminary mechanism research suggested that compound 10f could intercalate into calf thymus DNA to form a steady supramolecular complex which might block DNA replication to exert the powerful bioactivities.

  8. 天麻营繁茎被蜜环菌侵染过程中细胞结构的变化%The Changes of Cell Structure in the Courses of Armillaria mellea Penetrating the Nutritional Stems of Gastrodia elata

    Institute of Scientific and Technical Information of China (English)



    目的研究天麻营繁茎被蜜环菌侵染后细胞结构的变化,及天麻整个生长期的营养来源。方法作天麻营繁茎连续纵切片结合横切片观察;在天麻生长期切断其与菌材连接的蜜环菌索,测量新生麻生长情况。结果蜜环菌索侵入天麻营繁茎后,分成多个分枝的菌丝通道,菌丝突破通道形成菌丝流,向外侵入皮层细胞形成菌丝结,向内直接侵入大型细胞被天麻消化作为营养;切断天麻与菌材连接的蜜环菌索,新生麻就停止生长。结论蜜环菌索侵入天麻营繁茎后,菌丝结、突破菌丝通道的菌丝流,及大型细胞等三层细胞层呈片状环周包围了整个营繁茎,菌丝通道是天麻整个生长期营养的补给线。%Objective To study the cell structure changes of Gastrodia elata after Armillaria mellea infection as well as the nutritional resource of this important medicinal plant. Method Observation of the serial sections on the nutritional stems of G.elata, and measure new corms of G.elata when cut off the old corms connection with rhizomorph of A. mellea. Result After G. elata nutritional stems infected by A.mellea, the rhizomorph of A. mellea separate into several hyphal layers and penetrate hyphal stream which infect into the cells of cortex layer in the direction of outside and infect directly into layer cells in the direction of inside, these hypha can be used the nutrition of G. elata the new corms will be stop growth if cut off the rhizomorph of A. mellea connected with G. elata. Conclusion After the rhizomorph of A. mellea infected G. elata nutritional stems, hyphal coils and the hyphal stream will breakthrough passage cells as well as large cells surround the whole nutritional stems, so the passage cells is the key nutritional resource in the whole growth period of G. elata.

  9. Assessing high wind energy penetration

    DEFF Research Database (Denmark)

    Tande, J.O.


    In order to convincingly promote installing wind power capacity as a substantial part of the energy supply system, a set of careful analyses must be undertaken. This paper applies a case study concentrated on assessing the cost/benefit of high wind energy penetration. The case study considers...... expanding the grid connected wind power capacity in Praia, the capital of Cape Verde. The currently installed 1 MW of wind power is estimated to supply close to 10% of the electric energy consumption in 1996. Increasing the wind energy penetration to a higher level is considered viable as the project...... with the existing wind power, supply over 30% of the electric consumption in 1996. Applying the recommended practices for estimating the cost of wind energy, the life-cycle cost of this 2.4 MW investment is estimated at a 7% discount rate and a 20 year lifetime to 0.26 DKK/kW h....

  10. IPMN penetration of the stomach. (United States)

    Nakano, Masakazu; Tominaga, Keiichi; Watanabe, Hidetaka; Kanke, Kazunari; Tamano, Masaya; Hiraishi, Hideyuki


    An 83-year old Japanese man was transferred to our hospital due to a 1-week history of melena and signs of disordered awareness. Esophagogastroduodenoscopy showed a villous tumor associated with massive white mucous discharge in the posterior wall of the gastric corpus, where pathologically identified mucin-producing epithelium with nuclear atypia had developed into a papillary form. An abdominal enhanced computed tomography scan demonstrated communication between the dilated main pancreatic duct and the gastric lumen. Based on these findings, we reached a diagnosis of gastric penetration by an intraductal papillary mucinous neoplasm (IPMN) of the main pancreatic duct. IPMN is partly characterized by expansive mucinous growth that may result in penetration into adjacent organs.

  11. In vitro human skin penetration model for organophosphorus compounds with different physicochemical properties. (United States)

    Thors, L; Koch, B; Koch, M; Hägglund, L; Bucht, A


    A flow-through diffusion cell was validated for in vitro human epidermal penetration studies of organophosphorus compounds (OPCs) applied by infinite dosing. By testing OPCs with similar molecular weight but different physicochemical properties, it was shown that hydrophilic and lipophilic properties are major determinants for the penetration rate. Lipophilic OPCs displayed maximum cumulative penetration in the 20-75% agent concentration range whereas the hydrophilic OPCs displayed maximum cumulative penetration at 10 or 20% agent concentration. Low penetration was observed for all agents at 1% agent concentration or when applied as neat agents. The impact of the receptor solution composition was evaluated by comparing the penetration using receptor solutions of different ratios of ethanol and water. For diluted OPCs, a high concentration of ethanol in the receptor solution significantly increased the penetration compared to lower concentrations. When OPCs were applied as neat agents, the composition of the receptor solution only affected the penetration for one of four tested compounds. In conclusion, the flow-through diffusion cell was useful for examining the penetration of OPCs through the epidermal membrane. It was also demonstrated that the penetration rates of OPCs are strongly influenced by dilution in water and the receptor fluid composition.

  12. Penetration Testing in Wireless Networks


    Lamichhane, Shree


    This thesis illustrates the security measures and mechanisms behind the encryption and decryption of data while transmitting data in a wireless network. Furthermore, this thesis describes and demonstrates several security threats in a wireless network that are widely experienced. It also explains shortly the evolution of the widely implemented IEEE 802.11 standard and its amendments. Kali Linux tools were used to perform a penetration test in a WPA secured test network. In-formation on th...

  13. Transconjunctival penetration of mitomycin C


    Velpandian T; Sihota Ramanjit; Sinha Ankur; Gupta Viney


    Aims: The study was performed to estimate transconjunctival penetration of mitomycin C (MMC) to Tenon′s tissue following application over the intact conjunctiva before routine trabeculectomy. Settings and Design: Institution-based case series. Materials and Methods: In 41 eyes of 41 patients, MMC (0.4 mg/ml for 3 min) was applied over the intact conjunctiva before beginning trabeculectomy. Tenon′s capsule directly beneath the site of application was excised during trabecule...

  14. Cardiac Penetrating Injuries and Pseudoaneurysm

    Institute of Scientific and Technical Information of China (English)

    CHEN Shifeng


    Objective To discuss the early diagnosis and treatment of cardiac penetrating injuries and pseudoaneurysm. Methods 18 cases of cardiac penetrating injuries, in which 2 cases were complicated with pseudoaneurysm, were diagnosed by emergency operation and color Doppler echocardiography between May 1973 and Dec. 2001 in our hospital. The basis for emergency operation is the injured path locating in cardiac dangerous zone, severe shock or pericardial tamponade. ResultsAmong 18 cases of this study, 17 cases underwent emergency operation. During the operation, 11 cases were found injured in right ventricle, 2 cases were found injured in right atrium, 1 case was found injured in pulmonary artery,4 cases were found injured in left ventricle, 2 cases were found complicated with pseudoaneurysm. 17cases underwent cardiac repair including 1 case of rupture of aneurysm. 1 case underwent elective aneurysm resection. In whole group, 15 cases survived(83.33% ), 3 cases died( 16.67%). The cause of death is mainly hemorrhagic shock. Conclusion Highly suspicious cardiac penetrating injuries or hemopericaridium should undergo direct operative exploration. Pseudoaneurysm should be resected early,which can prevent severe complications.

  15. Locating a buried earth penetrator

    Energy Technology Data Exchange (ETDEWEB)

    Caffey, T.W.H.


    The purpose of this work was to assist the recovery of a buried earth penetrator by locating the vertical projection of the penetator upon the surface within a horizontal radius error of one meter. The penetrator will carry a small coil which is driven by an alternating current to form a magnetic dipole. Five measurements of the magnetic field vector upon the surface of the earth are shown to be sufficient for determining not only the xyz-coordinates of the dipole, but also the orientation of the dipole axis. The theory, computation process, and field tests are comprehensively described. Results of 26 field tests with the dipole at 9 different combinations of location and orientation are given. Average radial and vertical location errors are 0.27 m and -0.05 m, respectively, while the mean errors in the tilt and orientation angles of the dipole axis are 3 degrees and 8 degrees, respectively. The results are applied to the design of a locating system for a Pershing II penetrator which contains a recessed, rear-mounted coil.

  16. Correlation Between Cone Penetration Rate And Measured Cone Penetration Parameters In Silty Soils

    DEFF Research Database (Denmark)

    Poulsen, Rikke; Nielsen, Benjaminn Nordahl; Ibsen, Lars Bo


    penetration tests with varying penetration rates conducted at a test site where the subsoil primary consists of sandy silt. It is shown how a reduced penetration rate influences the cone penetration measurements e.g. the cone resistance, pore pressure, and sleeve friction.......This paper shows, how a change in cone penetration rate affects the cone penetration measurements, hence the cone resistance, pore pressure, and sleeve friction in silty soil. The standard rate of penetration is 20 mm/s, and it is generally accepted that undrained penetration occurs in clay while...... drained penetration occurs in sand. When lowering the penetration rate, the soil pore water starts to dissipate and a change in the drainage condition is seen. In intermediate soils such as silty soils, the standard cone penetration rate may result in a drainage condition that could be undrained...

  17. The production process for water penetrated brick

    Institute of Scientific and Technical Information of China (English)



    Waste penetrated brick, which is a green building material with good water penetration, high strength, lower firing temperature, lower production cost, good appearance and good structure, can holding ground water lever. This article analysis the production process and related factor for water penetrated brick:proper particle size distribution, proper shaping method, proper press and proper firing can ensure to produce good quality water penetrated brick.

  18. Penetration Testing: A Roadmap to Network Security



    Network penetration testing identifies the exploits and vulnerabilities those exist within computer network infrastructure and help to confirm the security measures. The objective of this paper is to explain methodology and methods behind penetration testing and illustrate remedies over it, which will provide substantial value for network security Penetration testing should model real world attacks as closely as possible. An authorized and scheduled penetration testing will probably detected ...

  19. Ethical Dilemmas and Dimensions in Penetration Testing


    Faily, Sharmal; McAlaney, John; Iacob, C


    Penetration testers are required to attack systems to evaluate their security, but without engaging in unethical behaviour while doing so. Despite work on hacker values and studies into security practice, there is little literature devoted to the ethical pressures associated with penetration testing. This paper presents several ethical dilemmas and dimensions associated with penetration testing;\\ud these shed light on the ethical positions taken by Penetration testers, and help identify poten...

  20. Network Penetration Testing and Research (United States)

    Murphy, Brandon F.


    This paper will focus the on research and testing done on penetrating a network for security purposes. This research will provide the IT security office new methods of attacks across and against a company's network as well as introduce them to new platforms and software that can be used to better assist with protecting against such attacks. Throughout this paper testing and research has been done on two different Linux based operating systems, for attacking and compromising a Windows based host computer. Backtrack 5 and BlackBuntu (Linux based penetration testing operating systems) are two different "attacker'' computers that will attempt to plant viruses and or NASA USRP - Internship Final Report exploits on a host Windows 7 operating system, as well as try to retrieve information from the host. On each Linux OS (Backtrack 5 and BlackBuntu) there is penetration testing software which provides the necessary tools to create exploits that can compromise a windows system as well as other operating systems. This paper will focus on two main methods of deploying exploits 1 onto a host computer in order to retrieve information from a compromised system. One method of deployment for an exploit that was tested is known as a "social engineering" exploit. This type of method requires interaction from unsuspecting user. With this user interaction, a deployed exploit may allow a malicious user to gain access to the unsuspecting user's computer as well as the network that such computer is connected to. Due to more advance security setting and antivirus protection and detection, this method is easily identified and defended against. The second method of exploit deployment is the method mainly focused upon within this paper. This method required extensive research on the best way to compromise a security enabled protected network. Once a network has been compromised, then any and all devices connected to such network has the potential to be compromised as well. With a compromised

  1. Kali Linux wireless penetration testing essentials

    CERN Document Server

    Alamanni, Marco


    This book is targeted at information security professionals, penetration testers and network/system administrators who want to get started with wireless penetration testing. No prior experience with Kali Linux and wireless penetration testing is required, but familiarity with Linux and basic networking concepts is recommended.

  2. 7 CFR 2902.14 - Penetrating lubricants. (United States)


    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Penetrating lubricants. 2902.14 Section 2902.14... Items § 2902.14 Penetrating lubricants. (a) Definition. Products formulated to provide light lubrication..., will give a procurement preference for qualifying biobased penetrating lubricants. By that...

  3. In vitro dermal penetration study of carbofuran, carbosulfan, and furathiocarb

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Kwang-Hyeon [Department of Pharmacology, Inje University, College of Medicine, 614-735, Busan (Korea); Kim, Jeong-Han [School of Agricultural Biotechnology, Seoul National University, 441-744, Suwon (Korea)


    In this study, the dermal penetration rate of carbofuran, carbosulfan, and furathiocarb has been measured with rat abdominal skin using the static diffusion cell. The technical grades of three compounds were applied at different doses on skin surface mounted in static diffusion cell and incubated at 32 C for 48 h with shaking. The same procedures were carried out with furathiocarb EC (emulsifiable concentrate) and WP (wettable powder). At regular intervals, the receptor fluid in cell was sampled and analyzed by HPLC. Only carbofuran was found in carbosulfan- or furathiocarb-treated samples, suggesting they converted into carbofuran while passing through the skin layer. The quantity of insecticide penetrating skin increased with time and applied dose. The skin penetration rate increased with the water solubility of insecticides. The dermal penetration rates of carbofuran, furathiocarb, and carbosulfan were determined as 1.05 {mu}g/cm{sup 2} per h (r{sup 2}=0.991), 0.46 {mu}g/cm{sup 2} per h (r{sup 2}=0.984) and 0.14 {mu}g/cm{sup 2} per h (r{sup 2}=0.967), respectively. There was no significant difference in rate of skin penetration between furathiocarb EC (1.42 {mu}g/cm{sup 2} per h, r{sup 2}=0.988) and WP (1.35 {mu}g/cm{sup 2} per h, r{sup 2}=0.982), while furathiocarb technical grade showed a lower skin penetration rate. In vitro models may be used to predict percutaneous absorption and are useful in selecting safer formulations for field application of pesticide. (orig.)

  4. FAA Fluorescent Penetrant Activities - An Update

    Energy Technology Data Exchange (ETDEWEB)

    Moore, D.G.


    The Federal Aviation Administration's Airworthiness Assurance NDI Validation Center (AANC) is currently characterizing low cycle fatigue specimens that will support the needs of penetrant manufacturers, commercial airline industry and the Federal Aviation Administration. The main focus of this characterization is to maintain and enhance the evaluation of penetrant inspection materials and apply resources to support the aircraft community needs. This paper discusses efforts to-date to document the Wright Laboratory penetrant evaluation process and characterize penetrant brightness readings in the initial set of sample calibration panels using Type 1 penetrant.

  5. Web penetration testing with Kali Linux

    CERN Document Server

    Muniz, Joseph


    Web Penetration Testing with Kali Linux contains various penetration testing methods using BackTrack that will be used by the reader. It contains clear step-by-step instructions with lot of screenshots. It is written in an easy to understand language which will further simplify the understanding for the user.""Web Penetration Testing with Kali Linux"" is ideal for anyone who is interested in learning how to become a penetration tester. It will also help the users who are new to Kali Linux and want to learn the features and differences in Kali versus Backtrack, and seasoned penetration testers

  6. Penetration testing with Raspberry Pi

    CERN Document Server

    Muniz, Joseph


    If you are looking for a low budget, small form-factor remotely accessible hacking tool, then the concepts in this book are ideal for you. If you are a penetration tester who wants to save on travel costs by placing a low-cost node on a target network, you will save thousands by using the methods covered in this book. You do not have to be a skilled hacker or programmer to use this book. It will be beneficial to have some networking experience; however, it is not required to follow the concepts covered in this book.

  7. Penetration Testing: A Roadmap to Network Security

    CERN Document Server

    Naik, Nitin A; Khamitkar, Santosh D; Kalyankar, Namdeo V


    Network penetration testing identifies the exploits and vulnerabilities those exist within computer network infrastructure and help to confirm the security measures. The objective of this paper is to explain methodology and methods behind penetration testing and illustrate remedies over it, which will provide substantial value for network security Penetration testing should model real world attacks as closely as possible. An authorized and scheduled penetration testing will probably detected by IDS (Intrusion Detection System). Network penetration testing is done by either or manual automated tools. Penetration test can gather evidence of vulnerability in the network. Successful testing provides indisputable evidence of the problem as well as starting point for prioritizing remediation. Penetration testing focuses on high severity vulnerabilities and there are no false positive.

  8. In situ characterization of projectile penetration into sand targets (United States)

    Borg, John P.; Sable, Peter; Sandusky, Harold; Felts, Joshua


    This work presents the results from dynamic penetration experiments in which long rod projectiles were launched into Ottawa sand at velocities ranging from 90 m/s to 350 m/s. A unique aspect of these experiments was that the sand targets were visually accessible, which allowed for the penetration event recorded using high-speed digital photography. The images were processed using two different correlation methods. In addition, stress measurements of the transmitted waveforms were simultaneously collected from a piezoelectric load cell that was buried in the sand at various locations relative to the shot line. The results indicate that impact results in two waves: one similar to a detached bow shock and one near the projectile that forms force chains. Grains are damaged and broken by the force chains which allows the projectile to penetrate the target.

  9. Evaluation of nicotinamide microemulsion on the skin penetration enhancement. (United States)

    Boonme, Prapaporn; Boonthongchuay, Chalida; Wongpoowarak, Wibul; Amnuaikit, Thanaporn


    This study purposed to evaluate a microemulsion containing nicotinamide for its characteristics, stability, and skin penetration and retention comparing with a solution of nicotinamide in 2:1 mixture of water and isopropyl alcohol (IPA). The microemulsion system was composed of 1:1 mixture of Span80 and Tween80 as a surfactant mixture, isopropyl palmitate (IPP) as an oil phase, and 2:1 mixture of water and IPA as an aqueous phase. Nicotinamide microemulsion was prepared by dissolving the active in the aqueous phase before simply mixing with the other components. It was determined for its characteristics and stability under various conditions. The skin penetration and retention studies of nicotinamide microemulsion and solution were performed by modified Franz diffusion cells, using newborn pig skin as the membrane. The results showed that nicotinamide microemulsion could be obtained as clear yellowish liquid, was water-in-oil (w/o) type, possessed Newtonian flow, and exhibited physicochemical stability when kept at 4 °C and room temperature (≈30 ± 2 °C) during 3 months. From the skin penetration data, the microemulsion could enhance the skin penetration of nicotinamide comparing with the solution. Additionally, nicotinamide microemulsion could provide much higher amount of skin retention than that of skin penetration, resulting in suitability for a cosmeceutical product.

  10. Influence of massage and occlusion on the ex vivo skin penetration of rigid liposomes and invasomes

    DEFF Research Database (Denmark)

    Trauer, S.; Richter, H.; Kuntsche, Judith


    Liposomes are frequently described as drug delivery systems for dermal and transdermal applications. Recently, it has been shown that particulate substances penetrate effectively into hair follicles and that the follicular penetration depth can be increased by massaging the skin, which simulates...... the in vivo movement of hairs in the hair follicles. In the present study, massage was applied to skin mounted to Franz diffusion cells. By means of confocal laser scanning microscopy, the influence of massage and occlusion on the follicular penetration depths of rigid and flexible liposomes loaded...... with a hydrophilic and lipophilic dye was investigated. The application of massage increased follicular penetration significantly. Occlusion resulted in an increased follicular penetration depth only for rigid liposomes, whereas invasomes did not penetrate more effectively if occlusion was applied. The results...

  11. Blockade of Airway Inflammation by Kaempferol via Disturbing Tyk-STAT Signaling in Airway Epithelial Cells and in Asthmatic Mice


    Ju-Hyun Gong; Daekeun Shin; Seon-Young Han; Sin-Hye Park; Min-Kyung Kang; Jung-Lye Kim; Young-Hee Kang


    Asthma is characterized by bronchial inflammation causing increased airway hyperresponsiveness and eosinophilia. The interaction between airway epithelium and inflammatory mediators plays a key role in the asthmatic pathogenesis. The in vitro study elucidated inhibitory effects of kaempferol, a flavonoid found in apples and many berries, on inflammation in human airway epithelial BEAS-2B cells. Nontoxic kaempferol at ≤20  μ M suppressed the LPS-induced IL-8 production through the TLR4 activat...

  12. Effects of soap-water wash on human epidermal penetration. (United States)

    Zhu, Hanjiang; Jung, Eui-Chang; Phuong, Christina; Hui, Xiaoying; Maibach, Howard


    Skin decontamination is a primary interventional method used to decrease dermal absorption of hazardous contaminants, including chemical warfare agents, pesticides and industrial pollutants. Soap and water wash, the most common and readily available decontamination system, may enhance percutaneous absorption through the "wash-in effect." To understand better the effect of soap-water wash on percutaneous penetration, and provide insight to improving skin decontamination methods, in vitro human epidermal penetration rates of four C(14) -labeled model chemicals (hydroquinone, clonidine, benzoic acid and paraoxon) were assayed using flow-through diffusion cells. Stratum corneum (SC) absorption rates of these chemicals at various hydration levels (0-295% of the dry SC weights) were determined and compared with the results of the epidermal penetration study to clarify the effect of SC hydration on skin permeability. Results showed accelerated penetration curves of benzoic acid and paraoxon after surface wash at 30 min postdosing. Thirty minutes after washing (60 min postdosing), penetration rates of hydroquinone and benzoic acid decreased due to reduced amounts of chemical on the skin surface and in the SC. At the end of the experiment (90 min postdosing), a soap-water wash resulted in lower hydroquinone penetration, greater paraoxon penetration and similar levels of benzoic acid and clonidine penetration compared to penetration levels in the non-wash groups. The observed wash-in effect agrees with the enhancement effect of SC hydration on the SC chemical absorption rate. These results suggest SC hydration derived from surface wash to be one cause of the wash-in effect. Further, the occurrence of a wash-in effect is dependent on chemical identity and elapsed time between exposure and onset of decontamination. By reducing chemical residue quantity on skin surface and in the SC reservoir, the soap-water wash may decrease the total quantity of chemical absorbed in the

  13. The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures. (United States)

    Meloni, Bruno P; Craig, Amanda J; Milech, Nadia; Hopkins, Richard M; Watt, Paul M; Knuckey, Neville W


    Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to

  14. Cable Braid Electromagnetic Penetration Model.

    Energy Technology Data Exchange (ETDEWEB)

    Warne, Larry K. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Langston, William L. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Basilio, Lorena I. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States); Johnson, W. A. [Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)


    The model for penetration of a wire braid is rigorously formulated. Integral formulas are developed from energy principles and reciprocity for both self and transfer immittances in terms of potentials for the fields. The detailed boundary value problem for the wire braid is also setup in a very efficient manner; the braid wires act as sources for the potentials in the form of a sequence of line multipoles with unknown coefficients that are determined by means of conditions arising from the wire surface boundary conditions. Approximations are introduced to relate the local properties of the braid wires to a simplified infinite periodic planar geometry. This is used in a simplified application of reciprocity to be able to treat nonuniform coaxial geometries including eccentric interior coaxial arrangements and an exterior ground plane.

