Slow release and delivery of antisense oligonucleotide drug by self-assembled peptide amphiphile nanofibers

Antisense oligonucleotides provide a promising therapeutic approach for several disorders including cancer. Chemical stability, controlled release, and intracellular delivery are crucial factors determining their efficacy. Gels composed of nanofibrous peptide network have been previously suggested as carriers for controlled delivery of drugs to improve stability and to provide controlled release, but have not been used for oligonucleotide delivery. In this work, a self-assembled peptide nanofibrous system is formed by mixing a cationic peptide amphiphile (PA) with Bcl-2 antisense oligodeoxynucleotide (ODN), G3139, through electrostatic interactions. The self-assembly of PA-ODN gel was characterized by circular dichroism, rheology, atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM and SEM images revealed establishment of the nanofibrous PA-ODN network. Due to the electrostatic interactions between PA and ODN, ODN release can be controlled by changing PA and ODN concentrations in the PA-ODN gel. Cellular delivery of the ODN by PA-ODN nanofiber complex was observed by using fluorescently labeled ODN molecule. Cells incubated with PA-ODN complex had enhanced cellular uptake compared to cells incubated with naked ODN. Furthermore, Bcl-2 mRNA amounts were lower in MCF-7 human breast cancer cells in the presence of PA-ODN complex compared to naked ODN and mismatch ODN evidenced by quantitative RT-PCR studies. These results suggest that PA molecules can control ODN release, enhance cellular uptake and present a novel efficient approach for gene therapy studies and oligonucleotide based drug delivery.     

Murine Borrelia arthritis is highly dependent on ASC and caspase-1, but independent of NLRP3

Introduction

The protein platform called the NOD-like-receptor -family member (NLRP)-3 inflammasome needs to be activated to process intracellular caspase-1. Active caspase-1 is able to cleave pro-Interleukin (IL)-1β, resulting in bioactive IL-1β. IL-1β is a potent proinflammatory cytokine, and thought to play a key role in the pathogenesis of Lyme arthritis, a common manifestation of Borrelia burgdorferi infection. The precise pathways through which B. burgdorferi recognition leads to inflammasome activation and processing of IL-1β in Lyme arthritis has not been elucidated. In the present study, we investigated the contribution of several pattern recognition receptors and inflammasome components in a novel murine model of Lyme arthritis.

Methods

Lyme arthritis was elicited by live B. burgdorferi, injected intra-articularly in knee joints of mice. To identify the relevant pathway components, the model was applied to wild-type, NLRP3-/-, ASC-/-, caspase-1-/-, NOD1-/-, NOD2-/-, and RICK-/- mice. As a control, TLR2-/-, Myd88-/- and IL-1R-/- mice were used. Peritoneal macrophages and bone marrow-derived macrophages were used for in vitro cytokine production and inflammasome activation studies. Joint inflammation was analyzed in synovial specimens and whole knee joints. Mann-Whitney U tests were used to detect statistical differences.

Results

We demonstrate that ASC/caspase-1-driven IL-1β is crucial for induction of B. burgdorferi-induced murine Lyme arthritis. In addition, we show that B. burgdorferi-induced murine Lyme arthritis is less dependent on NOD1/NOD2/RICK pathways while the TLR2-MyD88 pathway is crucial.

Conclusions

Murine Lyme arthritis is strongly dependent on IL-1 production, and B. burgdorferi induces inflammasome-mediated caspase-1 activation. Next to that, murine Lyme arthritis is ASC- and caspase-1-dependent, but NLRP3, NOD1, NOD2, and RICK independent. Also, caspase-1 activation by B. burgdorferi is dependent on TLR2 and MyD88. Based on present results indicating that IL-1 is one of the major mediators in Lyme arthritis, there is a rationale to propose that neutralizing IL-1 activity may also have beneficial effects in chronic Lyme arthritis.     

Treatment-associated polymorphisms in protease are significantly associated with higher viral load and lower CD4 count in newly diagnosed drug-naive HIV-1 infected patients

Background

Bone marrow stromal cell antigen 2 (BST-2) is a cellular factor that restricts the egress of viruses such as human immunodeficiency virus (HIV-1) from the surface of infected cells, preventing infection of new cells. BST-2 is variably expressed in most cell types, and its expression is enhanced by cytokines such as type I interferon alpha (IFN-??). In this present study, we used the beta-retrovirus, mouse mammary tumor virus (MMTV) as a model to examine the role of mouse BST-2 in host infection in vivo.

Results

By using RNA interference, we show that loss of BST-2 enhances MMTV replication in cultured mammary tumor cells and in vivo. In cultured cells, BST-2 inhibits virus accumulation in the culture medium, and co-localizes at the cell surface with virus structural proteins. Furthermore, both scanning electron micrograph (SEM) and transmission electron micrograph (TEM) show that MMTV accumulates on the surface of IFN??-stimulated cells.

Conclusions

Our data provide evidence that BST-2 restricts MMTV release from naturally infected cells and that BST-2 is an antiviral factor in vivo.     

