Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae

The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected

Energy Transfer from Magnetic Iron Oxide Nanoparticles: Implications for Magnetic Hyperthermia

Magnetic iron oxide nanoparticles (IONPs) have gained momentum in the field of biomedical applications. They can be remotely heated via alternating magnetic fields, and such heat can be transferred from the IONPs to the local environment. However, the microscopic mechanism of heat transfer is still debated. By X-ray total scattering experiments and first-principles simulations, we show how such heat transfer can occur. After establishing structural and microstructural properties of the maghemite phase of the IONPs, we built a maghemite model functionalized with aminoalkoxysilane, a molecule used to anchor (bio)molecules to oxide surfaces. By a linear response theory approach, we reveal that a resonance mechanism is responsible for the heat transfer from the IONPs to the surroundings. Heat transfer occurs not only via covalent linkages with the IONP but also through the solvent hydrogen-bond network. This result may pave the way to exploit the directional control of the heat flow from the IONPs to the anchored molecules─i.e., antibiotics, therapeutics, and enzymes─for their activation or release in a broader range of medical and industrial applications

Antimicrobial activity of nanoconjugated glycopeptide antibiotics and their effect on Staphylococcus Aureus biofilm

In the era of antimicrobial resistance, the use of nanoconjugated antibiotics is regarded as a promising approach for preventing and fighting infections caused by resistant bacteria, including those exacerbated by the formation of difficult-to-treat bacterial biofilms. Thanks to their biocompatibility and magnetic properties, iron oxide nanoparticles (IONPs) are particularly attractive as antibiotic carriers for the targeting therapy. IONPs can direct conjugated antibiotics to infection sites by the use of an external magnet, facilitating tissue penetration and disturbing biofilm formation. As a consequence of antibiotic localization, a decrease in its administration dosage might be possible, reducing the side effects to non-targeted organs and the risk of antibiotic resistance spread in the commensal microbiota. Here, we prepared nanoformulations of the ‘last-resort’ glycopeptides teicoplanin and vancomycin by conjugating them to IONPs via surface functionalization with (3-aminopropyl) triethoxysilane (APTES). These superparamagnetic NP-TEICO and NP-VANCO were chemically stable and NP-TEICO (better than NP-VANCO) conserved the typical spectrum of antimicrobial activity of glycopeptide antibiotics, being effective against a panel of staphylococci and enterococci, including clinical isolates and resistant strains. By a combination of different methodological approaches, we proved that NP-TEICO and, although to a lesser extent, NP-VANCO were effective in reducing biofilm formation by three methicillin-sensitive or resistant Staphylococcus aureus strains. Moreover, when attracted and concentrated by the action of an external magnet, NP-TEICO exerted a localized inhibitory effect on S. aureus biofilm formation at low antibiotic concentration. Finally, we proved that the conjugation of glycopeptide antibiotics to IONPs reduced their intrinsic cytotoxicity toward a human cell line. Copyright © 2021 Berini, Orlandi, Gamberoni, Martegani, Armenia, Gornati, Bernardini and Marinelli

Bottom Effect in Atomic Force Microscopy Nanomechanics

In this work, the influence of the rigid substrate on the determination of the sample Young''s modulus, the so-called bottom-effect artifact, is demonstrated by an atomic force microscopy force-spectroscopy experiment. The nanomechanical properties of a one-component supported lipid membrane (SLM) exhibiting areas of two different thicknesses are studied: While a standard contact mechanics model (Sneddon) provides two different elastic moduli for these two morphologies, it is shown that Garcia''s bottom-effect artifact correction yields a unique value, as expected for an intrinsic material property. Remarkably, it is demonstrated that the ratio between the contact radius (and not only the indentation) and the sample thickness is the key parameter addressing the relevance of the bottom-effect artifact. The experimental results are validated by finite element method simulations providing a solid support to Garcia''s theory. The amphiphilic nature of the investigated material is representative of several kinds of lipids, suggesting that the results have far reaching implications for determining the correct Young''s modulus of SLMs. The generality of Garcia''s bottom-effect artifact correction allows its application to every kind of supported soft film

Enzyme activation by alternating magnetic field: Importance of the bioconjugation methodology