  15. Ethical hacking and penetration testing guide

    CERN Document Server

    Baloch, Rafay


    Requiring no prior hacking experience, Ethical Hacking and Penetration Testing Guide supplies a complete introduction to the steps required to complete a penetration test, or ethical hack, from beginning to end. You will learn how to properly utilize and interpret the results of modern-day hacking tools, which are required to complete a penetration test. The book covers a wide range of tools, including Backtrack Linux, Google reconnaissance, MetaGooFil, dig, Nmap, Nessus, Metasploit, Fast Track Autopwn, Netcat, and Hacker Defender rootkit. Supplying a simple and clean explanation of how to effectively utilize these tools, it details a four-step methodology for conducting an effective penetration test or hack.Providing an accessible introduction to penetration testing and hacking, the book supplies you with a fundamental understanding of offensive security. After completing the book you will be prepared to take on in-depth and advanced topics in hacking and penetration testing. The book walks you through each ...


    Institute of Scientific and Technical Information of China (English)

    王顺; 戚秀杰; 韩迪


    Objective To probe into the function mechanism of penetration therapy with head electrical acupuncture on Parkinson's disease.Methods Microinjection of 6-hydroxydopamin(6-OHDA)on the left corpus striatum was adopted to prepare rotation model of Parkinson's disease in rat.Penetration therapy with head electrical acupuncture was administered in treatment.Normal group,sham-operation group,model group and penetration therapy group were set up.①Immunohistochemical(IHC)method was used to test the morphology and count of positive cell of tyrosine hydroxylase(TH).②RT-PCR technology was used to detect the expression of nestin mRNA of neural stem cell(NSC).Results ①Compared with model group,in penetration therapy group,the expressions of TH-positive neurons in immune response were increased in areal densitv(AD),numerical density(ND)and integrating optic density(P<0.05).②Compared with model group,in penetration therapy group,the expression of nestin mRNA was increased(P<0.05).Conclusion Penetration therapy with head electrical acupuncture promotes the proliferation of endogenous neural stem cells in substantia nigra of rat model of Parkinson's disease.%目的:探讨头部电针透穴疗法治疗帕金森病的作用机制.方法:以6-羟基多巴胺左侧纹状体微量注射法制备偏侧帕金森病大鼠旋转模型,采用头部电针透穴疗法进行治疗,设立正常组、假手术组、模型组和透针组:①以免疫组织化学方法检测酪氨酸羟化酶阳性细胞的形态、数量;②以RT-PCR技术检测神经干细胞巢蛋白mRNA的表达.结果:①透针组与模型组比较络氨酸羟化酶免疫反应阳性神经元在面密度、数密度及积分光密度方面表达增加(P<0.05);②透针组与模型组比较巢蛋白mRNA表达增加(P<0.05).结论:头部电针透穴疗法能促进帕金森病模型大鼠黑质内源性神经干细胞的增殖.

  17. Respirator Testing Using Virus Aerosol: Comparison between Viability Penetration and Physical Penetration. (United States)

    Zuo, Zhili; Kuehn, Thomas H; Pui, David Y H


    Viability, fluorescence (particle volume), photometric, viral RNA, and particle number penetration of MS2 bacteriophage through filter media used in three different models of respirators were compared to better understand the correlation between viability and physical penetration. Although viability and viral RNA penetration were better represented by particle volume penetration than particle number penetration, they were several-fold lower than photometric penetration, which was partially due to the difference in virus survival between upstream and downstream aerosol samples. Results suggest that the current NIOSH photometer-based test method can be used as a quick means to roughly differentiate respirators with different performance against virus aerosols.

  18. A Multiple Scattering Theory for Proton Penetration

    Institute of Scientific and Technical Information of China (English)

    YANG Dai-Lun; WU Zhang-Wen; JIANG Steve-Bin; LUO Zheng-Ming


    @@ We extend the electron small-angle multiple scattering theory to proton penetration. After introducing the concept of narrow energy spectra, the proton energy loss process is included in the proton deep penetration theory. It precisely describes the whole process of proton penetration. Compared to the Monte Carlo method,this method maintains the comparable precision and possesses much higher computational efficiency. Thus, it shows the real feasibility of applying this algorithm to proton clinical radiation therapy.

  19. In-place HEPA filter penetration test

    Energy Technology Data Exchange (ETDEWEB)

    Bergman, W.; Wilson, K.; Elliott, J. [Lawrence Livermore National Lab., CA (United States)] [and others


    We have demonstrated the feasibility of conducting penetration tests on high efficiency particulate air (HEPA) filters as installed in nuclear ventilation systems. The in-place penetration test, which is designed to yield equivalent penetration measurements as the standard DOP efficiency test, is based on measuring the aerosol penetration of the filter installation as a function of particle size using a portable laser particle counter. This in-place penetration test is compared to the current in-place leak test using light scattering photometers for single HEPA filter installations and for HEPA filter plenums using the shroud method. Test results show the in-place penetration test is more sensitive than the in-place leak test, has a similar operating procedure, but takes longer to conduct. Additional tests are required to confirm that the in-place penetration test yields identical results as the standard dioctyl phthalate (DOP) penetration test for HEPA filters with controlled leaks in the filter and gasket and duct by-pass leaks. Further development of the procedure is also required to reduce the test time before the in-place penetration test is practical. 14 refs., 14 figs., 3 tabs.

  20. Developmental Exposure To 2,3,7,8 Tetrachlorodibenzo-p-Dioxin Attenuates Later-Life Notch1-Mediated T Cell Development and Leukemogenesis


    Ahrenhoerster, Lori S.; Leuthner, Tess C.; Tate, Everett R.; Lakatos, Peter A.; Laiosa, Michael D.


    Over half of T-cell acute lymphoblastic leukemia (T-ALL) patients have activating mutations in the Notch gene. Moreover, the contaminant 2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is a known carcinogen that mediates its toxicity through the aryl hydrocarbon receptor (AHR), and crosstalk between activated AHR and Notch signaling pathways has previously been observed. Given the importance of Notch signaling in thymocyte development and T-ALL disease progression, we hypothesized that the activat...

  1. Preparation of a new cell-penetrating peptides as gene vector%新型穿膜肽作为基因运输载体的实验研究

    Institute of Scientific and Technical Information of China (English)

    田菊; 王志刚; 任建丽; 张清凤


    目的 人工设计合成穿膜肽新序列(CGRLWMRWYSPWARRYGC),并于体外考察其作为基因载体的潜力.方法 采用Na_芴甲氧羰基(Fmoc)法固相合成穿膜肽新序列,并用异硫氰酸荧光素(Frrc)标记N端.高效液相色谱及质谱仪检测其纯度及相对分子质量,流式细胞仪及共聚焦显微镜观察其穿膜能力,荧光显微镜和流式细胞仪观察其携带增强型绿色荧光蛋白质粒( pEGFP)进入细胞表达情况,MTT评价穿膜肽与质粒复合物的细胞毒性,琼脂糖凝胶电泳检测穿膜肽与质粒的亲和力及对质粒的保护作用.结果 合成的穿膜肽纯度为96.13%,相对分子质量为2 348.42,流式细胞仪检测显示荧光阳性细胞平均约占98.81%,共聚焦显微镜可见荧光标记的穿膜肽分布于细胞内;穿膜肽携带质粒在人脐静脉内皮细胞中的表达率平均约为11.44%;不同电荷比的复合物对细胞的活性没有明显影响(P>0.05);琼脂糖凝胶电泳显示当穿膜肽与质粒的电荷比大于2:1时,质粒不能迁移出来,电荷比为4:1、8:1和16:1的复合物能够抵抗DNA酶Ⅰ的消化作用,以8:1最为明显.结论 人工设计合成的新型穿膜肽能够作为一种潜在的基因运输载体.%Objective To design and synthesize a new sequence of cell-penetrating peptide (CPP) , CGRLWMRWYSPWARRYGC, and to investigate its potentiality as gene vector in vitro. Method The new sequence of CPP was synthesized by the solid-phase synthetic method with florenl methyoxycarbonyl ami no acids. N-terminal fluorescein isothiocyanate-labeled peptide was also synthesized. The purity and molecular weight of CPP were determined using HPLC and MALDI-TOF-MS. The cell-penetrating activity was examined with flow cytometry and confocal laser scanning microscopy. Transfection effect of CPPs-mediated enhanced green fluores-cence protein plasmid (pEGFP) in HUVEC was observed using fluorescent microscopy and flow cytometry. The cytotoxicity of

  2. Intracellular Protein Delivery for Treating Breast Cancer (United States)


    Rationally designed non-covalent protein nanocapsules, incorporating copper -free “click chemistry” moieties, polyethylene glycol (PEG) units, redox...chain variable fragment and luteinizing hormone releasing hormone (LHRH) peptide , which result in specific and efficient accumulation within tumor...nanocapsules with specific cancer cell targeting ligands; Task 3. Preparing and testing of MMP activatable cell penetrating peptides (ACCPs)-coupled

  3. Influence of jet thrust on penetrator penetration when studying the structure of space object blanket

    Directory of Open Access Journals (Sweden)

    N. A. Fedorova


    Full Text Available The article presents the calculation-and-theory-based research results to examine the possibility for using the jet thrust impulse to increase a penetration depth of high-velocity penetrator modules. Such devices can be used for studies of Earth surface layer composition, and in the nearest future for other Solar system bodies too. Research equipment (sensors and different instruments is housed inside a metal body of the penetrator with a sharpened nose that decreases drag force in soil. It was assumed, that this penetrator is additionally equipped with the pulse jet engine, which is fired at a certain stage of penetrator motion into target.The penetrator is considered as a rigid body of variable mass, which is subjected to drag force and reactive force applied at the moment the engine fires. A drag force was represented with a binomial empirical law, and penetrator nose part was considered to be conical. The jet thrust force was supposed to be constant during its application time. It was in accordance with assumption that mass flow and flow rate of solid propellant combustion products were constant. The amount of propellant in the penetrator was characterized by Tsiolkovsky number Z, which specifies the ratio between the fuel mass and the penetrator structure mass with no fuel.The system of equations to describe the penetrator dynamics was given in dimensionless form using the values aligned with penetration of an equivalent inert penetrator as the time and penetration depth scales. Penetration dynamics of penetrator represented in this form allowed to eliminate the influence of penetrator initial mass and its cross-section diameter on the solution results. The lack of such dependency is convenient for comparing the calculation results since they hold for penetrators of various initial masses and cross-sections.To calculate the penetration a lunar regolith was taken as a soil material. Calculations were carried out for initial velocities of

  4. Influence of massage and occlusion on the ex vivo skin penetration of rigid liposomes and invasomes. (United States)

    Trauer, Sindy; Richter, Heike; Kuntsche, Judith; Büttemeyer, Rolf; Liebsch, Manfred; Linscheid, Michael; Fahr, Alfred; Schäfer-Korting, Monika; Lademann, Jürgen; Patzelt, Alexa


    Liposomes are frequently described as drug delivery systems for dermal and transdermal applications. Recently, it has been shown that particulate substances penetrate effectively into hair follicles and that the follicular penetration depth can be increased by massaging the skin, which simulates the in vivo movement of hairs in the hair follicles. In the present study, massage was applied to skin mounted to Franz diffusion cells. By means of confocal laser scanning microscopy, the influence of massage and occlusion on the follicular penetration depths of rigid and flexible liposomes loaded with a hydrophilic and lipophilic dye was investigated. The application of massage increased follicular penetration significantly. Occlusion resulted in an increased follicular penetration depth only for rigid liposomes, whereas invasomes did not penetrate more effectively if occlusion was applied. The results confirm that massage is an important tool for increasing follicular penetration in ex vivo studies using Franz diffusion cells. Occlusion may reduce the efficacy of follicular penetration depending on the specific liposomal preparation. Rigidity in particular appears to be a relevant parameter.

  5. Efficient Calculation of Earth Penetrating Projectile Trajectories (United States)


    CALCULATION OF EARTH PENETRATING PROJECTILE TRAJECTORIES by Daniel F . Youch September 2006 Thesis Advisor: Joshua Gordis... Daniel F . Youch 5. FUNDING NUMBERS 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Naval Postgraduate School Monterey, CA 93943-5000 8. PERFORMING...EFFICIENT CALCULATION OF EARTH PENETRATING PROJECTILE TRAJECTORIES Daniel F . Youch Lieutenant Commander, United States Navy B.S., Temple

  6. Penetrating Moral Education in English Teaching

    Institute of Scientific and Technical Information of China (English)



    Middle school students’ moral education is not ful y implemented or even ignored in the present English teaching,so in this paper,the importance of penetrating the cultivation of middle school students’ moral education in English teaching is analyzed,the strategies of penetrating moral education are discussed and the significance is also pointed out.

  7. Endoscopic biopsy: Duodenal ulcer penetrating into liver

    Institute of Scientific and Technical Information of China (English)

    Baybora Kircali; Tülay Saricam; Aysegul Ozakyol; Eser Vardareli


    @@ TO THE EDITOR We have read with interest the recent report by E Kayacetin and S Kayacetin of Gastric ulcer penetrating to liver diagnosed by endoscopic biopsy[1] since we diagnosed the duodenal ulcer which penetrated into liver similarly. This is a rather unusual case because of the fifth case in the literature and responding to medical therapy.

  8. Quantitative penetration testing with item response theory

    NARCIS (Netherlands)

    Arnold, Florian; Pieters, Wolter; Stoelinga, Mariëlle


    Existing penetration testing approaches assess the vulnerability of a system by determining whether certain attack paths are possible in practice. Thus, penetration testing has so far been used as a qualitative research method. To enable quantitative approaches to security risk management, including

  9. Quantitative penetration testing with item response theory

    NARCIS (Netherlands)

    Pieters, W.; Arnold, F.; Stoelinga, M.I.A.


    Existing penetration testing approaches assess the vulnerability of a system by determining whether certain attack paths are possible in practice. Therefore, penetration testing has thus far been used as a qualitative research method. To enable quantitative approaches to security risk management, in

  10. Quantitative penetration testing with item response theory

    NARCIS (Netherlands)

    Arnold, Florian; Pieters, Wolter; Stoelinga, Mariëlle


    Existing penetration testing approaches assess the vulnerability of a system by determining whether certain attack paths are possible in practice. Thus, penetration testing has so far been used as a qualitative research method. To enable quantitative approaches to security risk management, including

  11. Binding and interstitial penetration of liposomes within avascular tumor spheroids. (United States)

    Kostarelos, Kostas; Emfietzoglou, Dimitris; Papakostas, Alexandros; Yang, Wei-Hong; Ballangrud, Ase; Sgouros, George


    The liposomal delivery of cancer therapeutics, including gene therapy vectors, is an area of intense study. Poor penetration of liposomes into interstitial tumor spaces remains a problem, however. In this work, the penetration of different liposomal formulations into prostate carcinoma spheroids was examined. Spheroid penetration was assessed by confocal microscopy of fluorescently labeled liposomes. The impact of liposomal surface charge, mean diameter, lipid bilayer fluidity and fusogenicity on spheroid penetration was examined. A variety of different liposome systems relevant to clinical or preclinical protocols have been studied, including classical zwitterionic (DMPC:chol) and sterically stabilized liposomes (DMPC:chol:DOPE-PEG2000), both used clinically, and cationic liposomes (DMPC:DOPE:DC-chol and DOTAP), forming the basis of the vast majority of nonviral gene transfer vectors tested in various cancer trials. Surface interactions between strongly cationic vesicles and the tumor cells led to an electrostatically derived binding-site barrier effect, inhibiting further association of the delivery systems with the tumor spheroids (DMPC:DC-chol). However, inclusion of the fusogenic lipid DOPE and use of a cationic lipid of lower surface charge density (DOTAP instead of DC-chol) led to improvements in the observed intratumoral distribution characteristics. Sterically stabilized liposomes did not interact with the tumor spheroids, whereas small unilamellar classical liposomes exhibit extensive distribution deeper into the tumor volume. Engineering liposomal delivery systems with a relatively low charge molar ratio and enhanced fusogenicity, or electrostatically neutral liposomes with fluid bilayers, offered enhanced intratumoral penetration. This study shows that a delicate balance exists between the strong affinity of delivery systems for the tumor cells and the efficient penetration and distribution within the tumor mass, similar to previous work studying

  12. Mathematical Based Calculation of Drug Penetration Depth in Solid Tumors

    Directory of Open Access Journals (Sweden)

    Hamidreza Namazi


    Full Text Available Cancer is a class of diseases characterized by out-of-control cells’ growth which affect cells and make them damaged. Many treatment options for cancer exist. Chemotherapy as an important treatment option is the use of drugs to treat cancer. The anticancer drug travels to the tumor and then diffuses in it through capillaries. The diffusion of drugs in the solid tumor is limited by penetration depth which is different in case of different drugs and cancers. The computation of this depth is important as it helps physicians to investigate about treatment of infected tissue. Although many efforts have been made on studying and measuring drug penetration depth, less works have been done on computing this length from a mathematical point of view. In this paper, first we propose phase lagging model for diffusion of drug in the tumor. Then, using this model on one side and considering the classic diffusion on the other side, we compute the drug penetration depth in the solid tumor. This computed value of drug penetration depth is corroborated by comparison with the values measured by experiments.

  13. Transconjunctival penetration of mitomycin C

    Directory of Open Access Journals (Sweden)

    Velpandian T


    Full Text Available Aims: The study was performed to estimate transconjunctival penetration of mitomycin C (MMC to Tenon′s tissue following application over the intact conjunctiva before routine trabeculectomy. Settings and Design: Institution-based case series. Materials and Methods: In 41 eyes of 41 patients, MMC (0.4 mg/ml for 3 min was applied over the intact conjunctiva before beginning trabeculectomy. Tenon′s capsule directly beneath the site of application was excised during trabeculectomy and was homogenized, centrifuged and MMC concentrations were analyzed using high-performance liquid chromatography (HPLC. Statistical Analysis Used: Statistical analysis was performed using stata0 8.0 version software (STATA Corporation, Houston, TX, USA. In this study, P -values less than 0.05 were considered as statistically significant. Results: The average weight of the sample of Tenon′s tissue excised was 5.51 ± 4.42 mg (range: 0.9-17.1 and the average estimated MMC concentration found to be present in Tenon′s tissue using HPLC was 18.67 ± 32.36 x 10−6 moles/kg of the tissue (range: 0.38-197.05 x 10−6 . In 36 of the 41 patients (87.80%, the MMC concentration reached above 2 x 10−6 moles/kg of the tissue concentration required to inhibit human conjunctival fibroblasts. Conclusions: Mitomycin C does permeate into the subconjunctival tissue after supraconjunctival application for 3 min. Application of MMC over the conjunctiva may be a useful alternative to subconjunctival or subscleral application during routine trabeculectomy and as an adjunct for failing blebs.

  14. Simulations of a plasmoid penetrating a magnetic barrier

    Energy Technology Data Exchange (ETDEWEB)

    Gunell, H; Koepke, M [Department of Physics, West Virginia University, Morgantown, WV 26506-6315 (United States); Hurtig, T [Swedish Defence Research Agency, Grindsjoen Research Centre, SE-147 25 Tumba (Sweden); Nilsson, H [Swedish Institute of Space Physics, PO Box 812, SE-981 28 Kiruna (Sweden); Brenning, N [Division of Space and Plasma Physics, School of Electrical Engineering, Royal Institute of Technology, SE-100 44 Stockholm (Sweden)], E-mail:


    Plasma structures, here typified by the term 'plasmoids', in the solar wind impacting on the magnetopause, i.e. the boundary between the solar wind and the Earth's magnetosphere, can penetrate this boundary and be injected into the magnetosphere. This can happen either by expulsion of the magnetic field from the structure and subsequent diffusion of the magnetic field into the structure or by the formation of a polarization electric field that lets the plasma structure E x B-drift into the earth's magnetic field. In both cases a collisionless resistivity is required at some stage of the process. While magnetic expulsion requires electromagnetic models for its description, polarization can be modelled electrostatically and both processes can be, and have been, studied in laboratory experiments. We present three-dimensional electrostatic particle-in-cell simulations that reproduce large-amplitude waves, in the lower-hybrid range, that have been observed in laboratory experiments. Lower-hybrid waves have also been seen at the magnetopause of the earth. We consider the implications for spacecraft-based studies of magnetopause penetration, and suggest that the search for penetrating plasma structures should emphasize cases in which the interplanetary magnetic field is oriented northwards, as this configuration is less likely for reconnection. The application of theoretical predictions to the magnetopause environment shows that a plasma structure penetrating via polarization needs to be small, i.e. less than 10-100 km wide for typical parameters, and that wave processes at the magnetopause are needed to create such small structures. A larger structure can penetrate by means of magnetic expulsion.

  15. Modeling pollutant penetration across building envelopes

    Energy Technology Data Exchange (ETDEWEB)

    Liu, De-Ling; Nazaroff, William W.


    As air infiltrates through unintentional openings in building envelopes, pollutants may interact with adjacent surfaces. Such interactions can alter human exposure to air pollutants of outdoor origin. We present modeling explorations of the proportion of particles and reactive gases (e.g., ozone) that penetrate building envelopes as air enters through cracks and wall cavities. Calculations were performed for idealized rectangular cracks, assuming regular geometry, smooth inner crack surface and steady airflow. Particles of 0.1-1.0 {micro}m diameter are predicted to have the highest penetration efficiency, nearly unity for crack heights of 0.25 mm or larger, assuming a pressure difference of 4 Pa or greater and a flow path length of 3 cm or less. Supermicron and ultrafine particles are significantly removed by means of gravitational settling and Brownian diffusion, respectively. In addition to crack geometry, ozone penetration depends on its reactivity with crack surfaces, as parameterized by the reaction probability. For reaction probabilities less than {approx}10{sup -5}, penetration is complete for cracks heights greater than 1 mm. However, penetration through mm scale cracks is small if the reaction probability is {approx}10{sup -4} or greater. For wall cavities, fiberglass insulation is an efficient particle filter, but particles would penetrate efficiently through uninsulated wall cavities or through insulated cavities with significant airflow bypass. The ozone reaction probability on fiberglass fibers was measured to be 10{sup -7} for fibers previously exposed to high ozone levels and 6 x 10{sup -6} for unexposed fibers. Over this range, ozone penetration through fiberglass insulation would vary from >90% to {approx}10-40%. Thus, under many conditions penetration is high; however, there are realistic circumstances in which building envelopes can provide substantial pollutant removal. Not enough is yet known about the detailed nature of pollutant penetration

  16. Sphere impact and penetration into wet sand

    KAUST Repository

    Marston, J. O.


    We present experimental results for the penetration of a solid sphere when released onto wet sand. We show, by measuring the final penetration depth, that the cohesion induced by the water can result in either a deeper or shallower penetration for a given release height compared to dry granular material. Thus the presence of water can either lubricate or stiffen the granular material. By assuming the shear rate is proportional to the impact velocity and using the depth-averaged stopping force in calculating the shear stress, we derive effective viscosities for the wet granular materials.

  17. Sphere impact and penetration into wet sand (United States)

    Marston, J. O.; Vakarelski, I. U.; Thoroddsen, S. T.


    We present experimental results for the penetration of a solid sphere when released onto wet sand. We show, by measuring the final penetration depth, that the cohesion induced by the water can result in either a deeper or shallower penetration for a given release height compared to dry granular material. Thus the presence of water can either lubricate or stiffen the granular material. By assuming the shear rate is proportional to the impact velocity and using the depth-averaged stopping force in calculating the shear stress, we derive effective viscosities for the wet granular materials.

  18. Earthquakes induced by deep penetrating bombing?

    Institute of Scientific and Technical Information of China (English)

    Serguei Yu. Balassanian


    The data of M≥5 earthquakes occurred in one year before and after 4 deep penetrating bombs in the region within 500 km and 1 000 km from the shooting site are presented. The 4 bombs are those happened in 1999 Kosovo of Yugoslavia, the 1991 Baghdad of Iraq, the 2001 Tora Bora of Afghanistan, and the 2003 Kirkuk of Iraq, respectively. The data indicate that the deep penetrating bombs may have remotely triggered some earthquakes. The deep penetrating bombs in seismically active regions should be forbidden.

  19. Simulated meteoroid penetration of reusable surface insulation (United States)

    Lehman, J. K.; Christensen, H. E.


    Meteoroid impact simulation test results on insulation tiles were used to determine penetration resistance of reusable surface insulation attached to simulated shuttle structures. The probability of no meteoroid damage to a typical shuttle orbiter was determined. Specimens were plasma jet tested to determine effects of various size meteoroid cavities on their thermal performance. None of the tiles failed catastrophically, but large, egg-shaped craters did result from meteoroid penetration. The metallic side of a specimen that was completely penetrated showed a petalled hole typical for thin gage shielded structures.

  20. Mechanisms of magnesium-induced vasodilation in cerebral penetrating arterioles. (United States)

    Murata, Takahiro; Dietrich, Hans H; Horiuchi, Tetsuyoshi; Hongo, Kazuhiro; Dacey, Ralph G


    We investigated in cerebral penetrating arterioles the signaling mechanisms and dose-dependency of extracellular magnesium-induced vasodilation and also its vasodilatory effects in vessels preconstricted with agonists associated with delayed cerebral vasospasm following SAH. Male rat penetrating arterioles were cannulated. Their internal diameters were monitored. To investigate mechanisms of magnesium-induced vasodilation, inhibitors of endothelial function, potassium channels and endothelial impairment were tested. To simulate cerebral vasospasm we applied several spasmogenic agonists. Increased extracellular magnesium concentration produced concentration-dependent vasodilation, which was partially attenuated by non-specific calcium-sensitive potassium channel inhibitor tetraethylammonium, but not by other potassium channel inhibitors. Neither the nitric oxide synthase inhibitor L-NNA nor endothelial impairment induced by air embolism reduced the dilation. Although the magnesium-induced vasodilation was slightly attenuated by the spasmogen ET-1, neither application of PF2α nor TXA2 analog effect the vasodilation. Magnesium induced a concentration- and smooth muscle cell-dependent dilation in cerebral penetrating arterioles. Calcium-sensitive potassium channels of smooth muscle cells may play a key role in magnesium-induced vasodilation. Magnesium also dilated endothelium-impaired vessels as well as vessels preconstricted with spasmogenic agonists. These results provide a fundamental background for the clinical use of magnesium, especially in treatment against delayed cerebral ischemia or vasospasm following SAH.

  1. Prediction of weld penetration extent in GTAW

    Institute of Scientific and Technical Information of China (English)

    Zhao Ming; Zhai Lei


    The governing equations of surface-deformation in fully-penetrated GTA W weld pool are derived. The solution scheme of the Lagrange multiplier, which makes the top sutface-deformation equation be coupled with that for the bottomsurface of a single weld pool, is given out. Through numerical analysis of weld pool surface-deformation in GTA W,it is found that there is clear correlation between the penetration extent and the Lagrange multiplier, in addition to the relationship between the penetration extent and the maximum depression and hump as well as the depressed volume of the top surface of weld pool. It lays good foundation for top-side sensing based weld penetration control.

  2. An Observational Investigation of Penetrative Convection

    DEFF Research Database (Denmark)

    Jensen, Niels Otto; Lenschow, D. H.


    Data taken during the Air Mass Transformation Experiment (AMTEX) by the NCAR Electra aircraft have proven useful for investigating the structure of thermals penetrating into the turbulent inversion layer which caps the convective mixed layer. Variances, covariances, spectra and cospectra...

  3. Temporary fire sealing of penetrations on TFTR

    Energy Technology Data Exchange (ETDEWEB)

    Hondorp, H.L.


    The radiation shielding provided for TFTR for D-D and D-T operation will be penetrated by numerous electrical and mechanical services. Eventually, these penetrations will have to be sealed to provide the required fire resistance, tritium sealability, pressure integrity and radiation attenuation. For the initial hydrogen operation, however, fire sealing of the penetrations in the walls and floor is the primary concern. This report provides a discussion of the required and desirable properties of a temporary seal which can be used to seal these penetrations for the hydrogen operation and then subsequently be removed and replaced as required for the D-D and D-T operations. Several candidate designs are discussed and evaluated and recommendations are made for specific applications.

  4. Penetration testing protecting networks and systems

    CERN Document Server

    Henry, Kevin M


    This book is a preparation guide for the CPTE examination, yet is also a general reference for experienced penetration testers, ethical hackers, auditors, security personnel and anyone else involved in the security of an organization's computer systems.

  5. Kali Linux wireless penetration testing beginner's guide

    CERN Document Server

    Ramachandran, Vivek


    If you are a security professional, pentester, or anyone interested in getting to grips with wireless penetration testing, this is the book for you. Some familiarity with Kali Linux and wireless concepts is beneficial.

  6. 3D Ground Penetrating Imaging Radar


    ECT Team, Purdue


    GPiR (ground-penetrating imaging radar) is a new technology for mapping the shallow subsurface, including society’s underground infrastructure. Applications for this technology include efficient and precise mapping of buried utilities on a large scale.

  7. Tissue-penetrating delivery of compounds and nanoparticles into tumors. (United States)

    Sugahara, Kazuki N; Teesalu, Tambet; Karmali, Priya Prakash; Kotamraju, Venkata Ramana; Agemy, Lilach; Girard, Olivier M; Hanahan, Douglas; Mattrey, Robert F; Ruoslahti, Erkki


    Poor penetration of drugs into tumors is a major obstacle in tumor treatment. We describe a strategy for peptide-mediated delivery of compounds deep into the tumor parenchyma that uses a tumor-homing peptide, iRGD (CRGDK/RGPD/EC). Intravenously injected compounds coupled to iRGD bound to tumor vessels and spread into the extravascular tumor parenchyma, whereas conventional RGD peptides only delivered the cargo to the blood vessels. iRGD homes to tumors through a three-step process: the RGD motif mediates binding to alphav integrins on tumor endothelium and a proteolytic cleavage then exposes a binding motif for neuropilin-1, which mediates penetration into tissue and cells. Conjugation to iRGD significantly improved the sensitivity of tumor-imaging agents and enhanced the activity of an antitumor drug.

  8. Penetrating facial injury by a wooden log


    Mohan, Sadanandan; Varghese, George; Kumar, Sanjay; Subramanian, Dinesh Pambungal


    Penetrating facial injuries are potentially dangerous and require emergency management because of the presence of vital structures in the face and it may be life threatening especially when the injury involves airway, major blood vessels, spinal cord and cervical spines. Penetrating injuries of facial region can occur due to missile injuries, blast injuries, accidental fall on sharp objects such as sticks or glass and motor vehicle accidents etc., Indications for immediate surgical management...

  9. Effect of penetrants on the aramid Nomex

    Indian Academy of Sciences (India)

    Anjana Jain; Kalyani Vijayan


    The response of Nomex sheets to some penetrants has been analyzed. The process of moisture uptake depends on the relative humidity (RH) of the ambient atmosphere and the initial characteristics of the polymer. In the case of common laboratory solvents, the uptake by the polymer shows an inverse dependence on the molar volume of the former. Although the calendered and the uncalendered Nomex sheets exhibit an overall similarity in their response to various penetrants, quantitative comparison reveals distinct differences.

  10. Magnetic field penetration of erosion switch plasmas (United States)

    Mason, Rodney J.; Jones, Michael E.; Grossmann, John M.; Ottinger, Paul F.


    Computer simulations demonstrate that the entrainment (or advection) of magnetic field with the flow of cathode-emitted electrons can constitute a dominant mechanism for the magnetic field penetration of erosion switch plasmas. Cross-field drift in the accelerating electric field near the cathode starts the penetration process. Plasma erosion propagates the point for emission and magnetic field injection along the cathode toward the load-for the possibility of rapid switch opening.

  11. 穿膜肽引导的体外表达转录因子蛋白Sox2进入红鳍东方鲀精巢细胞系%Intracellular delivery of the expressed fusion protein Sox2 with cell penetrating peptides to cultured Takifugu rupies spermary cells

    Institute of Scientific and Technical Information of China (English)

    杨秀霞; 侯雪宁; 徐彬; 郝萧; 姜国建; 樊廷俊


    为了探索适用于体外培养的鱼细胞外源基因转入方法,本研究通过构建红鳍东方鲀(Takifugu rubripes)转录因子Sox2的重组表达载体 pET32a(+)-Sox2-11R-6His,诱导表达并纯化得到了 C末端连接多聚精氨酸(11R)的重组蛋白Sox2-11R-6His,以其与红鳍东方鲀精巢细胞系细胞共孵育12 h后,光镜观察结合Western Blot检测发现重组蛋白进入细胞的效率与浓度呈剂量依赖关系且最佳孵育浓度为8μg/mL,当重组蛋白质量浓度达到10μg/mL 时,表现出明显的细胞毒性。对外源蛋白进行免疫荧光标记定位,发现重组蛋白分布于细胞质,部分进入到细胞核中。证明了穿膜肽11R可以有效运载转录因子重组蛋白至红鳍东方鲀的细胞系细胞中。本研究旨在将广泛应用于哺乳动物的细胞基因递送载体穿膜肽应用于鱼类细胞系细胞。%Research on exogenous gene function in cultured cell line cells is guaranteed by two biological processes, efficient gene transduction and errorless gene expression. Fish cells are not easy for exogenous gene transduction and expression depending on the gene delivery method used and the transgene promoter. Cell penetrating peptides (CPPs), such as the CPP of the human immunodeficiency virus type 1 TAT protein, poly-arginine (6–12 residues), and the CPP derived from flock house virus (FHV), are short cationic and/or amphipathic peptides that are able to transport various biological compounds such as peptides, proteins, and oligonucleotides into mammalian cells to modulate biological activities inside cells. Although it has been shown that CPPs can mediate efficient delivery into a wide variety of mam-malian cell types, the transduction of proteins to cultured fish cells is considered more challenging. In this work, the Sox2 gene from Takifugu rupies genomic DNA was cloned and subsequent expression was carried out in Escherichia coli in the form of recombinant proteins by introducing cell

  12. Simulation of Mine Electrical Penetration Technology

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-xin; YUE Jian-hua; LIU Shu-cai


    Based on the principle of electrical penetration, the reflection characteristics of collapse columns at different locations of a working face is numerically simulated by using a 3D finite element method. The data collected by the electrical penetration is processed and interpreted using "tunnel penetration" which is similar to radio wave penetration. Reflection characteristics of collapse columns at different locations below floors of coal seams are analyzed, providing a new paradigm and a theoretical foundation for processing and interpreting electrical penetration data. The tomography analysis is made based on data simulation and calculation results and alltransmitting-receiving points are analyzed for their corresponding maximum attenuation values and maximum absorption coefficients. On the basis of this, a new method for precisely interpreting the spatial positions of geological anomalous bodies is suggested. The simulation shows that 1) the detection result of both roof and floors of the working face by electrical penetration is a volumetric effect and 2) there exists a corresponding relation between the detection depth and the working face width, with the optimal detection depth within 40% of the working face width.

  13. Giant gastric ulcer penetrating into the pancreas. (United States)

    Fujihara, Shintaro; Mori, Hirohito; Nishiyama, Noriko; Kobara, Hideki; Masaki, Tsutomu


    A 57-year-old man developed haematemesis and was referred to our institution. His discomfort had begun 3 weeks earlier and localised to the upper abdomen. Abdominal CT showed a defect of gastric mucosa and gastric wall thickening. Oesophagogastroduodenoscopy showed that he had an active gastric ulcer, 40 mm in diameter, on the lesser curvature in the upper third of the corpus and the presence of a pancreatic body at the ulcer base, penetration to the pancreas. Open gastrectomy was performed with a preoperative diagnosis of localised peritonitis caused by penetration of the stomach into the pancreas. Histopathology of resected specimens showed a benign peptic ulcer, 40×40 mm in size, was found on the lesser curvature in the antrum, and this had penetrated through the pancreas. The most serious complications of PUD include haemorrhage, perforation, penetration, and gastric outlet obstruction. Approximately 7% of patients experience perforation, which occurs when an ulcer erodes through the wall and leaks air and digestive contents into the peritoneal cavity. Antral and duodenal ulcers can penetrate into the pancreas. We report a case of gastric ulcer penetrating into the pancreas.

  14. In vitro penetration of bleaching agents into the pulp chamber

    DEFF Research Database (Denmark)

    Benetti, Ana Raquel; Valera, M C; Mancini, M N G


    To investigate pulp chamber penetration of bleaching agents in teeth following restorative procedures.......To investigate pulp chamber penetration of bleaching agents in teeth following restorative procedures....

  15. Rapid penetration into granular media visualizing the fundamental physics of rapid earth penetration

    CERN Document Server

    Iskander, Magued


    Rapid Penetration into Granular Media: Visualizing the Fundamental Physics of Rapid Earth Penetration introduces readers to the variety of methods and techniques used to visualize, observe, and model the rapid penetration of natural and man-made projectiles into earth materials. It provides seasoned practitioners with a standard reference that showcases the topic's most recent developments in research and application. The text compiles the findings of new research developments on the subject, outlines the fundamental physics of rapid penetration into granular media, and assembles a com

  16. Overcoming the nail barrier: A systematic investigation of ungual chemical penetration enhancement. (United States)

    Brown, M B; Khengar, R H; Turner, R B; Forbes, B; Traynor, M J; Evans, C R G; Jones, S A


    This study investigated the in vitro nail permeability of penetrants of varying lipophilicity-caffeine (CF, logP -0.07), methylparaben (MP, logP 1.96) and terbinafine (TBF, logP 3.3) and the effect of 2 novel penetration enhancers (PEs), thioglycolic acid (TA) and urea hydrogen peroxide (urea H(2)O(2)) on their permeation. Studies were conducted using full thickness human nail clippings and ChubTur((R)) diffusion cells and penetrants were applied as saturated solutions. The rank order of steady-state penetrant flux through nails without PE application (MP>CF>TBF) suggested a greater sensitivity to penetrant molecular weight rather than logP. TA increased the flux of CF and MP approximately 4- and approximately 2-fold, respectively, whilst urea H(2)O(2) proved ineffective at enhancing permeability. The sequential application of TA followed by urea H(2)O(2) increased TBF and CF flux ( approximately 19- and approximately 4-fold, respectively) but reversing the application order of the PEs was only mildly effective at increasing just MP flux ( approximately 2-fold). Both nail PEs are likely to function via disruption of keratin disulphide bonds and the associated formation of pores that provide more 'open' drug transport channels. Effects of the PEs were penetrant specific, but the use of a reducing agent (TA) followed by an oxidising agent (urea H(2)O(2)) dramatically improved human nail penetration.

  17. Two methodologies for physical penetration testing using social engineering

    NARCIS (Netherlands)

    Dimkov, Trajce; Pieters, Wolter; Hartel, Pieter


    Penetration tests on IT systems are sometimes coupled with physical penetration tests and social engineering. In physical penetration tests where social engineering is allowed, the penetration tester directly interacts with the employees. These interactions are usually based on deception and if not

  18. Ex vivo penetration of low-level laser light through equine skin and flexor tendons. (United States)

    Duesterdieck-Zellmer, Katja F; Larson, Maureen K; Plant, Thomas K; Sundholm-Tepper, Andrea; Payton, Mark E


    OBJECTIVE To measure penetration efficiencies of low-level laser light energy through equine skin and to determine the fraction of laser energy absorbed by equine digital flexor tendons (superficial [SDFT] and deep [DDFT]). SAMPLE Samples of skin, SDFTs, and DDFTs from 1 metacarpal area of each of 19 equine cadavers. PROCEDURES A therapeutic laser with wavelength capabilities of 800 and 970 nm was used. The percentage of energy penetration for each wavelength was determined through skin before and after clipping and then shaving of hair, through shaved skin over SDFTs, and through shaved skin, SDFTs, and DDFTs (positioned in anatomically correct orientation). Influence of hair color; skin preparation, color, and thickness; and wavelength on energy penetration were assessed. RESULTS For haired skin, energy penetration was greatest for light-colored hair and least for dark-colored hair. Clipping or shaving of skin improved energy penetration. Light-colored skin allowed greatest energy penetration, followed by medium-colored skin and dark-colored skin. Greatest penetration of light-colored skin occurred with the 800-nm wavelength, whereas greatest penetration of medium- and dark-colored skin occurred with the 970-nm wavelength. As skin thickness increased, energy penetration of samples decreased. Only 1% to 20% and 0.1% to 4% of energy were absorbed by SDFTs and DDFTs, respectively, depending on skin color, skin thickness, and applied wavelength. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that most laser energy directed through equine skin was absorbed or scattered by the skin. To achieve delivery of energy doses known to positively affect cells in vitro to equine SDFTs and DDFTs, skin preparation, color, and thickness and applied wavelength must be considered.

  19. Wireless Network Penetration Testing and Security Auditing

    Directory of Open Access Journals (Sweden)

    Wang Shao-Long


    Full Text Available IEEE802.11 wireless wireless networks have security issues that are vulnerable to a variety of attacks. Due to using radio to transport data, attackers can bypass firewalls, sniff sensitive information, intercept packets and send malicious packets. Security auditing and penetration testing is expected to ensure wireless networks security. The contributions of this work are analyzed the vulnerability and types of attacks pertaining to IEEE 802.11 WLAN, performed well known attacks in a laboratory environment to conduct penetration tests to confirm whether our wireless network is hackable or not. WAIDPS is configured as auditing tool to view wireless attacks, such as WEP/WPA/WPA2 cracking, rouge access points, denial of service attack. WAIDPS is designed to detect wireless intrusion with additional features. Penetration testing and auditing will mitigate the risk and threatening to protect WALN.

  20. Opportunities for high wind energy penetration

    DEFF Research Database (Denmark)

    Tande, J.O.; Hansen, J.C.


    Wind power is today a mature technology, which at windy locations, is economically competitive to conventional power generation technologies. This and growing global environmental concerns have led governments to encourage and plan for wind energy development, a typical aim being 10% of electricity...... consumption. The successful operation of the three major power systems of Cape Verde, with a total wind energy penetration of about 15% since December 1994, demonstrates that power systems can be operated with high penetration of wind energy by adding simple control and monitoring systems only. Thorough...... analyses conclude that expanding to even above 15% wind energy penetration in the Cape Verde power systems is economical. Worldwide, numerous locations with favorable wind conditions and power systems similar to the Capeverdean provide good opportunities for installing wind farms and achieving high wind...

  1. Diagnosis and treatment of penetrating anorectal wounds

    Institute of Scientific and Technical Information of China (English)

    LIU Xin-sheng; HUI Xi-zeng; ZHANG Yang-de; LI Kun


    Objective:To research the diagnosis and effective treatment of penetrating anorectal wounds.Methods: Retrospective analysis was done in 16 cases of penetrating anorectal wounds from 1985 to 2004.Debridement and suture of anorectal and vesical wounds,effective diversion of fecal and urinary stream and sufficient presacral drainage were performed in all cases.Results: All the 16 cases were cured. Among them, 2cases with infection in presacral space were cured by sufficient drainage after operation, one case was cured by secondary repair after anal sphincter was repaired unsuccessfully and one case with rectovesical fistula was cured with conservative treatment. None of them suffered from complications such as anal stenosis, dysuria or importence etc.Conclusions: For penetrating anorectal wound, to master early recognition of concomitant injures, to select appropriate surgical intervention and to strengthen perioperative treatment are the keys to improve the curative effects.

