Peptide-Mediated Cellular Delivery of Oligonucleotide-Based Therapeutics In Vitro: Quantitative Evaluation of Overall Efficacy Employing Easy to Handle Reporter Systems

Cellular uptake of therapeutic oligonucleotides and subsequent intracellular trafficking to their target sites represents the major technical hurdle for the biological effectiveness of these potential drugs. Accordingly, laboratories worldwide focus on the development of suitable delivery systems. Among the different available non-viral systems like cationic polymers, cationic liposomes and polymeric nanoparticles, cell-penetrating peptides (CPPs) represent an attractive concept to bypass the problem of poor membrane permeability of these charged macromolecules. While uptake per se in most cases does not represent the main obstacle of nucleic acid delivery in vitro, it becomes increasingly apparent that intracellular trafficking is the bottleneck. As a consequence, in order to optimize a given delivery system, a side-by-side analysis of nucleic acid cargo internalized and the corresponding biological effect is required to determine the overall efficacy. In this review, we will concentrate on peptide-mediated delivery of siRNAs and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. To illustrate current limitations of non-viral nucleic acid delivery systems, we present own data as an example and discuss options of how to enhance trafficking of molecules entrapped in cellular compartments

Structural characterization of a first-generation articulated-truss joint for space crane application

A first-generation space crane articulated-truss joint was statically and dynamically characterized in a configuration that approximated an operational environment. The articulated-truss joint was integrated into a test-bed for structural characterization. Static characterization was performed by applying known loads and measuring the corresponding deflections to obtain load-deflection curves. Dynamic characterization was performed using modal testing to experimentally determine the first six mode shapes, frequencies, and modal damping values. Static and dynamic characteristics were also determined for a reference truss that served as a characterization baseline. Load-deflection curves and experimental frequency response functions are presented for the reference truss and the articulated-truss joint mounted in the test-bed. The static and dynamic experimental results are compared with analytical predictions obtained from finite element analyses. Load-deflection response is also presented for one of the linear actuators used in the articulated-truss joint. Finally, an assessment is presented for the predictability of the truss hardware used in the reference truss and articulated-truss joint based upon hardware stiffness properties that were previously obtained during the Precision Segmented Reflector (PSR) Technology Development Program

Open and reproducible science practices in psychoneuroendocrinology: Opportunities to foster scientific progress

This perspective article was written by invitation of the editors in chief as a summary and extension of the symposium entitled Psychoneuroendocrine Research in the Era of the Replication Crisis which was held at the virtual meeting of the International Society of Psychoneuroendocrinology 2021. It highlights the opportunities presented by the application of open and reproducible scientific practices in psychoneuroendocrinology (PNE), an interdisciplinary field at the intersection of psychology, endocrinology, immunology, neurology, and psychiatry. It conveys an introduction to the topics preregistration, registered reports, quantifying the impact of equally-well justifiable analysis decisions, and open data and scripts, while emphasizing ‘selfish’ reasons to adopt such practices as individual researcher. Complementary to the call for adoption of open science practices, we highlight the need for methodological best practice guidelines in the field of PNE, which could further contribute to enhancing replicability of results. We propose concrete steps for future actions and provide links to additional resources for those interested in adopting open and reproducible science practices in future studies

French vocabulary in Encore Tricolore: do pupils have a chance?

British learners acquire very little vocabulary in their foreign languages,compared to pupils elsewhere in Europe, particularly learners of English as aforeign language. Could the materials used for teaching help explain thisdifference? An analysis of the vocabulary loading of a textbook for French as aforeign language commonly used in Britain, Encore Tricolore (Mascie-Taylor andHonnor, 2001, Cheltenham, UK, Nelson Thornes), was carried out with thisquestion in mind. An analysis of the vocabulary suggests that it is not introducedand practised in a way that is conducive to building a sufficiently large vocabularyto reach level B1 of the Common European Framework of Reference forLanguages (CEFR)

Phase-separation of binary fluids in shear flow: a numerical study

The phase-separation kinetics of binary fluids in shear flow is studied numerically in the framework of the continuum convection-diffusion equation based on a Ginzburg-Landau free energy. Simulations are carried out for different temperatures both in d=2 and in d=3. Our results confirm the qualitative picture put forward by the large-N limit equations studied in \cite{noi}. In particular, the structure factor is characterized by the presence of four peaks whose relative oscillations give rise to a periodic modulation of the behavior of the rheological indicators and of the average domains sizes. This peculiar pattern of the structure factor corresponds to the presence of domains with two characteristic thicknesses whose relative abundance changes with time.Comment: 6 pages, 11 figures in .gif forma

Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum

BACKGROUND: Sulphadoxine-pyrimethamine has been abandoned as first- or second-line treatment by most African malaria endemic countries in favour of artemisinin-based combination treatments, but the drug is still used as intermittent preventive treatment during pregnancy. However, resistance to sulphadoxine-pyrimethamine has been increasing in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, it is important to monitor such markers. METHODS: This paper reports a novel sensitive, semi-quantitative and specific real-time PCR and melting curve analysis (MCA) assay using fluorescence resonance energy transfer (FRET) for the detection of DHPS-540, an important predictor for SP resistance. FRET/MCA was evaluated using 78 clinical samples from malaria patients and compared to PCR-RFLP. RESULTS: Sixty-two samples were in perfect agreement between both assays. One sample showed a small wild type signal with FRET/MCA that indicates a polyclonal infection. Four samples were not able to generate enough material in both assays to distinguish mutant from wild-type infection, six samples gave no signal in PCR-RFLP and five samples gave no amplification in FRET/MCA. CONCLUSION: FRET/MCA is an effective tool for the identification of SNPs in drug studies and epidemiological surveys on resistance markers in general and DHPS-540 mutation in particular

Interaction model for magnetic holes in a ferrofluid layer

Nonmagnetic spheres confined in a ferrofluid layer (magnetic holes) present dipolar interactions when an external magnetic field is exerted. The interaction potential of a microsphere pair is derived analytically, with a precise care for the boundary conditions along the glass plates confining the system. Considering external fields consisting of a constant normal component and a high frequency rotating in-plane component, this interaction potential is averaged over time to exhibit the average interparticular forces acting when the imposed frequency exceeds the inverse of the viscous relaxation time of the system. The existence of an equilibrium configuration without contact between the particles is demonstrated for a whole range of exciting fields, and the equilibrium separation distance depending on the structure of the external field is established. The stability of the system under out-of-plane buckling is also studied. The dynamics of such a particle pair is simulated and validated by experiments.Comment: 15 pages, 11 figures (18 with subfigures). to appear in Phys. Rev.

An in-vitro screening assay for the detection of inhibitors of proinflammatory cytokine synthesis: a useful tool for the development of new antiarthritic and disease modifying drugs

AbstractObjective This work targets the development of a new tool to help develop new anticytokine drugs that prevent or reduce the progression of arthritic diseases. The specific aim of our study was to establish a fast and reliable in vitro screening assay of cytokine synthesis inhibitors (TNFα, IL-1β) which shows better correlation with enzyme assays than previously reported in vitro assays. The test system should be able to detect p38-MAP kinase inhibitors.Material and methods Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation from human EDTA-potassium whole blood. Cells were adjusted at 1×106 cells/ml. PBMCs were stimulated with lipopolysaccharide (LPS; E. coli serotype 026:B6: 1μg/ml) in the presence of test compound (10−5–10−8M) for 4h at 37°C in a 5% CO2-incubator. Induced TNFα and IL-1β protein were measured by ELISA.Results The following are representative examples of inhibitors which effect cytokine synthesis. Corticoid Dexamethasone inhibits IL-1β and TNFα synthesis at IC50 of 38nM and 25nM, respectively. ERK1/ERK2 inhibitor U0126 effects cytokine synthesis at IC50 of 0.34μM for IL-1β production and 0.26μM for TNFα synthesis.p38-MAP kinase inhibitor SB 203580 inhibits IL-1β- and TNF-α-synthesis (IC50sof 0.052μM and 0.46μM) in the same degree as p38-MAP kinase activity (IC50: 0.34μM). Same results could be shown for SB 210313, which had same efficacy on IL-1β and TNFα biosynthesis (IC50's: 1.88μM and 1.01μM) and on p38-MAP kinase (IC50: 6.85μM). Also for SB 202190 this correlation in inhibition of IL-1β and TNFα synthesis (IC50's: 0.055μM and 1.01μM) and p38-MAP kinase inhibition (IC50: 0.088μM) could be shown.Conclusion This study shows the screening assay using PBMCs stimulated with LPS for IL-1β and TNFα synthesis is a reliable test system for the quantification of the effectiveness of new drugs modulating IL-1β and TNFα synthesis which is mainly mediated by p38-MAP Kinase. These assay allows fast detection of IL-1β and TNFα synthesis inhibitors with different modes of action, including p38-MAP kinase inhibitors. The results obtained with our in-vitro screening assay show good correlation with results from enzyme assays. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved