BackgroundBuilding reference libraries of DNA barcodes is relatively straightforward when specifically designed primers are available to amplify the COI-5P region from a relatively narrow taxonomic group (e.g. single class or single order). DNA barcoding marine communities have been comparatively harder to accomplish due to the broad taxonomic diversity and lack of consistently efficient primers. Although some of the so-called “universal” primers have been relatively successful, they still fail to amplify COI-5P of many marine animal groups, while displaying random success even among species within each group. Here we propose a new pair of primers designed to enhance amplification of the COI-5P region in a wide range of marine organisms.ResultsAmplification tests conducted on a wide range of marine animal taxa, rendered possible the first–time sequencing of DNA barcodes from eight separated phyla (Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nemertea and Platyhelminthes), comprising a total of 14 classes, 28 orders, 57 families, 68 genus and 76 species.ConclusionsThese primers demonstrated to be highly cost-effective, which is of key importance for DNA barcoding procedures, such as for building comprehensive DNA barcode libraries of marine communities, where the processing of a large numbers of specimens from a wide variety of marine taxa is compulsory.

The effect of nanoparticle size, shape, and surface properties on cellular uptake has been extensively investigated for its basic science and translational implications. Recently, softness is emerging as a design parameter for modulating the interaction of nanoparticles with cells and the biological microenvironment. Here, circular, quadrangular, and elliptical polymeric nanoconstructs of different sizes are realized with a Young's modulus ranging from ∼100 kPa (soft) to 10 MPa (rigid). The interaction of these nanoconstructs with professional phagocytic cells is assessed via confocal microscopy and flow cytometry analyses. Regardless of the size and shape, softer nanoconstructs evade cellular uptake up to 5 times more efficiently, by bone-marrow-derived monocytes, as compared to rigid nanoconstructs. Soft circular and quadrangular nanoconstructs are equally uptaken by professional phagocytic cells (<15%); soft elliptical particles are more avidly internalized (<60%) possibly because of the larger size and elongated shape, whereas over 70% of rigid nanoconstructs of any shape and size are uptaken. Inhibition of actin polymerization via cytochalasin D reduces the internalization propensity for all nanoconstruct types. High-resolution live cell microscopy documents that soft nanoconstructs mostly establish short-lived (<30 s) interactions with macrophages, thus diminishing the likelihood of recognition and internalization. The bending stiffness is identified as a discriminating factor for internalization, whereby particles with a bending stiffness slightly higher than cells would more efficiently oppose internalization as compared to stiffer or softer particles. These results confirm that softness is a key parameter in modulating the behavior of nanoparticles and are expected to inspire the design of more efficient nanoconstructs for drug delivery, biomedical imaging, and immunomodulatory therapies.

Gold nanoparticles functionalized with Gd(3+) chelates displaying fast water exchange, superb pH stability and inertness towards transmetalation with Zn(2+) have been prepared and characterized as a new high relaxivity (29 mM(-1) s(-1), 30 MHz, 25 °C) contrast agent potentially safe for in vivo MRI applications. The Lipari-Szabo treatment for internal rotation was used to evaluate the effect of linker flexibility on the relaxivity of the gold nanoparticles. The effect of fast water exchange on the relaxivity of gold nanoparticles functionalized with Gd(3+) chelates is also addressed in this communication.

Tissue plasminogen activator (tPA) is the sole approved therapeutic molecule for the treatment of acute ischemic stroke. Yet, only a small percentage of patients could benefit from this life-saving treatment because of medical contraindications and severe side effects, including brain hemorrhage, associated with delayed administration. Here, a nano therapeutic agent is realized by directly associating the clinical formulation of tPA to the porous structure of soft discoidal polymeric nanoconstructs (tPA-DPNs). The porous matrix of DPNs protects tPA from rapid degradation, allowing tPA-DPNs to preserve over 70 % of the tPA original activity after 3 h of exposure to serum proteins. Under dynamic conditions, tPA-DPNs dissolve clots more efficiently than free tPA, as demonstrated in a microfluidic chip where clots are formed mimicking in vivo conditions. At 60 min post treatment initiation, the clot area reduces by half (57 + 8 %) with tPA-DPNs, whereas a similar result (56 + 21 %) is obtained only after 90 min for free tPA. In murine mesentery venules, the intravenous administration of 2.5 mg/kg of tPA-DPNs resolves almost 90 % of the blood clots, whereas a similar dose of free tPA successfully recanalize only about 40 % of the treated vessels. At about 1/10 of the clinical dose (1.0 mg/kg), tPA-DPNs still effectively dissolve 70 % of the clots, whereas free tPA works efficiently only on 16 % of the vessels. In vivo, discoidal tPA-DPNs outperform the lytic activity of 200 nm spherical tPA-coated nanoconstructs in terms of both percentage of successful recanalization events and clot area reduction. The conjugation of tPA with preserved lytic activity, the deformability and blood circulating time of DPNs together with the faster blood clot dissolution would make tPA-DPNs a promising nanotool for enhancing both potency and safety of thrombolytic therapies.

No abstract