  2. Penetrating power of resonant electromagnetic induction imaging (United States)

    Guilizzoni, Roberta; Watson, Joseph C.; Bartlett, Paul; Renzoni, Ferruccio


    The possibility of revealing the presence and identifying the nature of conductive targets is of central interest in many fields, including security, medicine, industry, archaeology and geophysics. In many applications, these targets are shielded by external materials and thus cannot be directly accessed. Hence, interrogation techniques are required that allow penetration through the shielding materials, in order for the target to be identified. Electromagnetic interrogation techniques represent a powerful solution to this challenge, as they enable penetration through conductive shields. In this work, we demonstrate the power of resonant electromagnetic induction imaging to penetrate through metallic shields (1.5-mm-thick) and image targets (having conductivities σ ranging from 0.54 to 59.77 MSm-1) concealed behind them.

  3. Proniosomes for Penetration Enhancement in Transdermal System

    Directory of Open Access Journals (Sweden)

    Samita Singla


    Full Text Available Over the last few years an inclusive research has been done over provesicular approach for transdermal drug delivery. Skin has a very tough diffusion barrier inhibiting penetration of drug moiety which is rate limiting barrier for penetration of drugs. There are several approaches that deal with penetration enhancement across the skin. Vesicular and provesicular systems are promising amongst them. Vesicular systems including (niosomes, ethosomes, transfersomes and liposomes are promising systems to cross this permeation barrier. But their major drawback is their unstability, which can be overcome by using provesicular approaches like proniosomes, protransfersomes and proliposomes. Provesicular approaches have been proposed to enhance the stability of vesicles. These approaches may overcome skin barrier properties and enhance percutaneous absorption. In these systems both type of drugs, hydrophilic and lipophillic drugs can be incorporated. The focus of this review is to bring together different aspects related to mechanism of skin permeation of provesicular system, proniosomes preparation and its applications as a carrier system

  4. Penetration of Projectiles in Composite Laminates

    Directory of Open Access Journals (Sweden)

    B. P. Patel


    Full Text Available This paper deals with the prediction of the penetration phenomenon of a cylindro-conical impactor on the ke;labepoxy-laminated composites using C eight-nded serendipity q&drilateral finite elementbased on first-order shear deformation theory (FSDT. Local as well as global deformations during impact is considered in the evaluation of indentation, penetration, and perforation phases. Local strainsduring impact have been evaluated using the hypothesis made from the available experimental observations of bulging during penetration. A detailed parametric study, considering various projectilesand target plate variables, has been carried out to find their effect on the response of the plate, and ballistic parameters, such as ballistic limit and absorbed energy.

  5. Penetration profile of microspheres in follicular targeting of terminal hair follicles. (United States)

    Toll, R; Jacobi, U; Richter, H; Lademann, J; Schaefer, H; Blume-Peytavi, U


    The transfollicular administration of pharmacologically active molecules is of current therapeutic interest, mainly with regard to delivery to specific sites of the hair follicle (HF) and the reduction of hepatic metabolism and systemic toxicity. HF are privileged pathways for specific molecules depending on formulations, which enter faster into these shunts than through the stratum corneum. The aim was to optimize the delivery of fluorescent microspheres into the HF, thereby, developing a standardized protocol for follicular targeting with microspheres. The number of HF showing penetration, as well as the depth of penetration, was determined. Freshly excised skin samples with terminal HF were divided into groups, with or without prior treatment with cyanoacrylate skin surface stripping-technique (CSSS). Thereafter microspheres at a size of 0.75-6.0 microm were applied according to the developed standardized protocol. Skin biopsies were obtained, shock-frozen, and sectioned in 5 microm slices. We demonstrated a selective penetration route of the microspheres into the HF. Optimal microsphere size proved to be approximately 1.5 microm, with a 55% rate of all HF, and with a maximum penetration depth of >2300 microm. Without previous CSSS treatment of the skin, the transfollicular microsphere penetration was below 27% with a maximum penetration depth of 1000 microm. Thus, the basis for follicular targeting of essential structures containing stem cells for keratinocytes, melanocytes, and mast cells has been laid.

  6. Large scale wind power penetration in Denmark

    DEFF Research Database (Denmark)

    Karnøe, Peter


    he Danish electricity generating system prepared to adopt nuclear power in the 1970s, yet has become the world's front runner in wind power with a national plan for 50% wind power penetration by 2020. This paper deploys a sociotechnical perspective to explain the historical transformation of "net...... expertise evolves and contributes to the normalization and large-scale penetration of wind power in the electricity generating system. The analysis teaches us how technological paths become locked-in, but also indicates keys for locking them out....

  7. Standard Penetration Test and Relative Density (United States)


    laboratorio ejecutados con un penetr6metro est’tico pequeno. INTRODUCTION One of the main problems encountered in subsoil e’xploration is in situ...would be more valid. REFERENCES Burmister, D. M. �), "The Grading-Density Relation of Gr•anular Materials ." Proc,.edings of the American Society for...Reclamation (1953), "Second Progress Report of Re- search of the Penetration Resistance Method of Subsurface Explora- tion," Report No. EM-356. Design and Construction Division, Earth Materials Laboratory, Denver,

  8. FREEFALL: A seabed penetrator flight code

    Energy Technology Data Exchange (ETDEWEB)

    Hickerson, J.


    This report presents a one-dimensional model and computer program for predicting the motion of seabed penetrators. The program calculates the acceleration, velocity, and depth of a penetrator as a function of time from the moment of launch until the vehicle comes to rest in the sediment. The code is written in Pascal language for use on a small personal computer. Results are presented as printed tables and graphs. A comparison with experimental data is given which indicates that the accuracy of the code is perhaps as good as current techniques for measuring vehicle performance. 31 refs., 12 figs., 5 tabs.


    Institute of Scientific and Technical Information of China (English)

    刘宇红; 曾衍钧; 许景锋; 张梅


    Transdermal drug delivery system (TDDS) is a new method for drug delivery. The analysis of plenty of experiments in vitro can lead to a suitable mathematical model for the description of the process of the drug's penetration through the skin, together with the important parameters that are related to the characters of the drugs.After the research work of the experiments data,a suitable nonlinear regression model was selected. Using this model, the most important parameter-penetrated coefficient of 20 drugs was computed.In the result one can find, this work supports the theory that the skin can be regarded as singular membrane.

  10. Operational experience of extreme wind penetrations

    Energy Technology Data Exchange (ETDEWEB)

    Estanqueiro, Ana [INETI/LNEG - National Laboratory for Energy and Geology, Lisbon (Portugal); Mateus, Carlos B. [Instituto de Meteorologia, Lisboa (Portugal); Pestana, Rui [Redes Energeticas Nacionais (REN), Lisboa (Portugal)


    This paper reports the operational experience from the Portuguese Power System during the 2009/2010 winter months when record wind penerations were observed: the instantaneous wind power penetration peaked at 70% of consumption during no-load periods and the wind energy accounted for more than 50% of the energy consumed for a large period. The regulation measures taken by the TSO are presented in the paper, together with the additional reserves operated for added system security. Information on the overall power system behavior under such extreme long-term wind power penetrations will also be addressed. (org.)

  11. Hybrid treatment of penetrating aortic ulcer

    Energy Technology Data Exchange (ETDEWEB)

    Lara, Juan Antonio Herrero; Martins-Romeo, Daniela de Araujo; Escudero, Carlos Caparros; Falcon, Maria del Carmen Prieto; Batista, Vinicius Bianchi, E-mail: [Unidade de Gestao Clinica (UGC) de Diagnostico por Imagem - Hosppital Universitario Virgen Macarena, Sevilha (Spain); Vazquez, Rosa Maria Lepe [Unit of Radiodiagnosis - Hospital Nuestra Senora de la Merced, Osuna, Sevilha (Spain)


    Penetrating atherosclerotic aortic ulcer is a rare entity with poor prognosis in the setting of acute aortic syndrome. In the literature, cases like the present one, located in the aortic arch, starting with chest pain and evolving with dysphonia, are even rarer. The present report emphasizes the role played by computed tomography in the diagnosis of penetrating atherosclerotic ulcer as well as in the differentiation of this condition from other acute aortic syndromes. Additionally, the authors describe a new therapeutic approach represented by a hybrid endovascular surgical procedure for treatment of the disease. (author)

  12. Modeling the effect of dynamic surfaces on membrane penetration (United States)

    van Lehn, Reid; Alexander-Katz, Alfredo


    The development of nanoscale materials for targeted drug delivery is an important current pursuit in materials science. One task of drug carriers is to release therapeutic agents within cells by bypassing the cell membrane to maximize the effectiveness of their payload and minimize bodily exposure. In this work, we use coarse-grained simulations to study nanoparticles (NPs) grafted with hydrophobic and hydrophilic ligands that rearrange in response to the amphiphilic lipid bilayer. We demonstrate that this dynamic surface permits the NP to spontaneously penetrate to the bilayer midplane when the surface ligands are near an order-disorder transition. We believe that this work will lead to the design of new drug carriers capable of non-specifically accessing cell interiors based solely on their dynamic surface properties. Our work is motivated by existing nanoscale systems such as micelles, or NPs grafted with highly mobile ligands or polymer brushes.

  13. 分期实施异体角膜缘干细胞移植与穿透性角膜移植术治疗严重化学伤%A staged penetrating keratoplasty following limbal stem cells allograft in severe chemical injury leading to successful restoration of the ocular surface anatomy

    Institute of Scientific and Technical Information of China (English)

    Ali Hassan Alashwal; Raja Azmi Mohd Noor; Mohtar Ibrahim


    A 62-year-old man presented with severe bilateral ocular surface chemical injury and history of failed penetrating keratoplasty of right eye in 1996. Visual acuity was hand movement in right eye and light perception in left eye. Staged procedures of limbal stem cells allograft followed by penetrating keratoplasty have been done and resulted in good ocular surface restoration and rehabilitation of vision in right eye.%有1位于1996年实施右眼穿透性角膜移植术失败的双眼严重眼表化学伤的患者,62岁,右眼视力手动,左眼视力光感,于2006-09-16/2007-02-07分别对右眼实施了异体角膜缘干细胞移植术和穿透性角膜移植术,经过术后5mo的药物治疗观察,最终获得了右眼最佳矫正视力为6/30,角膜移植片透明,眼表得以重建,并未发现明显排斥迹象的良好效果.

  14. Penetration resistance according to penetration rate, cone base size and different soil conditions

    Directory of Open Access Journals (Sweden)

    Daniel Dias Valadão Junior


    Full Text Available This study aimed to evaluate the effect of penetration rate and the size of the cone base on the resistance to penetration under different soil moistures and soil bulk density. The experimental design was completely randomized in a 4x2x2x2 factorial arrangement, with the factors, soil bulk density of 1.0; 1.2; 1.4 and 1.6 Mg m-3, soil moisture at the evaluation of 0.16 and 0.22 kg kg-1, penetration rates of 0.166 and 30 mm s-1 and areas of the cone base of 10.98 and 129.28 mm² resulting in 32 treatments with 8 replicates. To ensure greater uniformity and similarity to field conditions, samples passed through cycles of wetting and drying. Only the interaction of the four factors was not significant. Resistance values varied with the density of the soil, regardless of moisture and penetration rate. Soil penetration resistance was influenced by the size of the cone base, with higher values for the smallest base independent of moisture and soil bulk density. The relationship between resistance to penetration and moisture is not always linear, once it is influenced by soil bulk density. Reduction in the area of the cone leads to an increase in the soil resistance to penetration.

  15. A novel 3D-layered electrochemical-thermal coupled model strategy for the nail-penetration process simulation (United States)

    Liang, Guozhou; Zhang, Yiming; Han, Qi; Liu, Zhaoping; Jiang, Zhen; Tian, Shuang


    The safety is one of the persisting concerns related with Lithium-ion (Li-ion) batteries, among which the internal short-circuit is the most dangerous abusive situation and has always been the root cause for several catastrophic accidents in recent years. In this work, a 3D-layered electrochemical-thermal coupled model is employed to investigate the nail-penetration process in a Li-ion cell. The model is based on multilayer construction of a cell, and an effective strategy to evaluate the short-circuit area equivalent resistance (i.e. the equivalent resistance of short-circuit area that is caused by nail-penetration) during the penetration process is proposed. The developed model is proved to have capability of estimating the thermal runaway time, as well as the temperature distribution during nail-penetration process. It is also found that the active material loss during the nail-penetration process can be reconstructed by utilizing the developed model, which could provide understandings about the side reactions inside the cell during the nail-penetration process. The present study provides some insights about the nail-penetration process, and can be treated as a useful tool that helps the design of Li-ion cells for improving safety.

  16. Penetration of amalgam constituents into dentine

    NARCIS (Netherlands)

    Scholtanus, Johannes D.; Ozcan, Mutlu; Huysmans, Marie-Charlotte D. N. J. M.


    Objectives: Amalgam restorations are replaced by adhesively placed composite resin restorations at an increasing rate. After the removal of amalgam dentine often shows marked dark discoloration that is attributed to the penetration of corrosion products from overlying amalgams. it is questioned whet

  17. Penetration of amalgam constituents into dentine.

    NARCIS (Netherlands)

    Scholtanus, J.D.; Ozcan, M.; Huysmans, M.C.D.N.J.M.


    OBJECTIVES: Amalgam restorations are replaced by adhesively placed composite resin restorations at an increasing rate. After the removal of amalgam dentine often shows marked dark discoloration that is attributed to the penetration of corrosion products from overlying amalgams. It is questioned whet

  18. Limited cerebrospinal fluid penetration of docetaxel

    NARCIS (Netherlands)

    ten Tije, Albert J; Loos, Walter J; Zhao, Ming; Baker, Sharyn D; Enting, Roelien H; van der Meulen, Hans; Verweij, Jaap; Sparreboom, Alex; Enting, Roeline


    Our purpose was to investigate the cerebrospinal fluid (CSF) penetration of docetaxel in cancer patients. Docetaxel was administered as a 1-h infusion at a dose of 75 mg/m2 to two patients with metastatic breast cancer and leptomeningeal carcinomatosis. CSF samples were obtained using a lumbar punct

  19. Computed tomographic findings in penetrating peptic ulcer

    Energy Technology Data Exchange (ETDEWEB)

    Madrazo, B.L.; Halpert, R.D.; Sandler, M.A.; Pearlberg, J.L.


    Four cases of peptic ulcer penetrating the head of the pancreas were diagnosed by computed tomography (CT). Findings common to 3 cases included (a) an ulcer crater, (b) a sinus tract, and (c) enlargement of the head of the pancreas. Unlike other modalities, the inherent spatial resolution of CT allows a convenient diagnosis of this important complication of peptic ulcer disease.

  20. Subsurface investigation with ground penetrating radar (United States)

    Ground penetrating radar (GPR) data was collected on a small test plot at the OTF/OSU Turfgrass Research & Education Facility in Columbus, Ohio. This test plot was built to USGA standards for a golf course green, with a constructed sand layer just beneath the surface overlying a gravel layer, that i...

  1. Planetary penetrators: Their origins, history and future (United States)

    Lorenz, Ralph D.


    Penetrators, which emplace scientific instrumentation by high-speed impact into a planetary surface, have been advocated as an alternative to soft-landers for some four decades. However, such vehicles have yet to fly successfully. This paper reviews in detail, the origins of penetrators in the military arena, and the various planetary penetrator mission concepts that have been proposed, built and flown. From the very limited data available, penetrator developments alone (without delivery to the planet) have required ˜$30M: extensive analytical instrumentation may easily double this. Because the success of emplacement and operation depends inevitably on uncontrollable aspects of the target environment, unattractive failure probabilities for individual vehicles must be tolerated that are higher than the typical '3-sigma' (99.5%) values typical for spacecraft. The two pathways to programmatic success, neither of which are likely in an austere financial environment, are a lucky flight as a 'piggyback' mission or technology demonstration, or with a substantial and unprecedented investment to launch a scientific (e.g. seismic) network mission with a large number of vehicles such that a number of terrain-induced failures can be tolerated.

  2. Penetration of albendazole sulphoxide into hydatid cysts.


    D. L. Morris; Chinnery, J B; Georgiou, G.; Stamatakis, G; Golematis, B.


    The penetration of albendazole sulphoxide, the principal metabolite of albendazole into hydatid cysts (E granulosus) was measured by means of in vitro animal and clinical studies. The drug freely diffuses across the parasitic membranes. Cyst/serum concentrations of 22% were achieved in patients, longer pre-operative therapy produced higher concentrations.

  3. Penetration of lipid monolayers by psychoactive drugs

    NARCIS (Netherlands)

    Demel, R.A.; Deenen, L.L.M. van


    The ability of a number of psychoactive drugs to penetrate lipid monolayers of varying composition was examined, and the following observation were made: (1) The increase in surface pressure of a monomolecular film appeared to depend on the chemical nature of the lipid as well as on the initial film

  4. Good penetration of moxifloxacin into human abscesses. (United States)

    Sauermann, Robert; Karch, Rudolf; Kjellsson, Maria C; Feurstein, Thomas; Püspök, Andreas; Langenberger, Herbert; Böhmdorfer, Michaela; Jäger, Walter; Zeitlinger, Markus


    Abscesses are often treated with antibiotics in addition to incision or when incision is unfeasible, but accurate information about antibiotic abscess penetration in humans is missing. This study aimed at evaluating the penetration of moxifloxacin into human abscesses. After administration of a single dose of 400 mg moxifloxacin, drug concentrations were measured in 10 differently located abscesses at incision, and in plasma over 8 h. At incision performed 0.9-4.8 h after administration, moxifloxacin concentrations in abscesses ranged from ≤0.01 to 9.2 mg/l (1.9 ± 3.4 mg/l), indicating pronounced drug accumulation in some abscesses. The degree of abscess penetration could not be explained by covariates like the ratio of surface area to volume or pH of abscesses, or by moxifloxacin plasma concentrations. Concluding, moxifloxacin was detectable in most abscesses and may be a useful antibiotic for this indication. However, antibiotic abscess penetration was highly variable and unpredictable, suggesting surgical abscess incision whenever possible.

  5. The Basics of Hacking and Penetration Testing Ethical Hacking and Penetration Testing Made Easy

    CERN Document Server

    Engebretson, Patrick


    The Basics of Hacking and Penetration Testing serves as an introduction to the steps required to complete a penetration test or perform an ethical hack. You learn how to properly utilize and interpret the results of modern day hacking tools; which are required to complete a penetration test. Tool coverage will include, Backtrack Linux, Google, Whois, Nmap, Nessus, Metasploit, Netcat, Netbus, and more. A simple and clean explanation of how to utilize these tools will allow you  to gain a solid understanding of each of the four phases and prepare them to take on more in-depth texts and topi

  6. Energy spectra of He{sup +} ions penetrating thick biological targets

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Y.; Tan, C.; Zhang, J.; Liu, X.; Liu, J. [Department of Physics, Shandong University Jinan 250100 (China); Mu, Y.; Wang, R. [Department of Optoelectronics, Shandong University, Jinan 250100 (China); Yu, Z. [Institute of Plasma Physics, Academia Sinica, Heifei 230031 (China)


    Energy spectra of 500 keV-1MeV He{sup +} ion penetrating 50 {mu}m-100 {mu}m thick seed coat of maize, fruit peel of grape and of tomato, are measured. The results indicate that these thick biological targets, as seen by the penetrating ions, are inhomogeneous, and there are open paths, along which the incident ions can penetrate the targets easily. While most of the incident ions are stopped in the targets, some of the penetrating ions only lose a small fraction of their initial incident energy. The penetration energy spectra show a pure electronic stopping feature. Transmission electron microscope (TEM) micrographs taken from these samples with thickness of 30 {mu}m indicate that 150 keV electron beam from the TEM can penetrate the thick samples to give very good images with clear contrast. The electronic structures of {beta}-1,4 glucosan molecular chains, which is deemed as the most important constituent of the cell walls of seed coats and peels of fruits, are calculated to show the possible open-path directions which exist in biological samples. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  7. The release and transdermal penetration of baclofen formulated in a poloxamer lecithin organogel. (United States)

    Arnold, John J; Asbill, Scott


    The purpose of this study was to evaluate the in vitro release and ex vivo penetration of baclofen following incorporation into a 2% poloxamer lecithin organogel. Franz cells were utilized for both the release and penetration studies. Semi-permeable dialysis membranes were used as the model skin for the penetration study. Baclofen release and penetration at predetermined time points were assessed using high-performamce liquid chromatographic analysis. Results demonstrated that baclofen release from the poloxamer lecithin organogel was significantly higher than its penetration through porcine skin. The amount of baclofen released by the poloxamer lecithin organogel was linear up to 12 hours. Approximately 20% of applied drug was released over the duration of the study period. In comparison with drug released, the ex vivo penetration of baclofen through porcine skin was very low with only minute detectable quantities (significantly less than 1%) after the 12-hour study period. These results suggest that the request to include baclofen into a compounded poloxamer lecithin organogel should be approached cautiously by compounding pharmacists.

  8. Common gene therapy viral vectors do not efficiently penetrate sputum from cystic fibrosis patients.

    Directory of Open Access Journals (Sweden)

    Kaoru Hida

    Full Text Available Norwalk virus and human papilloma virus, two viruses that infect humans at mucosal surfaces, have been found capable of rapidly penetrating human mucus secretions. Viral vectors for gene therapy of Cystic Fibrosis (CF must similarly penetrate purulent lung airway mucus (sputum to deliver DNA to airway epithelial cells. However, surprisingly little is known about the rates at which gene delivery vehicles penetrate sputum, including viral vectors used in clinical trials for CF gene therapy. We find that sputum spontaneously expectorated by CF patients efficiently traps two viral vectors commonly used in CF gene therapy trials, adenovirus (d∼80 nm and adeno-associated virus (AAV serotype 5; d∼20 nm, leading to average effective diffusivities that are ∼3,000-fold and 12,000-fold slower than their theoretical speeds in water, respectively. Both viral vectors are slowed by adhesion, as engineered muco-inert nanoparticles with diameters as large as 200 nm penetrate the same sputum samples at rates only ∼40-fold reduced compared to in pure water. A limited fraction of AAV exhibit sufficiently fast mobility to penetrate physiologically thick sputum layers, likely because of the lower viscous drag and smaller surface area for adhesion to sputum constituents. Nevertheless, poor penetration of CF sputum is likely a major contributor to the ineffectiveness of viral vector based gene therapy in the lungs of CF patients observed to date.