Investigation of binding properties of dicationic styrylimidazo[1,2‐a]pyridinium dyes to human serum albumin by spectroscopic techniques

The binding interaction between two dicationic styrylimidazo[1,2‐a]pyridinium dyes and human serum albumin (HSA) was investigated at physiological conditions using fluorescence, UV–vis absorption, and circular dichroism (CD) spectroscopies. Analysis of the fluorescence titration data at different temperatures suggested that the fluorescence quenching mechanism of HSA by these dyes was static. The calculated thermodynamic parameters (ΔG°, ΔH° and ΔS°) indicated that hydrogen bonding and van der Waals forces played a major role in the formation of the dye–HSA complex. Binding distances (r) between dyes and HSA were calculated according to Förster's non‐radiative energy transfer theory. Studies of conformational changes of HSA using CD measurements indicate that the α‐helical content of the protein decreased upon binding of the dyes. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular and geographic analyses of vampire bat-transmitted cattle rabies in central Brazil

Background

Vampire bats are important rabies virus vectors, causing critical problems in both the livestock industry and public health sector in Latin America. In order to assess the epidemiological characteristics of vampire bat-transmitted rabies, the authors conducted phylogenetic and geographical analyses using sequence data of a large number of cattle rabies isolates collected from a wide geographical area in Brazil.

Methods

Partial nucleoprotein genes of rabies viruses isolated from 666 cattle and 18 vampire bats between 1987 and 2006 were sequenced and used for phylogenetic analysis. The genetic variants were plotted on topographical maps of Brazil.

Results

In this study, 593 samples consisting of 24 genetic variants were analyzed. Regional localization of variants was observed, with the distribution of several variants found to be delimited by mountain ranges which served as geographic boundaries. The geographical distributions of vampire-bat and cattle isolates that were classified as the identical phylogenetic group were found to overlap with high certainty. Most of the samples analyzed in this study were isolated from adjacent areas linked by rivers.

Conclusion

This study revealed the existence of several dozen regional variants associated with vampire bats in Brazil, with the distribution patterns of these variants found to be affected by mountain ranges and rivers. These results suggest that epidemiological characteristics of vampire bat-related rabies appear to be associated with the topographical and geographical characteristics of areas where cattle are maintained, and the factors affecting vampire bat ecology.

     

A preliminary investigation into the relationship between functional movement screen scores and athletic physical performance in female team sport athletes

There is little research investigating relationships between the Functional Movement Screen (FMS) and athletic performance in female athletes. This study analyzed the relationships between FMS (deep squat; hurdle step [HS]; in-line lunge [ILL]; shoulder mobility; active straight-leg raise [ASLR]; trunk stability push-up; rotary stability) scores, and performance tests (bilateral and unilateral sit-and-reach [flexibility]; 20-m sprint [linear speed]; 505 with turns from each leg; modified T-test with movement to left and right [change-of-direction speed]; bilateral and unilateral vertical and standing broad jumps; lateral jumps [leg power]). Nine healthy female recreational team sport athletes (age = 22.67 ± 5.12 years; height = 1.66 ± 0.05 m; body mass = 64.22 ± 4.44 kilograms) were screened in the FMS and completed the afore-mentioned tests. Percentage between-leg differences in unilateral sit-and-reach, 505 turns and the jumps, and difference between the T-test conditions, were also calculated. Spearman''s correlations (p ≤ 0.05) examined relationships between the FMS and performance tests. Stepwise multiple regressions (p ≤ 0.05) were conducted for the performance tests to determine FMS predictors. Unilateral sit-and-reach positive correlated with the left-leg ASLR (r = 0.704-0.725). However, higher-scoring HS, ILL, and ASLR related to poorer 505 and T-test performance (r = 0.722-0.829). A higher-scored left-leg ASLR related to a poorer unilateral vertical and standing broad jump, which were the only significant relationships for jump performance. Predictive data tended to confirm the correlations. The results suggest limitations in using the FMS to identify movement deficiencies that could negatively impact athletic performance in female team sport athletes.     

Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides

 We describe the synthesis and purification of two functional peptides, namely human insulin-like growth factor II (IGF-II) and Xenopus laevis magainin II in Hansenula polymorpha after their synthesis as hybrid proteins fused to the C terminus of endogenous amine oxidase. The hybrid genes, placed under control of the H. polymorpha alcohol oxidase promoter (P_AOX), were integrated into the genomic alcohol oxidase locus, yielding stable production strains. High-level synthesis of the fusion proteins, exceeding 20% of total cellular protein, was obtained when the transformed strains were grown in methanol-limited chemostat cultures; when expressed by itself, i.e. in the absence of the amine oxidase gene, IGF-II could not be recovered from crude cell extracts, probably as a result of rapid proteolytic degradation. Accumulation in peroxisomes did not significantly affect the IGF-II protein stability when expressed in the absence of the carrier protein. Apparently, fusion to the large (±78 kDa) amine oxidase carrier particularly stabilizes the peptides and prevents them from proteolysis. After partial purification, the fusion partners were readily separated by factor Xa treatment. Received: 16 June 1995 / Accepted: 20 September 1995