Polynucleotide: Adenosine glycosidase is the sole activity of ribosome-inactivating proteins on DNA

Polynucleotide: adenosine glycosidases (PNAG) are a class of plant and bacterial enzymes commonly known as ribosome-inactivating proteins (RIP). They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22]. Several activities on nucleic acids, other than depurination, have been attributed to PNAG: in particular modifications induced in circular plasmids, including linearisation and topological changes, and cleavage of guanidinic residues. Here we describe a chromatographic procedure to obtain nuclease-free PNAG by dye-chromatography onto Procion Red derivatized Sepharose®. Highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycolyase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 confirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distribution of the bound saporin-S6 molecules along the DNA chain is markedly variable, (iii) plasmids already digested with saporin-S6 do not appear fragmented or topologically modified. The observations here described demonstrate that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed antiviral protein from seeds and saporin-S6. These proteins belong to different families, suggesting that the findings here described may be generalized to all PNAG

Chitosan nanoparticles for lipophilic anticancer drug delivery: Development, characterization and in vitro studies on HT29 cancer cells

The aim of this study was to develop chitosan-based nanoparticles that could encapsulate lipophilic molecules and deliver them to cancer cells. Nanoparticles were prepared with different molar ratios of chitosan, hyaluronic acid and sulphobutyl-ether-\u3b2-cyclodextrin and with or without curcumin. The nanosystems were characterized in terms of their size, zeta potential, morphology, encapsulation efficiency and stability in different media. Intestinal epithelial and colorectal cancer cells were treated with unloaded nanoparticles in order to study their effect on cellular membrane organization and ROS production. Finally, in vitro assays on both cellular lines were performed in order to evaluate the ability of nanoparticles to promote curcumin internalization and to study their effect on cell proliferation and cell cycle. Results show that nanoparticles were positively charged and their size increased with the increasing amounts of the anionic excipient. Nanoparticles showed good encapsulation efficiency and stability in water. Unloaded nanoparticles led to a change in lipid organization in the cellular membrane of both cell lines, without inducing ROS generation. Confocal microscopy, cell proliferation and cell cycle studies allowed the selection of the best formulation to limit curcumin cytotoxicity in normal intestinal epithelial cells and to reduce cancer cell proliferation. The latter was the result of the increase of expression for genes involved in apoptosis

Novel mixed vesicles containing lactobacilli biosurfactants for vaginal delivery of econazole nitrate

Vulvovaginal candidiasis (VVC) is one of the most common cause of vaginitis affecting 70-75% of women at least once during their lives. The treatment of VVC involves the use of topical and oral azoles, such as econazole nitrate (EN), a broad-spectrum antifungal and lipophilic drug. The bioavailability and antifungal effects of econazole are limited by its poor water solubility [1]. In this regard, its incorporation in vesicular systems may improve its availability at the application site, thus reducing the necessary daily-dose and consequently increasing patient compliance. Phospholipid vesicles can be enriched with surfactant molecules in order to modify their functional properties and allow an efficient drug delivery. Traditionally, surfactants are produced by organic chemical reactions from petroleum feedstocks. Compared with synthetic surfactants, biosurfactants (BS) are \u201cgreen products\u201d that show several advantages such as lower toxicity and higher biodegradability [2]. BS are amphiphilic biological compounds produced extracellularly or as part of the cell membranes by a variety of microorganisms, including probiotic lactobacilli, and exhibit pronounced surface and emulsifying activities [3]. The first step of this work was to characterize BS produced by Lactobacillus gasseri BC9, isolated from the vagina of a healthy premenopausal woman, in terms of chemical structure and technological parameters. Secondly, the lipid film hydration method was employed for the formulation of EN- loaded and unloaded mixed vesicles (MV), containing BS (BS-MV) or Tween 80 (tween-MV). Vesicles were characterized in terms of morphology, size and zeta-potential. Stability, encapsulation efficiency, mucoadhesion properties and release of EN from mixed vesicles were also evaluated by in vitro studies. Moreover, the ability of MV to exert antimicrobial activity against C. albicans, the most frequent etiologic agent of VVC, was evaluated. BS produced by L. gasseri BC9 was composed by peptide-like molecules containing hydrocarbon chains, as shown by FT-IR spectrum, and possessed a high surface activity together with a low critical micelle concentration. Mixed vesicles presented a spherical shape (as confirmed by AFM images) and a diameter in the range of 226-337 nm (as confirmed by DLS measurements). Compared to tween-MV, loaded vesicles containing BS showed higher size and a lower zeta-potential value, probably due to the interaction between the drug and BS. This interaction also allowed the entrapment of a great amount of EN and sustained its release over the time. Furthermore, BS-MV showed good stability over the time and interesting mucoadhesive properties. Finally, the antimicrobial tests demonstrated the inhibitory activity against C. albicans of EN formulated in the MV. Additional investigations on mixed vesicles and their incorporation in a suitable gel vehicle should be considered to further evaluate their applicability in vaginal drug delivery. [1] Firooz A, Nafisi S, Maibach HI. Novel drug delivery strategies for improving econazole antifungal action. Int J Pharm. 2015; 495(1):599-607. [2] Vaz DA Gudi\uf1a, EJ, Alameda EJ, Teixeira JA, Rodrigues LR. Performance of a biosurfactant produced by a Bacillus subtilis strain isolated from crude oil samples as compared to commercial chemical surfactants. Colloids Surf B Biointerfaces. 2012; 89:167-74. [3] Van Hamme JD, Singh A, Ward OP. Physiological aspects. Part 1 in a series of papers devoted to surfactants in microbiology and biotechnology. Biotechnol. Adv. 2006; 24:604-620

Lengthening Patients Previously Treated for Massive Lower Limb Reconstruction for Bone Tumors with the PRECICE 2 Nail

The objective of this study was to determine the efficacy of the PRECICE 2® nail in the treatment of lower limb length discrepancy in patients with a history of bone tumors. This study reports on outcomes, complications, and the safety of the PRECICE 2 limb lengthening nail in a cohort of pediatric patients with limb length discrepancy after surgery for bone tumors. Seventeen patients were treated with intramedullary magnetic nails. The average patient age at the time of surgery was 19 (range 11–32). The PRECICE 2 nail was used on 14 femurs (6 retrograde and 8 anterograde) and 3 tibias. The average consolidation time was 141 days (range 50–360) with a mean CI of 31 ± 12 days/cm. The ASAMI bone score showed 14 (82%) excellent results, 1 (6%) good result, and 2 (12%) poor results. The ASAMI functional score showed 13 (84.6%) excellent results, 3 (11.5%) good results, and 1 (3.8%) fair result. Patients treated with chemotherapy for bone cancer did not show any increase in distraction time or consolidation time. A total of 3 (17%) problems, 1 obstacle (5.5%), and 1 complication (5.5%) were encountered in our case series. The PRECICE 2 nail allows for effective and accurate lengthening preserving the range of motion in patients treated for bone tumors