  9. Penetration and performance of isocyanate wood binders on selected wood species

    Directory of Open Access Journals (Sweden)

    Gruver, T. M., and


    Full Text Available The penetration and performance of polymeric diphenylmethane diisocyanate (pMDI wood binder was investigated according to three factors: substrate species (aspen, yellow-poplar, or southern yellow pine; anatomical bonding plane (radial or tangential; and moisture content (0%, 5%, or 12%. Compression shear block tests and fluorescence microscopy were used to examine bond performance and resin penetration. Statistically, each of the aforementioned factors impacted results. As moisture content increased, observed bond strengths and wood failure increased. Bond formation did not occur when the substrates were equilibrated to 0% moisture content, except for the radial bonding surfaces of pine, which did adhere. At 5 and 12% moisture contents, tangential bonding surfaces out-performed radial bonding surfaces. In terms of resin penetration, moisture content was clearly the most important variable. Little penetration was observed at 0% moisture content, while extensive resin penetration was observed at elevated moisture contents. Pine was the only wood species to exhibit resin flow through radial cells, possibly explaining the enhanced resin penetration depths observed in pine samples.

  10. Efficacy of sunlight-activatable porphyrin formulates on larvae of Anopheles gambiae M and S molecular forms and An. arabiensis: a potential novel biolarvicide for integrated malaria vector control. (United States)

    Fabris, Clara; Ouédraogo, Robert Kossivi; Coppellotti, Olimpia; Dabiré, Roch K; Diabaté, Abdoulaye; Di Martino, Piera; Guidolin, Laura; Jori, Giulio; Lucantoni, Leonardo; Lupidi, Giulio; Martena, Valentina; Sawadogo, Simon P; Soncin, Marina; Habluetzel, Annette


    Biolarvicides, such as microbial formulations based on Bacillus thuringiensis and B. sphaericus, have been found to be highly effective against mosquito larvae and are currently employed as eco-friendly alternatives to synthetic chemical insecticides for vector control. Recently, a porphyrin of natural origin has been suggested as a sunlight-activatable larvicide against the dengue vector Aedes aegypti. In order to validate the approach for the control of the malaria vector, we tested the photo-larvicidal activity of a novel porphyrin, namely meso-tri(N-methyl-pyridyl), mono(N-dodecyl-pyridyl)porphine, C12, associated with two specifically selected carriers, against Anopheles gambiae s.s. and An. arabiensis larvae, both laboratory reared and collected from malaria endemic sites in Burkina Faso. Both C12-porphyrin formulates, when administered to larvae at a 50μM porphyrin dose, were accumulated in the alimentary canal. Subsequent exposure of the porphyrin-loaded larvae to sunlight for short times (0.5-3h) led to a complete mortality. The high efficacy exhibited by a "foodstuff" porphyrin formulate also in the presence of typical larval food particles opens promising perspectives for the development of an effective photocidal larvicide.

  11. Exploring the Possibility of Cryopreservation of Feline and Canine Erythrocytes by Rapid Freezing with Penetrating and Non-Penetrating Cryoprotectants (United States)

    Pervushina, Olga; Hofmann, Nicola; Glasmacher, Birgit; Zhegunov, Gennadiy


    Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v) ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization. PMID:28072844

  12. CNS Active O-Linked Glycopeptides that Penetrate the BBB

    Directory of Open Access Journals (Sweden)

    Evan M Jones


    Full Text Available Naturally occurring glycopeptides and glycoproteins play important roles in biological processes. Glycosylation is one of the most common post-translational modifications in vivo. Glycopeptides are involved in cell signaling and sorting, providing cell surface markers for recognition. From the drug design and synthesis perspective, modification of a peptide through glycosylation results in increased bioavailability and bioactivity of glycopeptides in living systems with negligible toxicity of degradation products. Glycopeptide synthesis can be accomplished through incorporation of a glycosylated amino acid in solid phase peptide synthesis (SPPS to form the desired peptide, or via incorporation of sugar-amino acid moieties. Additionally, research indicates that glycosylation increases penetration of the blood-brain barrier (BBB by peptides, which may lead to novel therapeutics for neurological disorders. Recent applications of glycopeptides have focused on the in vivo central nervous system effects after peripheral administration of centrally active peptides modified with various carbohydrates.

  13. Vacuum Radiation Pressure Fluctuations and Barrier Penetration

    CERN Document Server

    Huang, Haiyan


    We apply recent results on the probability distribution for quantum stress tensor fluctuations to the problem of barrier penetration by quantum particles. The probability for large stress tensor fluctuations decreases relatively slowly with increasing magnitude of the fluctuation, especially when the quantum stress tensor operator has been averaged over a finite time interval. This can lead to large vacuum radiation pressure fluctuations on charged or polarizable particles, which can in turn push the particle over a potential barrier. The rate for this effect depends sensitively upon the details of the time averaging of the stress tensor operator, which might be determined by factors such as the shape of the potential. We make some estimates for the rate of barrier penetration by this mechanism and argue that in some cases this rate can exceed the rate for quantum tunneling through the barrier. The possibility of observation of this effect is discussed.

  14. Depleted Uranium Penetrators : Hazards and Safety

    Directory of Open Access Journals (Sweden)

    S. S. Rao


    Full Text Available The depleted uranium (DU alloy is a state-of-the-art material for kinetic energy penetrators due to its superior ballistic performance. Several countries use DU penetrators in their main battle tanks. There is no gamma radiation hazard to the crew members from stowage of DO rounds. Open air firing can result in environmental contamination and associated hazards due to airborne particles containing essentially U/sub 3/0/sub 8/ and UO/sub 2/. Inhalation of polluted air only through respirators or nose masks and refraining form ingestion of water or food materials from contaminated environment are safety measures for avoiding exposure to uranium and its toxicity. Infusion of sodium bicarbonate helps in urinary excretion of uranium that may have entered the body.



    Raghu; Santhosh Babu; Devender


    Penetrating trauma forms an important component of surgical emergencies. The importance of this category becomes further apparent when one realises that most of such trauma victims are essentially healthy people and in the prime of their life. Over the past century, great advances were made in the management of such wounds. The operative management replaced the expectant therapy and reduced mortality rates. So this study is being done to evaluate the various indications for early ...

  16. Engineering and Design: Standard Penetration Test (United States)


    Penetration TestDe Contract Number Grant Number Program Element Number Author(s) Project Number Task Number Work Unit Number Performing Organization Name...jsfi I I US* of drlXXi~ mucl -r*ueusiog In I Decrm8e8 hcrea8ea Zither Increases Increases Increeses Decrea8es lncreeaes Increases Increeaes Zither &creases DecrU8e* nem8*s hcruses Xnt-ses tieee Utber k~ee Enclosure 2

  17. Market penetration of new energy technologies

    Energy Technology Data Exchange (ETDEWEB)

    Packey, D.J.


    This report examines the characteristics, advantages, disadvantages, and, for some, the mathematical formulas of forecasting methods that can be used to forecast the market penetration of renewable energy technologies. Among the methods studied are subjective estimation, market surveys, historical analogy models, cost models, diffusion models, time-series models, and econometric models. Some of these forecasting methods are more effective than others at different developmental stages of new technologies.

  18. Pseudo skin factor due to partial penetration

    Energy Technology Data Exchange (ETDEWEB)

    Reynolds, A.C.; Chen, J.C.; Raghavan, R.


    In this study the authors examine the pseudo skin factor due to partial penetration in a two-layer reservoir when only one layer is open to flow. They show that the pseudo skin factor can be correlated as a unique function of three reservoir parameters and in some cases can be accurately correlated as a function of two reservoir parameters. They also present graphs and procedures for estimating the pseudo skin factor.

  19. Pseudo skin factor due to partial penetration

    Energy Technology Data Exchange (ETDEWEB)

    Reynolds, A.C.; Chen, J.C.; Raghavan, R.


    This study examines the pseudo skin factor due to partial penetration in a 2-layer reservoir when only one layer is open to flow. The pseudo skin factor can be correlated as a unique function of 3 reservoir parameters and in some cases can be correlated accurately as a function of 2 reservoir parameters. Graphs and procedures for estimating the pseudo skin factor are presented. 17 references.

  20. Deep penetration of light into biotissue (United States)

    Bearden, Edward D.; Wilson, James D.; Zharov, Vladimir P.; Lowery, Curtis L.


    The results of a study of deep (several centimeters) light penetration into biological tissue are presented in order to estimate its significance to potentially photosensitive structures and processes including the fetal eyes. In order to accomplish this goal, samples of various tissues (fat, muscle, and uterus) from surgical patients and autopsies were examined with a double integrating sphere arrangement to determine their optical properties. The results were implemented in a Monte Carlo modeling program. Next, optical fiber probes were inserted into the uterus and abdominal wall of patients undergoing laparoscopic procedures. The fibers were couples to a photomultiplier tube with intervening filters allowing measurements of light penetration at various wavelengths. To determine the feasibility of stimulation in utero, a xenon lamp and waveguide were used to transilluminate the abdomen of several labor patients. Light in the range of 630 to 670 nm where the eye sensitivity and penetration depth are well matched, will likely provide the best chance of visual stimulation. Fetal heart rate, fetal movement, and fetal magnetoencephalography (SQUID) and electroencephalography (EEG) were observed in different studies to determine if stimulation has occurred. Since internal organs and the fetus are completely dark adapted, the amount of light required to simulate in our opinion could be on the order of 10(superscript -8 Watts.

  1. Penetration in bimodal, polydisperse granular material

    KAUST Repository

    Kouraytem, N.


    We investigate the impact penetration of spheres into granular media which are compositions of two discrete size ranges, thus creating a polydisperse bimodal material. We examine the penetration depth as a function of the composition (volume fractions of the respective sizes) and impact speed. Penetration depths were found to vary between delta = 0.5D(0) and delta = 7D(0), which, for mono-modal media only, could be correlated in terms of the total drop height, H = h + delta, as in previous studies, by incorporating correction factors for the packing fraction. Bimodal data can only be collapsed by deriving a critical packing fraction for each mass fraction. The data for the mixed grains exhibit a surprising lubricating effect, which was most significant when the finest grains [d(s) similar to O(30) mu m] were added to the larger particles [d(l) similar to O(200 - 500) mu m], with a size ratio, epsilon = d(l)/d(s), larger than 3 and mass fractions over 25%, despite the increased packing fraction. We postulate that the small grains get between the large grains and reduce their intergrain friction, only when their mass fraction is sufficiently large to prevent them from simply rattling in the voids between the large particles. This is supported by our experimental observations of the largest lubrication effect produced by adding small glass beads to a bed of large sand particles with rough surfaces.

  2. Penetration in bimodal, polydisperse granular material (United States)

    Kouraytem, N.; Thoroddsen, S. T.; Marston, J. O.


    We investigate the impact penetration of spheres into granular media which are compositions of two discrete size ranges, thus creating a polydisperse bimodal material. We examine the penetration depth as a function of the composition (volume fractions of the respective sizes) and impact speed. Penetration depths were found to vary between δ =0.5 D0 and δ =7 D0 , which, for mono-modal media only, could be correlated in terms of the total drop height, H =h +δ , as in previous studies, by incorporating correction factors for the packing fraction. Bimodal data can only be collapsed by deriving a critical packing fraction for each mass fraction. The data for the mixed grains exhibit a surprising lubricating effect, which was most significant when the finest grains [ds˜O (30 ) μ m ] were added to the larger particles [dl˜O (200 -500 ) μ m ] , with a size ratio, ɛ =dl/ds , larger than 3 and mass fractions over 25%, despite the increased packing fraction. We postulate that the small grains get between the large grains and reduce their intergrain friction, only when their mass fraction is sufficiently large to prevent them from simply rattling in the voids between the large particles. This is supported by our experimental observations of the largest lubrication effect produced by adding small glass beads to a bed of large sand particles with rough surfaces.

  3. Penetrating spinal injuries and their management

    Directory of Open Access Journals (Sweden)

    A Kumar


    Full Text Available Penetrating spinal trauma due to missile/gunshot injuries has been well reported in the literature and has remained the domain of military warfare more often. Civic society′s recent upsurge in gunshot injuries has created a dilemma for the treating neurosurgeon in many ways as their management has always involved certain debatable and controversial issues. Both conservative and surgical management of penetrating spinal injuries (PSI have been practiced widely. The chief neurosurgical concern in these types of firearm injuries is the degree of damage sustained during the bullet traversing through the neural tissue and the after-effects of the same in long term. We had an interesting case of a penetrating bullet injury to cervical spine at C2 vertebral level. He was operated and the bullets were removed from posterior midline approach. Usually, the management of such cases differs from region to region depending on the preference of the surgeon but still certain common principles are followed world over. Thus, we realized the need to review the literature regarding spinal injuries with special emphasis on PSI and to study the recent guidelines for their treatment in light of our case.

  4. Penetration Testing Professional Ethics: a conceptual model and taxonomy


    Justin Pierce; Ashley Jones; Matthew Warren


    In an environment where commercial software is continually patched to correct security flaws, penetration testing can provide organisations with a realistic assessment of their security posture. Penetration testing uses the same principles as criminal hackers to penetrate corporate networks and thereby verify the presence of software vulnerabilities. Network administrators can use the results of a penetration test to correct flaws and improve overall security. The use of hacking techniques...

  5. Penetration Testing Curriculum Development in Practice

    Directory of Open Access Journals (Sweden)

    Chengcheng Li


    Full Text Available As both the frequency and the severity of network breaches have increased in recent years, it is essential that cybersecurity is incorporated into the core of business operations. Evidence from the U.S. Bureau of Labor Statistics (Bureau of Labor Statistics, 2012 indicates that there is, and will continue to be, a severe shortage of cybersecurity professionals nationwide throughout the next decade. To fill this job shortage we need a workforce with strong hands-on experience in the latest technologies and software tools to catch up with the rapid evolution of network technologies. It is vital that the IT professionals possess up-to-date technical skills and think and act one step ahead of the cyber criminals who are constantly probing and exploring system vulnerabilities. There is no perfect security mechanism that can defeat all the cyber-attacks; the traditional defensive security mechanism will eventually fail to the pervasive zero-day attacks. However, there are steps to follow to reduce an organization’s vulnerability to cyber-attacks and to mitigate damages. Active security tests of the network from a cyber-criminal’s perspective can identify system vulnerabilities that may lead to future breaches. “If you know yourself but not the enemy, for every victory gained you will also suffer a defeat. But if you know the enemy and know yourself, you need not fear the result of hundred battles” (Sun, 2013. Penetration testing is a discipline within cybersecurity that focuses on identifying and exploiting the vulnerabilities of a network, eventually obtaining access to the critical business information. The pentesters, the security professionals who perform penetration testing, or ethical hackers, break the triad of information security - Confidentiality, Integrity, and Accountability (CIA - as if they were a cyber-criminal. The purpose of ethical hacking or penetration testing is to know what the “enemy” can do and then generate a

  6. Ultrastructure and penetration of cell co-culture model of human skin equivalent%细胞共培养人体皮肤类似物超微结构观察及其渗透性测试

    Institute of Scientific and Technical Information of China (English)

    李辉; 王景雁; 林红梅; 于竞新; 章烨雯; 毛颖秋; 吴清


    目的:观察人体皮肤类似物模型形态结构,对其屏障功能进行初步评价。方法通过胶原蛋白包被成纤维细胞,制备真皮层,真皮层表面接种角质细胞,浸没培养1周后,气-液界面培养以促进角质细胞的分化形成角质层,3周后收获皮肤模型;通过肉眼观察、电镜观察皮肤类似物的宏观结构和超微结构;以吲哚美辛作为模型药物,进行体外透皮实验考察细胞共培养皮肤类似物模型的屏障功能。结果皮肤类似物形成与人类皮肤相似的真皮层和表皮层,表皮层表面形成完整的角质层,具有与人类皮肤类似的超微结构;24 h 内渗透性实验结果显示皮肤类似物屏障功能略低于大鼠腹部皮肤,但22~24 h,皮肤类似物对模型药的透过速率远小于大鼠腹部皮肤(约为其28%),且2种皮肤模型对吲哚美辛经皮渗透的稳态流速没有显著性差异(P >0.05)。结论皮肤类似物有与人体皮肤相类似的形态结构特征,与大鼠腹部皮肤相比对吲哚美辛有相似的屏障作用,有望替代动物皮肤成为经皮给药系统评价的新型皮肤模型。%Abatract:Objective To observe the structure of human skin equivalent (HSE)model in vitro,and to preliminarily assess the barrier function of HSE.Methods The HSE model was established by two steps:preparation of dermal layer and preparation of full-thickness skin.Keratinocytes were inoculated on the surface of dermal layer prepared by type 1 collagen coating fibroblasts.After submerged cultured for one week,air-liquid interface cultured for three weeks to enhance the proliferation of keratinocytes,the HSE model was obtained.Then the macroscopical structure and electon microscopical structure of HSE were studied.To investigate the barrier function of HSE model,indometacin was selected as the model drug for the penetration testing in vitro.Results The structures similar to dermal layer and

  7. Hybrid Collagenase Nanocapsules for Enhanced Nanocarrier Penetration in Tumoral Tissues. (United States)

    Villegas, María Rocío; Baeza, Alejandro; Vallet-Regí, María


    Poor penetration of drug delivery nanocarriers within dense extracellular matrices constitutes one of the main liabilities of current nanomedicines. The conjugation of proteolytic enzymes on the nanoparticle surface constitutes an attractive alternative. However, the scarce resistance of these enzymes against the action of proteases or other aggressive agents present in the bloodstream strongly limits their application. Herein, a novel nanodevice able to transport proteolytic enzymes coated with an engineered pH-responsive polymeric is presented. This degradable coat protects the housed enzymes against proteolytic attack at the same time that it triggers their release under mild acidic conditions, usually present in many tumoral tissues. These enzyme nanocapsules have been attached on the surface of mesoporous silica nanoparticles, as nanocarrier model, showing a significatively higher penetration of the nanoparticles within 3D collagen matrices which housed human osteosarcoma cells (HOS). This strategy can improve the therapeutic efficacy of the current nanomedicines, allowing a more homogeneous and deeper distribution of the therapeutic nanosystems in cancerous tissues.

  8. A SNARE-protein has opposing functions in penetration resistance and defence signalling pathways

    DEFF Research Database (Denmark)

    Zhang, Ziguo; Feechan, Angela; Pedersen, Carsten


    Penetration resistance is often the first line of defence against fungal pathogens. Subsequently induced defences are mediated by the programmed cell death (PCD) reaction pathway and the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signalling pathways. We previously demonstrated...

  9. Radon penetration of concrete slab cracks, joints, pipe penetrations, and sealants. (United States)

    Nielson, K K; Rogers, V C; Holt, R B; Pugh, T D; Grondzik, W A; de Meijer, R J


    Radon movement through 12 test slabs with different cracks, pipe penetrations, cold joints, masonry blocks, sealants, and tensile stresses characterized the importance of these anomalous structural domains. Diffusive and advective radon transport were measured with steady-state air pressure differences controlled throughout the deltaP = 0 to 60 Pa range. Diffusion coefficients (deltaP = 0) initially averaged 6.5 x 10(-8) m2 s(-1) among nine slabs with only 8% standard deviation, but increased due to drying by 0.16% per day over a 2-y period to an average of 2.0 x 10(-7) m2 s(-1). An asphalt coating reduced diffusion sixfold but an acrylic surface sealant had no effect. Diffusion was 42 times higher in solid masonry blocks than in concrete and was not affected by small cracks. Advective transport (deltaP permeability), pipe penetrations, and caulked gaps, but was significant for cracks, disturbed pipe penetrations, cold joints, masonry blocks, and concrete under tensile stress. Crack areas calculated to be as small as 10(-7) m2 significantly increased radon advection. Algebraic expressions predict air velocity and effective crack width from enhanced radon transport and air pressures. Masonry blocks, open cracks, and slab cold joints enhance radon penetration but stressed slabs, undisturbed pipe penetrations, and sealed cracks may not.

  10. An overview of skin penetration enhancers: penetration enhancing activity, skin irritation potential and mechanism of action

    Directory of Open Access Journals (Sweden)

    Sarunyoo Songkro


    Full Text Available Transdermal drug delivery has attracted considerable attention over the past 2-3 decades in regard of its many potentialadvantages. However, the role of the skin as a protective barrier renders skin absorption of most drugs problematic. Therefore,skin penetration enhancers are frequently used in the field of transdermal drug delivery in order to reversibly reduce thebarrier function of the stratum corneum, the outermost layer of the skin. To date, a wide range of chemical compounds havebeen shown to enhance the skin penetration of therapeutic drugs. This review presents a critical account of the most commonlyused chemical penetration enhancers (fatty acids and surfactants, and some newer classes of chemical enhancers (terpenes,polymers, monoolein, oxazolidinones, with emphasis on their efficacy, mechanism of action, and skin irritation potential. Thisreview also discusses the traditional and more recently developed methods for the screening and evaluation of chemical penetration enhancers, and addresses the continuing problems in the rational selection of a chemical penetration enhancer for a specific drug to be delivered via the transdermal route.

  11. Effects of Sizes and Conformations of Fish-Scale Collagen Peptides on Facial Skin Qualities and Transdermal Penetration Efficiency

    Directory of Open Access Journals (Sweden)

    Huey-Jine Chai


    Full Text Available Fish-scale collagen peptides (FSCPs were prepared using a given combination of proteases to hydrolyze tilapia (Oreochromis sp. scales. FSCPs were determined to stimulate fibroblast cells proliferation and procollagen synthesis in a time- and dose-dependent manner. The transdermal penetration capabilities of the fractionationed FSCPs were evaluated using the Franz-type diffusion cell model. The heavier FSCPs, 3500 and 4500 Da, showed higher cumulative penetration capability as opposed to the lighter FSCPs, 2000 and 1300 Da. In addition, the heavier seemed to preserve favorable coiled structures comparing to the lighter that presents mainly as linear under confocal scanning laser microscopy. FSCPs, particularly the heavier, were concluded to efficiently penetrate stratum corneum to epidermis and dermis, activate fibroblasts, and accelerate collagen synthesis. The heavier outweighs the lighter in transdermal penetration likely as a result of preserving the given desired structure feature.