Iron oxide nanoparticles (NPs) are attractive materials for enzyme immobilization and, thanks to their superparamagnetism, can be accessed by remote stimuli. This can be exploited to activate molecules that are not remotely actuable. Here, we demonstrate that thermophilic enzymes chemically linked to NPs can be activated in a \u201cwireless\u201d fashion by an external alternate magnetic field (AMF). To this aim, we have conjugated, with different binding strategies, the thermophilic enzymes \u3b1-amylase and L-aspartate oxidase to iron oxide NPs obtaining NP-enzyme systems with activities depending on the different orientations and stretching of the enzymes. Since enzyme activation occurs without a significant rise of the \u201coverall\u201d temperature of the systems, we have speculated a local NP-enzyme heating that does not immediately interest the rest of the solution that remains at relatively low temperature, low enough to allow non-thermophilic enzymes to work together with the NP-conjugated thermophilic enzymes. Nanoactuation of thermophilic enzymes by AMF has potential applications in different fields. Indeed, multi-enzymatic processes with enzymes with different temperature optima could be carried out in the same reaction pot and thermolabile products could be efficiently produced by thermophilic enzymes without suffering for the high temperatures. Moreover, our findings represent a proof of concept of the possibility to achieve a fine-tuning of the enzyme-NP system with the aim to intervene in cell metabolism

Prevalence of resistance mutations related to integrase inhibitor S/GSK1349572 in HIV-1 subtype B raltegravir-naive and -treated patients

Objectives To compare the frequency of previously in vitro-selected integrase mutations (T124A, T124A/S153F, S153Y, T124A/S153Y and L101I/T124A/S153Y) conferring resistance to S/GSK1349572 between HIV-1 subtype B integrase inhibitor (INI)-naive and raltegravir-treated patients. Methods Integrase sequences from 650 INI-naive patients and 84 raltegravir-treated patients were analysed. Results The T124A mutation alone and the combination T124A/L101I were more frequent in raltegravir-failing patients than in INI-naive patients (39.3% versus 24.5%, respectively, P = 0.005 for T124A and 20.2% versus 10.0%, respectively, P = 0.008 for T124A/L101I). The S153Y/F mutations were not detected in any integrase sequence (except for S153F alone, only detected in one INI-naive patient). Conclusions T124A and T124A/L101I, more frequent in raltegravir-treated patients, could have some effect on raltegravir response and their presence could play a role in the selection of other mutations conferring S/GSK1349572 resistance. The impact of raltegravir-mediated changes such as these on the virological response to S/GSK1349572 should be studied further

Remote activation of enzyme nanohybrids for cancer prodrug therapy controlled by magnetic heating

Herein, we have developed nanohybrids (nHs) to remotely activate a therapeutic enzyme for its use in Directed Enzyme Prodrug Therapy (DEPT). The coencapsulation of magnetic nanoparticles (MNPs) with horseradish peroxidase (HRP) using biomimetic silica as an entrapment matrix was optimized to obtain nanosized hybrids (∼150 nm) for remote activation of the therapeutic enzyme. HRP converts indole-3-acetic acid (3IAA) into peroxylated radicals, whereas MNPs respond to alternating magnetic fields (AMFs) becoming local hotspots. The AMF application triggered an increase in the bioconversion rate of HRP matching the activity displayed at the optimal temperature of the nHs (Topt = 50 °C) without altering the temperature of the reaction media. This showed that enzyme nanoactuation is possible with MNPs even if they are not covalently bound. After an extensive physicochemical/magnetic characterization, the spatial location of each component of the nH was deciphered, and an insulating role of the silica matrix was suggested as critical for introducing remote control over HRP. In vitro assays, using a human pancreatic cancer cell line (MIA PaCa-2), showed that only upon exposure to AMF and in the presence of the prodrug, the enzyme-loaded nHs triggered cell death. Moreover, in vivo experiments showed higher reductions in the tumor volume growth in those animals treated with nHs in the presence of 3IAA when exposed to AMF. Thus, this work demonstrates the feasibility of developing a spatiotemporally controlled DEPT strategy to overcome unwanted off-target effects

The genotypic false positive rate determined by V3 population sequencing can predict the burden of HIV-1 CXCR4-using species detected by pyrosequencing

The false-positive rate (FPR) is a percentage-score provided by Geno2Pheno-algorithm indicating the likelihood that a V3-sequence is falsely predicted as CXCR4-using. We evaluated the correlation between FPR obtained by V3 population-sequencing and the burden of CXCR4-using variants detected by V3 ultra-deep sequencing (UDPS) and Enhanced-Sensitivity Trofile assay (ESTA)