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

Cataloged from PDF version of article.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 deliver

Oligonucleotide delivery with cell surface binding and cell penetrating peptide amphiphile nanospheres

Cataloged from PDF version of article.A drug delivery system designed specifically for oligonucleotide therapeutics can ameliorate the problems associated with the in vivo delivery of these molecules. The internalization of free oligonudeotides is challenging, and cytotoxicity is the main obstacle for current transfection vehicles. To develop nontoxic delivery vehicles for efficient transfection of oligonudeotides, we designed a self-assembling peptide amphiphile (PA) nanosphere delivery system decorated with cell penetrating peptides (CPPs) containing multiple arginine residues (R-4 and R-8), and a cell surface binding peptide (KRSR), and report the efficiency of this system in delivering G-3129, a Bcl-2 antisense oligonucleotide (AON). PA/AON (peptide amphiphile/antisense oligonucleotide) complexes were characterized with regards to their size and secondary structure, and their cellular internalization efficiencies were evaluated. The effect of the number of arginine residues on the cellular internalization was investigated by both flow cytometry and confocal imaging, and the results revealed that uptake efficiency improved as the number of arginines in the sequence increased. The combined effect of cell penetration and surface binding property on the cellular internalization and its uptake mechanism was also evaluated by mixing R-8-PA and KRSR-PA. R-8 and R-8/KRSR decorated PAs were found to drastically increase the internalization of AONs compared to nonbioactive PA control. Overall, the KRSR-decorated self-assembled PA nanospheres were demonstrated to be noncytotoxic delivery vectors with high transfection rates and may serve as a promising delivery system for AONs

Heterotrophic ammonium removal by a novel hatchery isolate Acinetobacter calcoaceticus STB1

Cataloged from PDF version of article.A novel bacterial strain, STB1, was isolated from a commercial sea bass hatchery and found to display high heterotrophic ammonium removal characteristics at different concentrations of ammonium (NH4+-N). The species identity of STB1 was determined via 16S rRNA gene sequence analysis to be Acinetobacter calcoaceticus. We evaluated ammonium removal characteristics of STB1 at varying ammonium concentrations, and observed that STB1 can almost completely remove ammonium at low (50 mg l -1), and medium (100 mg l -1) concentrations within 72 h, while 45% ammonium removal was observed at a higher concentration (210 mg l -1) during the same period. Trace amount of the metabolized ammonium was converted to nitrite or nitrate and 22.16% of total nitrogen was incorporated into cell biomass, while 4.34% of total nitrogen was initially incorporated into cell biomass and subsequently released to the supernatant fraction in the 100 mg l -1 sample. Most of the remaining conversion products are expected to be gaseous denitrification products. Toxicological studies with Artemia salina (brine shrimp) nauplii revealed that STB1 strain is non-toxic to Artemia larvae, which suggests that STB1 can be safely and efficiently utilized in water quality enrichment in aquatic ecosystems. © 2012 Elsevier Ltd

Induction of triacylglycerol production in Chlamydomonas reinhardtii: Comparative analysis of different element regimes

Cataloged from PDF version of article.In this study, impacts of different element absence (nitrogen, sulfur, phosphorus and magnesium) and supplementation (nitrogen and zinc) on element uptake and triacylglycerol production was followed in wild type Chlamydomonas reinhardtii CC-124 strain. Macro- and microelement composition of C. reinhardtii greatly differed under element regimes studied. In particular, heavy metal quotas of the microalgae increased strikingly under zinc supplementation. Growth was suppressed, cell biovolume, carbohydrate, total neutral lipid and triacylglycerol levels increased when microalgae were incubated under these element regimes. Most of the intracellular space was occupied by lipid bodies under all nutrient starvations, as observed by confocal microscopy and transmission electron micrographs. Results suggest that sulfur, magnesium and phosphorus deprivations are superior to nitrogen deprivation for the induction triacylglycerol production in C. reinhardtii. On the other hand, FAME profiles of the nitrogen, sulfur and phosphorus deprived cells were found to meet the requirements of international standards for biodiesel. Crown Copyright (C) 2013 Published by Elsevier Ltd. All rights reserved

Toxicity of lanthanum oxide (La2O3) nanoparticles in aquatic environments

This study demonstrates the acute toxicity of lanthanum oxide nanoparticles (La2O3 NP) on two sentinel aquatic species, fresh-water microalgae Chlorella sp. and the crustacean Daphnia magna. The morphology, size and charge of the nanoparticles were systematically studied. The algal growth inhibition assay confirmed absence of toxic effects of La2O3 NP on Chlorella sp., even at higher concentration (1000 mg L-1) after 72 h exposure. Similarly, no significant toxic effects were observed on D. magna at concentrations of 250 mg L-1 or less, and considerable toxic effects were noted in higher concentrations (effective concentration [EC50] 500 mg L-1; lethal dose [LD50] 1000 mg L-1). In addition, attachment of La2O3 NP on aquatic species was demonstrated using microscopy analysis. This study proved to be beneficial in understanding acute toxicity in order to provide environmental protection as part of risk assessment strategies. © The Royal Society of Chemistry 2015

Efficient ammonium removal from aquatic environments by Acinetobacter calcoaceticus STB1 immobilized on an electrospun cellulose acetate nanofibrous web