  12. Penetration Testing Professional Ethics: a conceptual model and taxonomy

    Directory of Open Access Journals (Sweden)

    Justin Pierce


    Full Text Available In an environment where commercial software is continually patched to correct security flaws, penetration testing can provide organisations with a realistic assessment of their security posture. Penetration testing uses the same principles as criminal hackers to penetrate corporate networks and thereby verify the presence of software vulnerabilities. Network administrators can use the results of a penetration test to correct flaws and improve overall security. The use of hacking techniques, however, raises several ethical questions that centre on the integrity of the tester to maintain professional distance and uphold the profession. This paper discusses the ethics of penetration testing and presents our conceptual model and revised taxonomy.

  13. Effect of Liquid Penetrant Sensitivity on Probability of Detection (United States)

    Parker, Bradford H.


    The objective of the task is to investigate the effect of liquid penetrant sensitivity level on probability of crack detection (POD). NASA-STD-5009 currently requires the use of only sensitivity level 4 liquid penetrants. This requirement is based on the fact that the data generated in the NTIAC Nondestructive Evaluation (NDE) Capabilities Data Book was produced using only sensitivity level 4 penetrants. Many NDE contractors supporting NASA Centers routinely use sensitivity level 3 penetrants. Because of the new NASA-STD-5009 requirement, these contractors will have to either shift to sensitivity level 4 penetrants or perform formal POD demonstration tests to qualify their existing process.

  14. Mastering Kali Linux for advanced penetration testing

    CERN Document Server

    Beggs, Robert W


    This book provides an overview of the kill chain approach to penetration testing, and then focuses on using Kali Linux to provide examples of how this methodology is applied in the real world. After describing the underlying concepts, step-by-step examples are provided that use selected tools to demonstrate the techniques. If you are an IT professional or a security consultant who wants to maximize the success of your network testing using some of the advanced features of Kali Linux, then this book is for you. This book will teach you how to become an expert in the pre-engagement, management,

  15. Penetrating abdominal injuries in children in Nigeria. (United States)

    Ameh, E A; Nmadu, P T


    This is a report of a retrospective study of 24 children managed for penetrating abdominal injury over 10 years, and it represents 34% of all abdominal injuries in children in that period. Falls onto sharp objects within and around the home were responsible for ten of the injuries, seven were injured by animal horns and four were sporting injuries. Violence and road traffic accidents were uncommon. Most patients (67%) had evisceration of omentum or intestine, and one of these was found at laparotomy to have a jejuno-jejunal intussusception. Seven children had injury to hollow viscera. There were three deaths, one each from overwhelming sepsis, tetanus and haemorrhage.

  16. Penetrating wounds of the head and neck. (United States)

    Jahrsdoerfer, R A; Johns, M E; Cantrell, R W


    Wounding capability of bullets is primarily releated to velocity. Bullet mass and shape, and specific gravity of body tissues being struck by the missile, are lesser factors. Seventy cases of penetrating wounds of the head and neck were treated during a six-year period. Vascular injuries were more common with neck wounds, while face and head injuries (extracranial) were similar to maxillofacial trauma. It is recognized that hemorrhage at the base of the skull is difficult to treat, and contemporary training in temporal bone and base of skull surgery is mandatory for the critical management of these wounds.

  17. [Penetrating ocular trauma with intraocular foreign body]. (United States)

    Musat, O; Ochinciuc, Uliana; Gutu, Tatiana; Cristescu, T R; Coman, Corina


    We present the case of a 65 years old pacient which was admitted for the sudden decrease of visual acuity in the left eye, accompanied by ocular pain and conjunctival hiperemia, simptoms appeared after an ocular trauma. After the clinical and paraclinical examination we determined the diagnosis of OS: Penetrating ocular trauma with retention of a foreign body; posttraumatic cataract. Surgical treatement was warrented and we performed OS : Facoemulsification + PFK implant in sulcus + 23 Ga posterior vitrectomy + peeling of the posterior hyaloid membrane + extraction of the foreign body + LASER endofotocoagulation + transscleral cryotherapy + SF6 gas injection. The post-operatory evolution was favorable.

  18. Premium Efficiency Motors And Market Penetration Policy

    Energy Technology Data Exchange (ETDEWEB)

    Benhaddadi, Mohamed; Olivier, Guy


    This paper illustrates the induced enormous energy saving potential, permitted by using high-efficiency motors. Furthermore, the most important barriers to larger high-efficiency motors utilization are identified, and some incentives recommendations are given to overcome identified impediments. The authors consider that there is a strong case to enhance incentives policies for larger market penetration. The US Energy Policy Act and the Canadian Energy Efficient Act have lead to North American leadership on motor efficiency implementation. North America is not on the leading edge for energy saving and conservation. Motor efficiency is an exception that should be at least maintained.

  19. Kali Linux assuring security by penetration testing

    CERN Document Server

    Ali, Shakeel; Allen, Lee


    Written as an interactive tutorial, this book covers the core of Kali Linux with real-world examples and step-by-step instructions to provide professional guidelines and recommendations for you. The book is designed in a simple and intuitive manner that allows you to explore the whole Kali Linux testing process or study parts of it individually.If you are an IT security professional who has a basic knowledge of Unix/Linux operating systems, including an awareness of information security factors, and want to use Kali Linux for penetration testing, then this book is for you.

  20. Hacking with Kali practical penetration testing techniques

    CERN Document Server

    Broad, James


    Hacking with Kali introduces you the most current distribution of the de facto standard tool for Linux pen testing. Starting with use of the Kali live CD and progressing through installation on hard drives, thumb drives and SD cards, author James Broad walks you through creating a custom version of the Kali live distribution. You'll learn how to configure networking components, storage devices and system services such as DHCP and web services. Once you're familiar with the basic components of the software, you'll learn how to use Kali through the phases of the penetration testing lifecycle

  1. Retained crossbow bolt after penetrating facial trauma. (United States)

    Shah, Manan U; Sridhara, Shankar K; Wolf, Jeffrey S; Ambro, Bryan T


    We present an unusual case of a retained crossbow bolt in the maxillofacial area of a 31-year-old man. While crossbow injuries are rare, this case is of interest because otolaryngologists are often faced with treating retained foreign objects after penetrating facial trauma. These cases are difficult to manage because of the complexity and variety of injuries that can occur during both the initial trauma and the removal. We focus on the management of the bolt's removal and provide a brief discussion of the relevant literature on crossbow injuries to the head and neck.

  2. Aerodynamic Jump for Long Rod Penetrators


    Mark L. Bundy


    Aerodynamic jump for a non-spinning kinetic energy penetrator is neither a discontinuous change in the ,direction of motion at the origin of free night, nor is it the converse, i.e. a cumulativer~direc4on over a domain of infinite extent. Rather aerodynamic jump, for such a projectile, is a localised redirection of the centre of gravity motion, caused ~ the force of lift due to yaw over ther4latively short region from entry into free flight until the yaw reaches its first maximum. The primary...

  3. Suicide bomb attack causing penetrating craniocerebral injury

    Institute of Scientific and Technical Information of China (English)

    Manzar Hussain; Muhammad Ehsan Bari


    Penetrating cerebral injuries caused by foreign bodies are rare in civilian neurosurgical trauma,although there are various reports of blast or gunshot injuries in warfare due to multiple foreign bodies like pellets and nails.In our case,a 30-year-old man presented to neurosurgery clinic with signs and symptoms of right-sided weakness after suicide bomb attack.The skull X-ray showed a single intracranial nail.Small craniotomy was done and the nail was removed with caution to avoid injury to surrounding normal brain tissue.At 6 months' follow-up his right-sided power improved to against gravity.

  4. Effect of sodium lauryl sulfate (SLS) on in vitro percutaneous penetration of water, hydrocortisone and nickel

    DEFF Research Database (Denmark)

    Frankild, S; Andersen, Klaus Ejner; Nielsen, Gunnar


    as membrane in static in vitro penetration cells. Simultaneous application of SLS together with 1 of the tracer compounds showed, after 48 h, a significant dose-effect relationship between SLS concentration (0.25%, 2% and 10%) and penetration of tritiated water or nickel (p ... or hydrocortisone. Pretreatment of the skin with SLS for 2 h using 3 concentrations (0.25%, 4% and 10%) showed, after 48 h, a significant dose-effect relationship between SLS treatment and penetration of tritiated water or nickel (p .../damaging effect on the skin barrier. It should be kept in mind that the model uses a dead skin membrane without the barrier repair mechanisms of live skin....

  5. Strategies to increase drug penetration in solid tumors

    Directory of Open Access Journals (Sweden)

    Il-Kyu eChoi


    Full Text Available Despite significant improvement in modalities for treatment of cancer that led to a longer survival period, the death rate of patients with solid tumors has not changed during the last decades. Emerging studies have identified several physical barriers that limit the therapeutic efficacy of cancer therapeutic agents such as monoclonal antibodies, chemotherapeutic agents, antitumor immune cells, and gene therapeutics. Most solid tumors are of epithelial origin and, although malignant cells are de-differentiated, they maintain intercellular junctions, a key feature of epithelial cells, both in the primary tumor as well as in metastatic lesions. Furthermore, nests of malignant epithelial tumor cells are shielded by layers of extracellular matrix (ECM proteins (e.g. collagen, elastin, fibronectin, laminin whereby tumor vasculature rarely penetrates into the tumor nests. In this chapter, we will review potential strategies to modulate the ECM and epithelial junctions to enhance the intratumoral diffusion and/or to remove physical masking of target receptors on malignant cells. We will focus on peptides that bind to the junction protein desmoglein 2 (DSG2 and trigger intracellular signaling, resulting in the transient opening of intercellular junctions. Intravenous injection of these junction openers increased the efficacy and safety of therapies with monoclonal antibodies, chemotherapeutics, and T-cells in mouse tumor models and was safe in non-human primates. Furthermore, we will summarize approaches to transiently degrade ECM proteins or downregulate their expression. Among these approaches is the intratumoral expression of relaxin or decorin after adenovirus (Ad- or stem cell-mediated gene transfer. We will provide examples that relaxin- based approaches increase the antitumor efficacy of oncolytic viruses, monoclonal antibodies, and T-cells.

  6. Root cap removal increases root penetration resistance in maize (Zea mays L). (United States)

    Iijima, Morio; Higuchi, Toshifumi; Barlow, Peter W; Bengough, A Glyn


    The root cap assists the passage of the root through soil by means of its slimy mucilage secretion and by the sloughing of its outer cells. The root penetration resistance of decapped primary roots of maize (Zea mays L. cv. Mephisto) was compared with that of intact roots in loose (dry bulk density 1.0 g cm-3; penetration resistance 0.06 MPa) and compact soil (1.4 g cm-3; penetration resistance 1.0 MPa), to evaluate the contribution of the cap to decreasing the impedance to root growth. Root elongation rate and diameter were the same for decapped and intact roots when the plants were grown in loose soil. In compacted soil, however, the elongation rate of decapped roots was only about half that of intact roots, whilst the diameter was 30% larger. Root penetration resistances of intact and decapped seminal axis were 0.31 and 0.52 MPa, respectively, when the roots were grown in compacted soil. These results indicated that the presence of a root cap alleviates much of the mechanical impedance to root penetration, and enables roots to grow faster in compacted soils.

  7. Lipid peroxidation and water penetration in lipid bilayers

    DEFF Research Database (Denmark)

    Conte, Elena; Megli, Francesco Maria; Khandelia, Himanshu


    changes in the acyl chain order in the sub-polar region and at the methyl-terminal induced by lipid peroxidation were detected by X-band EPR. Concomitantly, the polarity and proticity of the membrane bilayer in those regions were investigated at W band in frozen samples. Analysis of the g(xx) and A......(zz) parameters revealed that OHPLPC, but mostly HpPLPC, induced a measurable increase in polarity and H-bonding propensity in the central region of the bilayer. Molecular dynamics simulation performed on 16-DSA in the PLPC-HpPLPC bilayer revealed that water molecules are statistically favored with respect...... to the hydroperoxide groups to interact with the nitroxide at the methyl-terminal, confirming that the H-bonds experimentally observed are due to increased water penetration in the bilayer. The EPR and MD data on model membranes demonstrate that cell membrane damage by oxidative stress cause alteration of water...

  8. Analysis of liquid penetration in paper structures by advanced imaging techniques (United States)

    Arthur, Beth Ann

    Ink penetration in paper is influenced by the structure of the interfiber and intrafiber void spaces and the surface characteristics of the fibers. This dissertation describes new techniques to determine the influence of the fiber surfaces and the cell wall internal structure on ink spreading and penetration. The location and penetration of ink is demonstrated by optical, scanning electron, confocal laser scanning, and transmission electron (TEM) microscopy methods. Ink penetration, as determined by each of these methods, is compared. The hemicelluloses of the fiber's internal void surfaces can be determined by immunochemical labeling in conjunction with TEM imaging. It is demonstrated through the use of primary monoclonal antibodies with specificity for hemicelluloses with a secondary colloidal gold marker. This technique provides a way to visualize the location of hemicelluloses inside the cell wall and on the surfaces of nanopores. Combining paper structure with fluid spreading and wicking models can identify the influence of fiber surfaces and the cell wall on drop absorption. Ink spreading coefficients for such modeling are determined through a series of designed experiments (DoE) and comparisons to a theoretical sessile drop. Application: Microscopic techniques used to determine biological and physical locations in plants on a fibrous level also can be used to study ink diffusion, water uptake, and other characteristics of fibrous material. Key Words: Immunolableing of hemicelluloses, DoE, drop spread modeling, ink diffusion.

  9. Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain.

    Directory of Open Access Journals (Sweden)

    Pranav Danthi


    Full Text Available Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein mu1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of mu1 indicates that the mu1 phi domain is sufficient to elicit a cell death response. To evaluate the contribution of the mu1 phi domain to the induction of apoptosis following reovirus infection, phi mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective phi mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

  10. Penetration of projectiles into granular targets (United States)

    Ruiz-Suárez, J. C.


    Energetic collisions of subatomic particles with fixed or moving targets have been very valuable to penetrate into the mysteries of nature. But the mysteries are quite intriguing when projectiles and targets are macroscopically immense. We know that countless debris wandering in space impacted (and still do) large asteroids, moons and planets; and that millions of craters on their surfaces are traces of such collisions. By classifying and studying the morphology of such craters, geologists and astrophysicists obtain important clues to understand the origin and evolution of the Solar System. This review surveys knowledge about crater phenomena in the planetary science context, avoiding detailed descriptions already found in excellent papers on the subject. Then, it examines the most important results reported in the literature related to impact and penetration phenomena in granular targets obtained by doing simple experiments. The main goal is to discern whether both schools, one that takes into account the right ingredients (planetary bodies and very high energies) but cannot physically reproduce the collisions, and the other that easily carries out the collisions but uses laboratory ingredients (small projectiles and low energies), can arrive at a synergistic intersection point.

  11. [Videothoracospy in thoracic trauma and penetrating injuries]. (United States)

    Lang-Lazdunski, L; Chapuis, O; Pons, F; Jancovici, R


    Videothoracoscopy represents a valid and useful approach in some patients with blunt chest trauma or penetrating thoracic injury. This technique has been validated for the treatment of clotted hemothorax or posttraumatic empyema, traumatic chylothorax, traumatic pneumothorax, in patients with hemodynamic stability. Moreover, it is probably the most reliable technique for the diagnosis of diaphragmatic injury. It is also useful for the extraction of intrathoracic projectiles and foreign bodies. This technique might be useful in hemodynamically stable patients with continued bleeding or for the exploration of patients with penetrating injury in the cardiac area, although straightforward data are lacking to confirm those indications. Thoracotomy or median sternotomy remain indicated in patients with hemodynamic instability or those that cannot tolerate lateral decubitus position or one-lung ventilation. Performing video-surgery in the trauma setting require expertise in both video-assisted thoracic surgery and chest trauma management. The contra-indications to videothoracoscopy and indications for converting the procedure to an open thoracotomy should be perfectly known by surgeons performing video-assisted thoracic surgery in the trauma setting. Conversion to thoracotomy or median sternotomy should be performed without delay whenever needed to avoid blood loss and achieve an adequate procedure.

  12. Ground penetrating radar for asparagus detection (United States)

    Seyfried, Daniel; Schoebel, Joerg


    Ground penetrating radar is a promising technique for detection of buried objects. Recently, radar has more and more been identified to provide benefits for a plurality of applications, where it can increase efficiency of operation. One of these fields is the industrial automatic harvesting process of asparagus, which is performed so far by cutting the soil ridge at a certain height including all the asparagus spears and subsequently sieving the latter out of the soil. However, the height where the soil is cut is a critical parameter, since a wrong value leads to either damage of the roots of the asparagus plants or to a reduced crop yield as a consequence of too much biomass remaining in the soil. In this paper we present a new approach which utilizes ground penetrating radar for non-invasive sensing in order to obtain information on the optimal height for cutting the soil. Hence, asparagus spears of maximal length can be obtained, while keeping the roots at the same time undamaged. We describe our radar system as well as the subsequent digital signal processing steps utilized for extracting the information required from the recorded radar data, which then can be fed into some harvesting unit for setting up the optimal cutting height.

  13. Foliar penetration enhanced by biosurfactant rhamnolipid. (United States)

    Liu, Haojing; Shao, Bing; Long, Xuwei; Yao, Yang; Meng, Qin


    With recent environmental and health concerns, biosurfactants have obtained increasing interest in replacing conventional surfactants for diverse applications. In agriculture, the use of surfactant in stimulating foliar uptake is mainly for wetting leaf surface, resisting deposition/evaporation, enhancing penetration across cuticular membrane (CM) and translocation. This paper aimed to address the improved foliar uptake by rhamnolipid (RL) in comparison with the currently used alkyl polyglucoside (APG). As found, compared with APG at 900mg/L (1×critical micellar concentration, CMC), RL at a much lower concentration of 50mg/L (1×CMC) showed much better wettability and surface activity, indicative of its high effectiveness as surfactants. Its performance on resistance to deposition and evaporation was at least as same as APG. Moreover, RL could significantly improve the penetration of herbicide glyphosate and other two small water-soluble molecules (phenol red and Fe(2+)) across CM at an equivalent efficiency as APG at 1×CMC. Finally, the greatly enhanced herbicidal actitivity of glyphosate on greenhouse plants confirmed that RL and APG could both enhance the foliar uptake including translocation. Overall, RL should be more applicable than APG in agriculture due to its more promising properties on health/environmental friendliness.

  14. Penetrating eye injuries from writing instruments

    Directory of Open Access Journals (Sweden)

    Kelly SP


    Full Text Available Simon P Kelly, Graham MB ReevesThe Royal Bolton Hospital, Bolton, UKPurpose: To consider the potential for ocular injury from writing implements by presenting four such cases, and to consider the incidence of such eye injuries from analysis of a national trauma database.Methods: The Home and Leisure Accident Surveillance System was searched for records of eye injuries from writing instruments to provide UK estimates of such injuries. Four patients with ocular penetrating injury from pens or pencils (especially when caused by children, and examined by the authors, are described which illustrate mechanisms of injury.Results: It is estimated that around 748 ocular pen injuries and 892 ocular pencil injuries of undetermined severity occurred annually in the UK during the database surveillance period 2000–2002. No eye injuries from swords, including toy swords and fencing foils, were reported.Conclusion: Ocular perforation sometimes occur from writing instruments that are thrown in the community, especially by children. Implications for policy and prevention are discussed. Non-specialists should have a low threshold for referring patients with eye injuries if suspicious of ocular penetration, even where caused by everyday objects, such as writing instruments.Keywords: eye injury, eye, children, mechanism, writing instruments, prevention

  15. Penetration through a wall: Is it reality?

    CERN Document Server

    Ivlev, B


    A tennis ball is not expected to penetrate through a brick wall since a motion under a barrier is impossible in classical mechanics. With quantum effects a motion of a particle through a barrier is allowed due to quantum tunneling. According to usual theories of tunneling, the particle density decays inside a classical barrier resulting in an extremely slow pentration process. However, there are no general laws forbidding fast motion through classical barriers. The problem addressed is investigation of unusual features o quantum tunneling through a classic static barrier which is at least two-dimensional. Here we show that penetration through such barrier can be not slow. When the barrier satisfies the certain conditions, a regime of quantum lens is possible with formation of caustics. De Broglie waves are reflected from the caustics, interfere, and result in a not small flux from under the barrier. This strongly contrasts to the usual scenario with a decaying under-barrier density. We construct a particular ...

  16. High-Penetration Photovoltaic Planning Methodologies

    Energy Technology Data Exchange (ETDEWEB)

    Gao, David Wenzhong [Alternative Power Innovations, LLC, Broomfield, CO (United States); Muljadi, Eduard [National Renewable Energy Lab. (NREL), Golden, CO (United States); Tian, Tian [National Renewable Energy Lab. (NREL), Golden, CO (United States); Miller, Mackay [National Renewable Energy Lab. (NREL), Golden, CO (United States)


    The main objective of this report is to provide an overview of select U.S. utility methodologies for performing high-penetration photovoltaic (HPPV) system planning and impact studies. This report covers the Federal Energy Regulatory Commission's orders related to photovoltaic (PV) power system interconnection, particularly the interconnection processes for the Large Generation Interconnection Procedures and Small Generation Interconnection Procedures. In addition, it includes U.S. state interconnection standards and procedures. The procedures used by these regulatory bodies consider the impacts of HPPV power plants on the networks. Technical interconnection requirements for HPPV voltage regulation include aspects of power monitoring, grounding, synchronization, connection to the overall distribution system, back-feeds, disconnecting means, abnormal operating conditions, and power quality. This report provides a summary of mitigation strategies to minimize the impact of HPPV. Recommendations and revisions to the standards may take place as the penetration level of renewables on the grid increases and new technologies develop in future years.

  17. Plasma penetration of the dayside magnetopause

    Energy Technology Data Exchange (ETDEWEB)

    Gunell, H.; Maggiolo, R. [Belgian Institute for Space Aeronomy, Avenue Circulaire 3, B-1180 Brussels (Belgium); Nilsson, H.; Stenberg, G.; Lundin, R. [Swedish Institute of Space Physics, P.O. Box 812, SE-981 28 Kiruna (Sweden); Hamrin, M. [Department of Physics, Umea University, SE-901 87 Umea (Sweden); Karlsson, T. [Space and Plasma Physics, Royal Institute of Technology (KTH), SE-100 44 Stockholm (Sweden); Andre, M. [Swedish Institute of Space Physics, Box 537, SE-751 21 Uppsala (Sweden); Dandouras, I. [Institut de Recherche en Astrophysique et Planetologie, UPS-CNRS, Toulouse (France)


    Data from the Cluster spacecraft during their magnetopause crossing on 25 January 2002 are presented. The magnetopause was in a state of slow non-oscillatory motion during the observational period. Coherent structures of magnetosheath plasma, here typified as plasmoids, were seen on closed magnetic field lines on the inside of the magnetopause. Using simultaneous measurements on two spacecraft, the inward motion of the plasmoids is followed from one spacecraft to the next, and it is found to be in agreement with the measured ion velocity. The plasma characteristics and the direction of motion of the plasmoids show that they have penetrated the magnetopause, and the observations are consistent with the concept of impulsive penetration, as it is known from theory, simulations, and laboratory experiments. The mean flux across the magnetopause observed was 0.2%-0.5% of the solar wind flux at the time, and the peak values of the flux inside the plasmoids reached approximately 20% of the solar wind flux.

  18. Feature Profile Simulations and Finite Penetration Depth (United States)

    Moroz, Paul; Moroz, Daniel


    In plasma materials processing, energetic ions, neutrals and UV photons typically penetrate deep inside solid materials breaking atomic bonds and displacing atoms on their paths. These important phenomena are rarely taken into consideration in processing simulation software, primarily because the proper penetration depths and the corresponding energy depositions, breaking bonds, and atom displacements are difficult and computationally expensive to compute. The FPS-3D feature profile simulator [1-2] is doing that computationally efficiently by utilizing tabulated results obtained with other methods. We discuss, compare, and present results of such simulations made with different methods, one of which is the molecular dynamics analysis. In general, molecular dynamics could be used for simulating materials processing, etching and deposition, but it is extremely computationally expensive to be used for large groups of atoms. In practice, molecular dynamics methods are too slow to be used for feature profile simulations. However, they could help in defining proper chemical reactions and corresponding rates to be used in an advanced feature profile simulator such as FPS-3D. We present results of FPS-3D simulations for Si and SiO2 etching in Ar/Cl2 and Ar/C4F6/O2 plasmas. [4pt] [1] P. Moroz, ``General Feature Profile Simulator FPS-3D,'' ECS Transactions, 35, 25 (2011). [0pt] [2] P. Moroz, ``Numerical Simulation of Feature Profile Evolution using FPS-3D,'' IEEE Transactions of Plasma Science, 39, 2804 (2011).

  19. Intraductal Papillary-Mucinous Neoplasm of the Pancreas Penetrating to the Stomach and the Common Bile Duct

    Directory of Open Access Journals (Sweden)

    Norihiro Goto


    Full Text Available Context Intraductal papillary mucinous neoplasm (IPMN of the pancreas occasionally penetrates to others organs. We present a case of IPMN penetrating to the stomach and the common bile duct. Case report A 75-year-old man was admitted to the hospital because of epigastric pain. Computed tomography (CT showed a papillary tumor protruding into the markedly dilated main pancreatic duct and splenic vein obstruction. The tumor was diagnosed as IPMN arising in the main duct, but he rejected surgery and he was followed without treatment. One year later, gastroduodenoscopy revealed gastropancreatic fistula and we were able to pass an endoscope through the fistula and directly examine the lumen of the main pancreatic duct and the papillary tumor adjacent to the fistula. Absence of malignant cells on histopathology suggested mechanical penetration rather than invasive penetration. CT showed splenic vein reperfusion due to decreased inner pressure of the main pancreatic duct. Two and a half years later, CT revealed biliopancreatic fistula formation. Endoscope biliary drainage was performed but failed. Despite jaundice, he is still ambulatory and seen in the clinic three years after the first admission. Conclusions We have experienced a case of IPMN penetrating to the stomach and the common bile duct that has taken a slow course. It represents the importance of distinguishing mechanical penetration from invasive penetration as well as mechanical splenic vein obstruction from splenic vein invasion.

  20. Radon penetration of concrete slab cracks, joints, pipe penetrations, and sealants

    NARCIS (Netherlands)

    Nielson, KK; Rogers, VC; Holt, RB; Pugh, TD; Grondzik, WA; deMeijer, RJ


    Radon movement through 12 test slabs with different cracks, pipe penetrations, cold joints, masonry blocks, sealants, and tensile stresses characterized the importance of these anomalous structural domains, Diffusive and advective radon transport were measured with steady-state air pressure differen

  1. Long-rod penetration:the transition zone between rigid and hydrodynamic penetration modes

    Institute of Scientific and Technical Information of China (English)

    Jian-feng LOU; Yan-geng ZHANG; Zheng WANG; Tao HONG; Xiao-li ZHANG; Shu-dao ZHANG


    Long-rod penetration in a wide range of velocity means that the initial impact velocity varies in a range from tens of meters per second to several kilometers per second. The long rods maintain rigid state when the impact velocity is low, the nose of rod deforms and even is blunted when the velocity gets higher, and the nose erodes and fails to lead to the consumption of long projectile when the velocity is very high due to instantaneous high pressure. That is, from low velocity to high velocity, the projectile undergoes rigid rods, deforming non-erosive rods, and erosive rods. Because of the complicated changes of the projectile, no well-established theoretical model and numerical simulation have been used to study the transition zone. Based on the analysis of penetration behavior in the transition zone, a phenomenological model to describe target resistance and a formula to calculate penetration depth in transition zone are proposed, and a method to obtain the boundary velocity of transition zone is determined. A combined theoretical analysis model for three response regions is built by analyzing the characteristics in these regions. The penetration depth predicted by this combined model is in good agreement with experimental result.

  2. Stylet penetration of Cacopsylla pyri; an electrical penetration graph (EPG) study

    NARCIS (Netherlands)

    Civolani, S.; Leis, M.; Grandi, G.; Garzo, E.; Pasqualini, E.; Musacchi, S.; Chicca, M.; Castaldelli, G.; Rossie, M.; Tjallingii, W.F.


    Detailed information on plant penetration activities by pear psylla Cacopsylla pyri L. (Hemiptera Psyllidae) is essential to study phytoplasma transmission of “Candidatus Phytoplasma pyri” responsible of pear decline disease (PD) and to trace and evaluate resistant traits in new pear tree selections

  3. Electromagnetic modelling of Ground Penetrating Radar responses to complex targets (United States)

    Pajewski, Lara; Giannopoulos, Antonis


    This work deals with the electromagnetic modelling of composite structures for Ground Penetrating Radar (GPR) applications. It was developed within the Short-Term Scientific Mission ECOST-STSM-TU1208-211013-035660, funded by COST Action TU1208 "Civil Engineering Applications of Ground Penetrating Radar". The Authors define a set of test concrete structures, hereinafter called cells. The size of each cell is 60 x 100 x 18 cm and the content varies with growing complexity, from a simple cell with few rebars of different diameters embedded in concrete at increasing depths, to a final cell with a quite complicated pattern, including a layer of tendons between two overlying meshes of rebars. Other cells, of intermediate complexity, contain pvc ducts (air filled or hosting rebars), steel objects commonly used in civil engineering (as a pipe, an angle bar, a box section and an u-channel), as well as void and honeycombing defects. One of the cells has a steel mesh embedded in it, overlying two rebars placed diagonally across the comers of the structure. Two cells include a couple of rebars bent into a right angle and placed on top of each other, with a square/round circle lying at the base of the concrete slab. Inspiration for some of these cells is taken from the very interesting experimental work presented in Ref. [1]. For each cell, a subset of models with growing complexity is defined, starting from a simple representation of the cell and ending with a more realistic one. In particular, the model's complexity increases from the geometrical point of view, as well as in terms of how the constitutive parameters of involved media and GPR antennas are described. Some cells can be simulated in both two and three dimensions; the concrete slab can be approximated as a finite-thickness layer having infinite extension on the transverse plane, thus neglecting how edges affect radargrams, or else its finite size can be fully taken into account. The permittivity of concrete can be

  4. Design and analysis of firewall-penetrated scheme based on trusted host

    Institute of Scientific and Technical Information of China (English)

    ZHANG Deng-yin; WANG Qian-qian; XU Jian


    On the basis of various firewall-penetrated technologies, a novel firewall-penetrated technology based on trusted host, which can realize the communication between penetrating host and protected host, is proposed in this paper.Meanwhile, the firewall-penetrated evaluating model and its formalized statement is also proposed by quantizing penetrated level, penetrated quality, and penetrated hiding. Finally, the firewall-penetrated technology based on trusted host is analyzed under the firewall-penetrated evaluating model, and the experimental results show that this technology has obvious penetrating effect and high penetrating quality.

  5. The full penetration hole as a stochastic process: controlling penetration depth in keyhole laser-welding processes (United States)

    Blug, A.; Abt, F.; Nicolosi, L.; Heider, A.; Weber, R.; Carl, D.; Höfler, H.; Tetzlaff, R.


    Although laser-welding processes are frequently used in industrial production the quality control of these processes is not satisfactory yet. Until recently, the "full penetration hole" was presumed as an image feature which appears when the keyhole opens at the bottom of the work piece. Therefore it was used as an indicator for full penetration only. We used a novel camera based on "cellular neural networks" which enables measurements at frame rates up to 14 kHz. The results show that the occurrence of the full penetration hole can be described as a stochastic process. The probability to observe it increases near the full penetration state. In overlap joints, a very similar image feature appears when the penetration depth reaches the gap between the sheets. This stochastic process is exploited by a closed-loop system which controls penetration depth near the bottom of the work piece ("full penetration") or near the gap in overlap joints ("partial penetration"). It guides the welding process at the minimum laser power necessary for the required penetration depth. As a result, defects like spatters are reduced considerably and the penetration depth becomes independent of process drifts such as feeding rate or pollution on protection glasses.

  6. Transdermal drug delivery of labetalol hydrochloride: Feasibility and effect of penetration enhancers

    Directory of Open Access Journals (Sweden)

    Saqib Zafar


    Full Text Available Objectives : The objective of this study is to investigate the feasibility of transdermal drug delivery of Labetalol Hydrochloride (LHCl and to study the effect of different penetration enhancers on the skin permeability of LHCl. Methods : The permeability experiments were conducted using a horizontal glass diffusion cell with a diffusional area of 2.37 cm-2 on albino rat skin. The effect of various penetration enhancers namely turpentine oil, dimethyl formamide (DMF, menthol, dimethyl sulfoxide, pine oil, and 2-pyrollidone, and the effect of the concentration of drug and enhancer in the donor phase on the skin permeability of LHCl was studied. Results : The apparent partition coefficient of the drug was found to be 6.95, suggesting it to be a lipophilic drug. The preliminary skin permeation studies revealed that the permeation of LHCL through albino rat skin was moderate (Kp = 6.490 Χ 10 -2 cm hr -1 from isotonic phosphate buffer of pH 7.4. An appreciable increase in the LHCl permeability coefficient was observed on using a co-solvent (ethanol 95% with the penetration enhancers in the donor phase. DMSO (10% v/v was found to be the most effective enhancer for Labetalol hydrochloride (Enhancement Factor = 1.165. An increase in the concentration of drug and enhancer in the donor cell accentuated the permeability coefficient of LHCl. Conclusions : It was concluded that LHCl could be delivered via the dermal route with the use of 10% DMSO as the penetration enhancer.

  7. Influence of hydroxypropyl-beta-cyclodextrin on transdermal penetration and photostability of avobenzone. (United States)

    Yang, Jing; Wiley, Cody J; Godwin, Donald A; Felton, Linda A


    The objective of the present study was to determine the effects of hydroxypropyl-beta-cyclodextrin (HPCD) complexation on the transdermal penetration and photostability of a model ultraviolet A (UVA) absorber, butyl methoxydibenzoylmethane (avobenzone), and to determine the influence of complexation on in vivo photoprotection. Avobenzone-HPCD complexation was demonstrated by differential scanning calorimetry. Formulations containing 0.12 mg/ml avobenzone and up to 30% (w/w) HPCD were prepared. Transdermal penetration was conducted using a modified Franz diffusion cell apparatus. As the concentration of HPCD was increased from 0% to 20%, transdermal permeation increased. Maximum flux occurred at 20% HPCD, where sufficient cyclodextrin was present to completely solubilize all avobenzone. When the concentration of HPCD was increased to 30%, transdermal penetration decreased, suggesting the formation of an avobenzone reservoir on the skin surface. Photostability of avobenzone was investigated under 100, 250, and 500 kJ/m2 UVA irradiation. The 30% HPCD formulation was the most photostable, followed by 20%, 10%, and 0% formulations. In vivo, the 30% HPCD formulation afforded the best photoprotection, as evidenced by the lowest extent of sunburn cell formation and edema induction. This work indicates that inclusion of HPCD in sunscreen formulations may enhance photoprotection by reducing both skin penetration and photodecomposition of UV absorbers.

  8. Civil engineering applications of ground penetrating radar

    CERN Document Server

    Pajewski, Lara


    This book, based on Transport and Urban Development COST Action TU1208, presents the most advanced applications of ground penetrating radar (GPR) in a civil engineering context, with documentation of instrumentation, methods, and results. It explains clearly how GPR can be employed for the surveying of critical transport infrastructure, such as roads, pavements, bridges, and tunnels, and for the sensing and mapping of underground utilities and voids. Detailed attention is also devoted to use of GPR in the inspection of geological structures and of construction materials and structures, including reinforced concrete, steel reinforcing bars, and pre/post-tensioned stressing ducts. Advanced methods for solution of electromagnetic scattering problems and new data processing techniques are also presented. Readers will come to appreciate that GPR is a safe, advanced, nondestructive, and noninvasive imaging technique that can be effectively used for the inspection of composite structures and the performance of diagn...

  9. Aerodynamic Jump for Long Rod Penetrators

    Directory of Open Access Journals (Sweden)

    Mark L. Bundy


    Full Text Available Aerodynamic jump for a non-spinning kinetic energy penetrator is neither a discontinuous change in the ,direction of motion at the origin of free night, nor is it the converse, i.e. a cumulativer~direc4on over a domain of infinite extent. Rather aerodynamic jump, for such a projectile, is a localised redirection of the centre of gravity motion, caused ~ the force of lift due to yaw over ther4latively short region from entry into free flight until the yaw reaches its first maximum. The primary objective of this paper is to provide answtfrs to the questions like what is aerodynamic jump, what liauses it, !lnd wh~t aspects df the flight trajectory does it refer to, or account for .

  10. Cephalic Tetanus from Penetrating Orbital Wound

    Directory of Open Access Journals (Sweden)

    Eloïse Guyennet


    Full Text Available Tetanus is a neurologic disorder caused by tetanospasmin, a protein toxin elaborated by Clostridium tetani. Cephalic tetanus is a localized form of the disease causing trismus and dysfunction of cranial nerves. We report the case of a man who presented with facial trauma, complete ophthalmoplegia, exophthalmos, areactive mydriasis, and periorbital hematoma. An orbital CT revealed air bubbles in the right orbital apex. The patient was given a tetanus toxoid booster and antibiotherapy. After extraction of a wooden foreign body, the patient developed right facial nerve palsy, disorders of swallowing, contralateral III cranial nerve palsy, and trismus. Only one case of cephalic tetanus from penetrating orbital wound has been reported in literature 20 years ago. When a patient presents with an orbital wound with ophthalmoplegia and signs of anaerobic infection, cephalic tetanus should be ruled out.

  11. Penetrating Stab Wound of the Right Ventricle

    Directory of Open Access Journals (Sweden)

    Onursal Buğra


    Full Text Available 18 years old male patient was admitted to our emergency unit with a penetrating stab wound to the right ventricle. A stab wound to the right ventricle was found to be 3 cm in diameter. The bleeding was controlled by insertion of a Foley catheter and inflation of the balloon. The stab wound had transected distal acute marginal side ofthe right coronary artery. A successful repair was performed with the use of a foley catheter and application of the Medtronic Octopus Tissue Stabilization System. The wound was closed with pledgeted mattress sutures. The distal acute marginal side of the right coronary artery was ligated. In this presentation, the surgical intervention method was reported and followed by a discussion of emergency surgical procedures of the heart.

  12. Plasma Bursts in Deep Penetration Laser Welding (United States)

    Mrňa, L.; Šarbort, M.

    We present an experimental study of the deep penetration laser welding process which aims to analyze the plasma plume oscillations on a short time scale. Using the high-speed camera we show that the plasma comes out of the keyhole in the form of short bursts rather than the continuous flow. We detect these bursts as the short-time intensity oscillations of light emissions coming from the plasma plume. We determine the period of bursts using the statistical signal processing methods and the short-time frequency analysis. Finally, we compare the characteristics of plasma bursts and the geometry of resulting welds carried out on a 2 kW Yb:YAG laser welding machine for the steel workpiece and various welding parameters settings.

  13. Penetration of tamoxifen citrate loaded ethosomes and liposomes across human skin: a comparative study with confocal laser scanning microscopy. (United States)

    Sarwa, Khomendra K; Suresh, Preeti K; Rudrapal, Mithun; Verma, Vinod K


    In the present study, ethosomal and liposomal formulations containing tamoxifen citrate were prepared and evaluated for their penetration properties in human cadaver skin using Franz diffusion cell and confocal laser scanning microscope (CLSM). The results clearly revealed that ethosomal vesicles showed a better drug permeation profile than that of liposomal vesicles. In addition, low fluorescence intensity in CLSM was recorded with liposomes as compared to ethosomes, indicating lower cumulative amount of drug permeation from liposomal vesicles. Furthermore, CLSM showed uniform fluorescence intensity across the entire depth of skin in ethosomal treatment, indicating high penetrability of ethosomal vesicles through human cadaver skin. In contrast, low penetrability of conventional liposomal vesicles was recorded as penetration was limited to the 7(th) section (i.e. upper epidermis layer) of skin as evident from visualization of intact liposomal vesicles in CLSM.

  14. In vitro human epidermal penetration of 1-bromopropane. (United States)

    Frasch, H Frederick; Dotson, G Scott; Barbero, Ana M


    1-Bromopropane (1-BP; CAS number 106-94-5), also known as n-propyl bromide, is a halogenated short-chain alkane used as an organic solvent with numerous commercial and industrial applications, including garment dry cleaning and vapor degreasing of metals. The purpose of this study was to determine the dermal absorption characteristics and corrosivity of 1-BP. Heat-separated human epidermal membranes were mounted on static diffusion cells. Different exposure scenarios were studied (infinite dose, finite dose, and transient exposure) using neat 1-BP and saturated aqueous solution as donor. Steady-state fluxes for infinite-dose neat 1-BP exposure averaged 625 to 960 μg cm(-2) h(-1). The finite-dose (10 μl/cm(2) = 13.5 mg/cm(2)) unoccluded donor resulted in penetration of <0.2% of the applied dose (22 μg/cm(2)). A 10-min transient exposure to infinite dose resulted in total penetration of 179 μg/cm(2). Steady-state 1-BP fluxes from neat application of a commercial dry cleaning solvent were similar (441 to 722 μg cm(-2) h(-1)). The permeability coefficient of 1-BP in water vehicle was 0.257 ± 0.141 cm/h. The absorption potential of 1-BP following dermal exposure is dependent upon the type and duration of exposure. Donor losses due to evaporation were approximately 500-fold greater than dermal absorption flux; evaporation flux was 420 mg cm(-2) h(-1). 1-BP is cytotoxic but not corrosive, based on results from a cultured reconstructed human epidermal model (EpiDerm Skin Corrosivity Test).

  15. Are capecitabine and the active metabolite 5-Fu CNS penetrable to treat breast cancer brain metastasis? (United States)

    Zhang, Jinqiang; Zhang, Lingli; Yan, Yumei; Li, Shaorong; Xie, Liang; Zhong, Wei; Lv, Jing; Zhang, Xiuhua; Bai, Yu; Cheng, Ziqiang


    Brain metastasis (BM) is increasingly diagnosed in Her2 positive breast cancer (BC) patients. Lack of effective treatment to breast cancer brain metastases (BCBMs) is probably due to inability of the current therapeutic agents to cross the blood-brain barrier. The central nervous system (CNS) response rate in BCBM patients was reported to improve from 2.6%-6% (lapatinib) to 20%-65% (lapatinib in combination with capecitabine). Lapatinib is a poor brain penetrant. In this study, we evaluated the CNS penetration of capecitabine and hoped to interpret the mechanism of the improved CNS response from the pharmacokinetic (PK) perspective. Capecitabine does not have antiproliferative activity and 5-fluorouracil (5-FU) is the active metabolite. Capecitabine was orally administered to mouse returning an unbound brain-to-blood ratio (Kp,uu,brain) at 0.13 and cerebrospinal fluid (CSF)-to-unbound blood ratio (Kp,uu,CSF) at 0.29 for 5-FU. Neither free brain nor CSF concentration of 5-FU can achieve antiproliferative concentration for 50% of maximal inhibition of cell proliferation of 4.57 µM. BCBM mice were treated with capecitabine monotherapy or in combination with lapatinib. The Kp,uu,brain value of 5-FU increased to 0.17 in the brain tumor in the presence of lapatinib, which is still far below unity. The calculated free concentration of 5-FU and lapatinib in the brain tumor did not reach the antiproliferative potency and neither treatment showed antitumor activity in the BCBM mice. The CNS penetration of 5-FU in human was predicted based on the penetration in preclinical brain tumor, CSF, and human PK and the predicted free CNS concentration was below the antiproliferative potency. These results suggest that CNS penetration of 5-FU and lapatinib are not desirable and development of a true CNS penetrable therapeutic agent will further improve the response rate for BCBM.