Cataloged from PDF version of article.A novel biocomposite material was developed by immobilizing an ammonia-oxidizing bacterial strain, Acinetobacter calcoaceticus STB1, on an electrospun porous cellulose acetate (CA) nanofibrous web. Ammonium removal characteristics of the STB1 immobilized CA nanofibrous web were determined at varying initial ammonium concentrations, and removal rates of 100%, 98.5% and 72% were observed within 48 h for 50 mg L-1, 100 mg L-1 and 200 mg L-1 samples, respectively. Most of the ammonia is inferred to be converted into nitrogen or is accumulated as bacterial biomass, as only trace amounts of ammonium were converted into nitrite or nitrate. Reusability test results indicate that, at an initial ammonium concentration of 100 mg L-1, bacteria-immobilized CA nanofibrous webs can be reused for at least 5 cycles. SEM images of the STB1/CA nanofibrous web after five cycles of reuse and rigorous washing demonstrate that bacterial biofilms strongly adhere to nanofiber surfaces

Highly senstivive determination of 2.4,6-Trinitrotolunene and Related Byproducts Using a Diol Functionalized Column for High Performance Liquid Chromatography

Cataloged from PDF version of article.In this work, a new detection method for complete separation of 2,4,6-trinitrotoluene (TNT); 2,4-dinitrotoluene (2,4-DNT); 2,6-dinitrotoluene (2,6-DNT); 2-aminodinitrotoluene (2-ADNT) and 4-aminodinitrotoluene (4-ADNT) molecules in high-performance liquid-chromatography (HPLC) with UV sensor has been developed using diol column. This approach improves on cost, time, and sensitivity over the existing methods, providing a simple and effective alternative. Total analysis time was less than 13 minutes including column re-equilibration between runs, in which water and acetonitrile were used as gradient elution solvents. Under optimized conditions, the minimum resolution between 2,4-DNT and 2,6-DNT peaks was 2.06. The recovery rates for spiked environmental samples were between 95-98%. The detection limits for diol column ranged from 0.78 to 1.17 μg/L for TNT and its byproducts. While the solvent consumption was 26.4 mL/min for two-phase EPA and 30 mL/min for EPA 8330 methods, it was only 8.8 mL/min for diol column. The resolution was improved up to 49% respect to two-phase EPA and EPA 8330 methods. When compared to C-18 and phenyl-3 columns, solvent usage was reduced up to 64% using diol column and resolution was enhanced approximately two-fold. The sensitivity of diol column was afforded by the hydroxyl groups on polyol layer, joining the formation of charge-transfer complexes with nitroaromatic compounds according to acceptor-donor interactions. Having compliance with current requirements, the proposed method demonstrates sensitive and robust separation. © 2014 Gumuscu et al

Fast Responding and Selective Near-IR Bodipy Dye for Hydrogen Sulfide Sensing

Cataloged from PDF version of article.A Bodipy based, highly selective probe for hydrogen sulfide has been designed, synthesized and demonstrated to detect H2S in living cells. In this design, the reduction of two arylazido groups change the charge transfer characteristics of the 3,5-distyryl substituents on the Bodipy core, producing a 20 nm bathochromic spectral shift in the absorption band, and quenching of the emission by 85% compared to the original intensity, through photoinduced electron transfer

Nitrogen and sulfur deprivation differentiate lipid accumulation targets of Chlamydomonas reinhardtii

Cataloged from PDF version of article.Nitrogen (N) and sulfur (S) have inter-related and distinct impacts on microalgal metabolism; with N starvation having previously been reported to induce elevated levels of the biodiesel feedstock material triacylglycerol (TAG), while S deprivation is extensively studied for its effects on biohydrogen production in microalgae.1,2 We have previously demonstrated that N- and S-starved cells of Chlamydomonas reinhardtii display different metabolic trends, suggesting that different response mechanisms exist to compensate for the absence of those two elements.3 We used C. reinhardtii CC-124 mt(-) and CC-125 mt(+) strains to test possible metabolic changes related to TAG accumulation in response to N and S deprivation, considering that gamete differentiation in this organism is mainly regulated by N.4 Our findings contribute to the understanding of microalgal response to element deprivation and potential use of element deprivation for biodiesel feedstock production using microalgae, but much remains to be elucidated on the precise contribution of both N and S starvation on microalgal metabolism. © 2012 Landes Bioscience

Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil

In this environmental-sample based study, rapid microbial-mediated degradation of 2,4,6-trinitrotoluene (TNT) contaminated soils is demonstrated by a novel strain, Achromobacter spanius STE 11. Complete removal of 100mgL-1 TNT is achieved within only 20h under aerobic conditions by the isolate. In thisbio-conversion process, TNT is transformed to 2,4-dinitrotoluene (7mgL-1), 2,6-dinitrotoluene (3mgL-1), 4-aminodinitrotoluene (49mgL-1) and 2-aminodinitrotoluene (16mgL-1) as the key metabolites. A. spanius STE 11 has the ability to denitrate TNT in aerobic conditions as suggested by the dinitrotoluene and NO3 productions during the growth period. Elemental analysis results indicate that 24.77mgL-1 nitrogen from TNT was accumulated in the cell biomass, showing that STE 11 can use TNT as its sole nitrogen source. TNT degradation was observed between pH 4.0-8.0 and 4-43°C; however, the most efficient degradation was at pH 6.0-7.0 and 30°C. © 2013 Elsevier Ltd