  16. Polymeric Nanocapsules and Lipid-Core Nanocapsules Have Diverse Skin Penetration. (United States)

    de Brum, Tainá L; Fiel, Luana A; Contri, Renata V; Guterres, Sílvia S; Pohlmann, Adriana R


    Biodegradable nanoparticles have been widely studied as drug carriers in order to increase drug solubility in aqueous media, modify biodistribution, target tissues and organs or control the drug release. Those nanoparticles are, in general, produced as liquid formulations to act as final dosage forms or as intermediate for solid or semi-solid products. Considering the dermatological applications, as medicines or cosmetics, different nanoparticles have been proposed to control the skin penetration of encapsulated lipophilic substances. A point rarely investigated is the penetration of the carrier itself into the skin, independent of the drug penetration profile. In this way, our objective was to correlate the flexibility of the biodegradable nanoparticles to the depth of their skin penetration. To minimize the impact of the chemical composition, the surface chemistry or the shape and size distribution on the results, two kinds of polymeric nanocapsules presenting diverse mechanical properties were produced using almost the same materials and their concentrations. The nanocapsules (NC) and the lipid-core nanocapsules (LNC) were prepared by solvent displacement using Rhodamine B-labeled polymer, oil and surfactants. The only difference in composition between them is the presence of sorbitan monostearate in the latter which was used to have a more rigid nanoparticle as previously reported. NC and LNC had, respectively, mean diameters of 178 and 180 nm and zeta potentials of -11 and -9 mV. The in vitro skin penetration was carried out using Franz cells (pig skin as membrane). Skin samples were observed by confocal laser scanning microscopy (CLSM). NC reached the dermis, while LNC was retained at the outermost layers of the skin. The result was in accordance with the flexibility previously determined for those nanocapsules, in a way that higher flexibility gives deeper penetration. NC can reach the dermis and LNC can act as reservoir systems at the epidermis.

  17. Hacking and penetration testing with low power devices

    CERN Document Server

    Polstra, Philip


    Hacking and Penetration Testing with Low Power Devices shows you how to perform penetration tests using small, low-powered devices that are easily hidden and may be battery-powered. It shows how to use an army of devices, costing less than you might spend on a laptop, from distances of a mile or more. Hacking and Penetration Testing with Low Power Devices shows how to use devices running a version of The Deck, a full-featured penetration testing and forensics Linux distribution, and can run for days or weeks on batteries due to their low power consumption. Author Philip Polstra shows how to

  18. Building virtual pentesting labs for advanced penetration testing

    CERN Document Server

    Cardwell, Kevin


    Written in an easy-to-follow approach using hands-on examples, this book helps you create virtual environments for advanced penetration testing, enabling you to build a multi-layered architecture to include firewalls, IDS/IPS, web application firewalls, and endpoint protection, which is essential in the penetration testing world. If you are a penetration tester, security consultant, security test engineer, or analyst who wants to practice and perfect penetration testing skills by building virtual pen testing labs in varying industry scenarios, this is the book for you. This book is ideal if yo

  19. Effect of Liquid Penetrant Sensitivity on Probability of Detection (United States)

    Parker, Bradford H.


    The objective of the task is to investigate the effect of liquid penetrant sensitivity level on probability of detection (POD) of cracks in various metals. NASA-STD-5009 currently requires the use of only sensitivity level 4 liquid penetrants for NASA Standard Level inspections. This requirement is based on the fact that the data used to establish the reliably detectable flaw sizes penetrant inspection was from studies performed in the 1970s using penetrant deemed to be equivalent only to modern day sensitivity level 4 penetrants. However, many NDE contractors supporting NASA Centers routinely use sensitivity level 3 penetrants. Because of the new NASA-STD-5009 requirement, these contractors will have to either shift to sensitivity level 4 penetrants or perform formal POD demonstration tests to qualify their existing process. We propose a study to compare the POD generated for two penetrant manufactures, Sherwin and Magnaflux, and for the two most common penetrant inspection methods, water washable and post emulsifiable, hydrophilic. NDE vendors local to GSFC will be employed. A total of six inspectors will inspect a set of crack panels with a broad range of fatigue crack sizes. Each inspector will perform eight inspections of the panel set using the combination of methods and sensitivity levels described above. At least one inspector will also perform multiple inspections using a fixed technique to investigate repeatability. The hit/miss data sets will be evaluated using both the NASA generated DOEPOD software and the MIL-STD-1823 software.

  20. Staged Penetrating Sclerokeratoplasty and Penetrating Keratoplasty for Management of Advanced Acquired Anterior Staphyloma

    Directory of Open Access Journals (Sweden)

    Enrique de la Torre-Gonzalez


    Full Text Available Herein we describe a staged surgical technique consisting of penetrating sclerokeratoplasty (PSKP followed by penetrating keratoplasty (PKP and present its clinical course and complications over two years of follow-up. A 23-year-old man presented with cosmetically unacceptable protrusion of the globe corresponding to the cornea and sclera. PSKP was performed transplanting a full-thickness beveled 13 mm corneoscleral tectonic graft. Hypotony developed subsequently and was successfully managed medically, however corneal graft failure occurred. After 15 months, a 7.5 mm PKP was performed for optical reasons, which subsequently remained clear with a healthy epithelium. In this particular case, cosmetic, tectonic, therapeutic, and optical requirements were met. PSKP is a surgical procedure which entails a high rate of complications but may be the only alternative when the main goal of intervention is restoration of the globe in complicated cases such as our patient.

  1. Penetration, Post-penetration Development, and Reproduction of Meloidogyne incognita on Cucumis melo var. texanus. (United States)

    Faske, T R


    Cucumis melo var. texanus, a wild melon commonly found in the southern United States and two accessions, Burleson Co. and MX 1230, expressed resistance to Meloidogyne incognita in preliminary experiments. To characterize the mechanism of resistance, we evaluated root penetration, post-penetration development, reproduction, and emigration of M. incognita on these two accessions of C. melo var. texanus. Additionally, we evaluated 22 accessions of C. melo var. texanus for their reaction against M. incognita in a greenhouse experiment. Fewer (P ≤ 0.05) J2 penetrated the root system of C. melo var. texanus accessions (Burleson Co. and MX 1230) and C. metuliferus (PI 482452) (resistant control), 7 days after inoculation (DAI) than in C. melo 'Hales Best Jumbo' (susceptible control). A delayed (P ≤ 0.05) rate of nematode development was observed at 7, 14, and 21 DAI that contributed to lower (P ≤ 0.05) egg production on both accessions and C. metuliferus compared with C. melo. Though J2 emigration was observed on all Cucumis genotypes a higher (P ≤ 0.05) rate of J2 emigration was observed from 3 to 6 DAI on accession Burleson Co. and C. metuliferus than on C. melo. The 22 accessions of C. melo var. texanus varied relative to their reaction to M. incognita with eight supporting similar levels of nematode reproduction to that of C. metuliferus. Cucumis melo var. texanus may be a useful source of resistance against root-knot nematode in melon.

  2. Ground Penetrating Radar Technologies in Ukraine (United States)

    Pochanin, Gennadiy P.; Masalov, Sergey A.


    Transient electromagnetic fields are of great interest in Ukraine. The following topics are studied by research teams, with high-level achievements all over the world: (i) Ultra-Wide Band/Short-pulse radar techniques (IRE and LLC "Transient Technologies", for more information please visit and; (ii) Ground Penetrating Radar (GPR) with stepped frequency sounding signals (IRE); (iii) Continuous-Wave (CW) radar with phase-shift keying signals (IRE); and (iv) Radio-wave interference investigation (Scientific and Technical Centre of The Subsurface Investigation, GPR applications are mainly in search works, for example GPR is often used to search for treasures. It is also used to identify leaks and diffusion of petroleum in soil, in storage areas, as well as for fault location of pipelines. Furthermore, GPR is used for the localization of underground utilities and for diagnostics of the technical state of hydro dams. Deeper GPR probing was performed to identify landslides in Crimea. Rescue radar with CW signal was designed in IRE to search for living people trapped under the rubble of collapsed buildings. The fourth version of this radar has been recently created, showing higher stability and noise immunity. Radio-wave interference investigation allows studying the soil down to tens of meters. It is possible to identify areas with increased conductivity (moisture) of the soil. LLC "Transient Technologies" is currently working with Shevchenko Kyiv University on a cooperation program in which the construction of a test site is one of the planned tasks. In the framework of this program, a GPR with a 300 MHz antenna was handed to the geological Faculty of the University. Employees of "Transient Technologies" held introductory lectures with a practical demonstration for students majoring in geophysics. The authors participated to GPR

  3. Penetration and Toxicity of Nanomaterials in Higher Plants

    Directory of Open Access Journals (Sweden)

    Giuseppe Chichiriccò


    Full Text Available Nanomaterials (NMs comprise either inorganic particles consisting of metals, oxides, and salts that exist in nature and may be also produced in the laboratory, or organic particles originating only from the laboratory, having at least one dimension between 1 and 100 nm in size. According to shape, size, surface area, and charge, NMs have different mechanical, chemical, electrical, and optical properties that make them suitable for technological and biomedical applications and thus they are being increasingly produced and modified. Despite their beneficial potential, their use may be hazardous to health owing to the capacity to enter the animal and plant body and interact with cells. Studies on NMs involve technologists, biologists, physicists, chemists, and ecologists, so there are numerous reports that are significantly raising the level of knowledge, especially in the field of nanotechnology; however, many aspects concerning nanobiology remain undiscovered, including the interactions with plant biomolecules. In this review we examine current knowledge on the ways in which NMs penetrate plant organs and interact with cells, with the aim of shedding light on the reactivity of NMs and toxicity to plants. These points are discussed critically to adjust the balance with regard to the risk to the health of the plants as well as providing some suggestions for new studies on this topic.

  4. Ice-Penetrating Robot for Scientific Exploration (United States)

    Zimmerman, Wayne; Carsey, Frank; French, Lloyd


    The cryo-hydro integrated robotic penetrator system (CHIRPS) is a partially developed instrumentation system that includes a probe designed to deeply penetrate the European ice sheet in a search for signs of life. The CHIRPS could also be used on Earth for similar exploration of the polar ice caps especially at Lake Vostok in Antarctica. The CHIRPS probe advances downward by a combination of simple melting of ice (typically for upper, non-compacted layers of an ice sheet) or by a combination of melting of ice and pumping of meltwater (typically, for deeper, compacted layers). The heat and electric power for melting, pumping, and operating all of the onboard instrumentation and electronic circuitry are supplied by radioisotope power sources (RPSs) and thermoelectric converters energized by the RPSs. The instrumentation and electronic circuitry includes miniature guidance and control sensors and an advanced autonomous control system that has fault-management capabilities. The CHIRPS probe is about 1 m long and 15 cm in diameter. The RPSs generate a total thermal power of 1.8 kW. Initially, as this power melts the surrounding ice, a meltwater jacket about 1 mm thick forms around the probe. The center of gravity of the probe is well forward (down), so that the probe is vertically stabilized like a pendulum. Heat is circulated to the nose by means of miniature pumps and heat pipes. The probe melts ice to advance in a step-wise manner: Heat is applied to the nose to open up a melt void, then heat is applied to the side to allow the probe to slip down into the melt void. The melt void behind the probe is allowed to re-freeze. Four quadrant heaters on the nose and another four quadrant heaters on the rear (upper) surface of the probe are individually controllable for steering: Turning on two adjacent nose heaters on the nose and two adjacent heaters on the opposite side at the rear causes melt voids to form on opposing sides, such that the probe descends at an angle from

  5. Two methodologies for physical penetration testing using social engineering

    NARCIS (Netherlands)

    Dimkov, Trajce; Pieters, Wolter; Hartel, Pieter


    During a penetration test on the physical security of an organization, if social engineering is used, the penetration tester directly interacts with the employees. These interactions are usually based on deception and if not done properly can upset the employees, violate their privacy or damage thei

  6. Shape optimization of high-speed penetrators: a review (United States)

    Ben-Dor, Gabi; Dubinsky, Anatoly; Elperin, Tov


    In spite of a large number of publications on shape optimization of penetrating projectiles there are no dedicated surveys of these studies. The goal of the present review is to close this gap. The review includes more than 50 studies published since 1980 and devoted to solving particular problems of shape optimization of high-speed penetrators. We analyze publications which employed analytical and numerical method for shape optimization of high-speed penetrators against concrete, metal, fiber-reinforced plastic laminate and soil shields. We present classification of the mathematical models used for describing interaction between a penetrator and a shield. The reviewed studies are summarized in the table where we display the following information: the model; indicate whether the model accounts for or neglects friction at the surface of penetrator; criterion for optimization (depth of penetration into a semi-infinite shield, ballistic limit velocity for a shield having a finite thickness, several criteria); class of considered shapes of penetrators (bodies of revolution, different classes of 3-D bodies, etc.); method of solution (analytical or numerical); in comments we present additional information on formulation of the optimization problem. The survey also includes discussion on certain methodological facets in formulating shape optimization problems for high-speed penetrators.

  7. Aorto-esophageal fistula secondary to penetrating atherosclerotic ulcer

    Directory of Open Access Journals (Sweden)

    Ranjana Gupta


    Full Text Available Aorto-esophageal fistula (AEF is a rare and life threatening condition, which can be rapidly fatal. More than half of such cases are secondary to aortic aneurysm rupture. There are only two previous reports describing AEF caused by penetrating atherosclerotic ulcer. We present multidetector computed tomography findings in a case of AEF secondary to penetrating atherosclerotic ulcer.

  8. Unsteady penetration of a target by a liquid jet. (United States)

    Uth, Tobias; Deshpande, Vikram S


    It is widely acknowledged that ceramic armor experiences an unsteady penetration response: an impacting projectile may erode on the surface of a ceramic target without substantial penetration for a significant amount of time and then suddenly start to penetrate the target. Although known for more than four decades, this phenomenon, commonly referred to as dwell, remains largely unexplained. Here, we use scaled analog experiments with a low-speed water jet and a soft, translucent target material to investigate dwell. The transient target response, in terms of depth of penetration and impact force, is captured using a high-speed camera in combination with a piezoelectric force sensor. We observe the phenomenon of dwell using a soft (noncracking) target material. The results show that the penetration rate increases when the flow of the impacting water jet is reversed due to the deformation of the jet-target interface--this reversal is also associated with an increase in the force exerted by the jet on the target. Creep penetration experiments with a constant indentation force did not show an increase in the penetration rate, confirming that flow reversal is the cause of the unsteady penetration rate. Our results suggest that dwell can occur in a ductile noncracking target due to flow reversal. This phenomenon of flow reversal is rather widespread and present in a wide range of impact situations, including water-jet cutting, needleless injection, and deposit removal via a fluid jet.

  9. A Study on Factors Affecting Airborne LiDAR Penetration

    Directory of Open Access Journals (Sweden)

    Wei-Chen Hsu


    Full Text Available This study uses data from different periods, areas and parameters of airborne LiDAR (light detection and ranging surveys to understand the factors that influence airborne LiDAR penetration rate. A discussion is presented on the relationships between these factors and LiDAR penetration rate. The results show that the flight height above ground level (AGL does not have any relationship with the penetration rate. There are some factors that should have larger influence. For example, the laser is affected by a wet ground surface by reducing the number of return echoes. The field of view (FOV has a slightly negative correlation with the penetration rate, which indicates that the laser incidence angle close to zero should achieve the best penetration. The vegetation cover rate also shows a negative correlation with the penetration rate, thus bare ground and reduced vegetation in the aftermath of a typhoon also cause high penetration rate. More return echoes could be extracted from the full-waveform system, thereby effectively improving the penetration rate. This study shows that full-waveform LiDAR is an effective method for increasing the number of surface reflected echoes. This study suggests avoiding LiDAR survey employment directly following precipitation to prevent laser echo reduction.

  10. 3D Numerical Simulation of Projectile Penetration into Concrete Target

    Institute of Scientific and Technical Information of China (English)


    Basing on the explicit instantaneous dynamics software MSC-Dytran and the general coupling arithmetic, the process of the projectile penetration into concrete target was simulated with the point-line-surface-body modeling method. Simulation results are in agreement with experimental results. The simulated data could provide design reference for the defense engineering construction and penetrator design.

  11. Quantitative Penetration Testing with Item Response Theory (extended version)

    NARCIS (Netherlands)

    Arnold, F.; Pieters, W.; Stoelinga, M.I.A.


    Existing penetration testing approaches assess the vulnerability of a system by determining whether certain attack paths are possible in practice. Thus, penetration testing has so far been used as a qualitative research method. To enable quantitative approaches to security risk management, including

  12. 40 CFR 1065.376 - Chiller NO2 penetration. (United States)


    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Chiller NO2 penetration. 1065.376... CONTROLS ENGINE-TESTING PROCEDURES Calibrations and Verifications Nox and N2o Measurements § 1065.376 Chiller NO2 penetration. (a) Scope and frequency. If you use a chiller to dry a sample upstream of a...

  13. Quantitative penetration testing with item response theory (extended version)

    NARCIS (Netherlands)

    Arnold, Florian; Pieters, Wolter; Stoelinga, Mariëlle


    Existing penetration testing approaches assess the vulnerability of a system by determining whether certain attack paths are possible in practice. Therefore, penetration testing has thus far been used as a qualitative research method. To enable quantitative approaches to security risk management, in

  14. Storage Possibilities for Enabling Higher Wind Energy Penetration

    DEFF Research Database (Denmark)

    Swierczynski, Maciej Jozef; Teodorescu, Remus; Rodriguez, Pedro


    As the installed worldwide wind energy capacity increases about 30% annually and Kyoto protocol that came in force in 2005, wind penetration level in power system is considered to significantly increase in near future. Due to increased penetration and nature of the wind, especially its...

  15. Ground penetrating radar for underground sensing in agriculture: a review (United States)

    Liu, Xiuwei; Dong, Xuejun; Leskovar, Daniel I.


    Belowground properties strongly affect agricultural productivity. Traditional methods for quantifying belowground properties are destructive, labor-intensive and pointbased. Ground penetrating radar can provide non-invasive, areal, and repeatable underground measurements. This article reviews the application of ground penetrating radar for soil and root measurements and discusses potential approaches to overcome challenges facing ground penetrating radar-based sensing in agriculture, especially for soil physical characteristics and crop root measurements. Though advanced data-analysis has been developed for ground penetrating radar-based sensing of soil moisture and soil clay content in civil engineering and geosciences, it has not been used widely in agricultural research. Also, past studies using ground penetrating radar in root research have been focused mainly on coarse root measurement. Currently, it is difficult to measure individual crop roots directly using ground penetrating radar, but it is possible to sense root cohorts within a soil volume grid as a functional constituent modifying bulk soil dielectric permittivity. Alternatively, ground penetrating radarbased sensing of soil water content, soil nutrition and texture can be utilized to inversely estimate root development by coupling soil water flow modeling with the seasonality of plant root growth patterns. Further benefits of ground penetrating radar applications in agriculture rely on the knowledge, discovery, and integration among differing disciplines adapted to research in agricultural management.

  16. Penetrance of Hypertrophic Cardiomyopathy in Children and Adolescents

    DEFF Research Database (Denmark)

    Jensen, Morten K; Havndrup, Ole; Christiansen, Michael;


    The penetrance of hypertrophic cardiomyopathy (HCM) during childhood and adolescence has been only sparsely described. We studied the penetrance of HCM and the short- and long-term outcomes of clinical screening and predictive genetic testing of child relatives of patients with HCM....

  17. The Impact of IPv6 on Penetration Testing

    NARCIS (Netherlands)

    Ottow, Christiaan; Vliet, van Frank; Boer, de Pieter-Tjerk; Pras, Aiko


    In this paper we discuss the impact the use of IPv6 has on remote penetration testing of servers and web applications. Several modifications to the penetration testing process are proposed to accommodate IPv6. Among these modifications are ways of performing fragmentation attacks, host discovery and

  18. Impact and Penetration of Nanoparticle Suspension Drops into Porous Membranes (United States)

    Sahu, Rakesh; Yarin, Alexander; Pourdeyhimi, Behnam


    The impacts and dynamic penetration of drops with suspended nanoparticles into porous membranes are studied experimentally and theoretically. This type of penetration is radically different from the wettability-driven imbibition. Two types of membranes are used in the experiments: (i) glass fiber filter membrane (wettable) and (ii) PTFE depth filter (non-wettable). The nanoparticle entrainment and deposition inside the membrane bulk is used to mostly visualize the ultimate penetration fronts of the carrier fluid by observing the cut cross-sections of the filter membranes, albeit also provides an insight into potentially new applications like circuit printing on nonwovens. The experimental results demonstrate that during the dynamic focusing responsible for water penetration into micro- and nanopores, water can penetrate into a non-wettable porous medium (PTFE). Water also penetrates by the same focusing mechanism into the wettable glass fiber membrane, where it additionally spreads on a much longer time scale due to the wettability-driven flow. A theory explaining dynamic penetration of liquid into porous medium after drop impact is proposed. It is used to explain and predict water penetration into the non-wettable media after drop impact, and the results are compared with the experimental data. The work was supported by the Nonwovens Cooperative Research Center (NCRC).

  19. 40 CFR 1065.365 - Nonmethane cutter penetration fractions. (United States)


    ... fractions. 1065.365 Section 1065.365 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Measurements § 1065.365 Nonmethane cutter penetration fractions. (a) Scope and frequency. If you use a FID... penetration fractions of methane, PFCH4, and ethane, PF C2H6. As detailed in this section, these...


    Institute of Scientific and Technical Information of China (English)


    How the outer substance could penetrate through the skin lies in the stratum corneum, because it is the main barrier in the multi-layers of the skin. Supposing the keratin cell with a special geometry as tetrakaidecahedron, the penetration property of stratum corneum was the key problem which was numerically simulated with finite element method. At first the discretization of the stratum corneum region was given in two steps: first, the discretization of the keratin cell; second, the discretization of fattiness that surrounds the keratin. Then there was the work of numerical simulation. In this procedure, the finite element method and the multi-grid method were used. The former was to obtain the discretization of basic elements; the latter was to decrease the high frequency error. At last the visualization of the numerical simulation was shown.