Unveiling the interaction between DNA G-quadruplexes and RG-rich peptides.

G-quadruplexes are non-canonical DNA secondary structures formed within guanine-rich strands that play important roles in various biological processes, including gene regulation, telomere maintenance and DNA replication. The biological functions and formation of these DNA structures are strictly controlled by several proteins that bind and stabilize or resolve them. Many G-quadruplex-binding proteins feature an arginine and glycine-rich motif known as the RGG or RG-rich motif. Although this motif plays a crucial role in the recognition of such non-canonical structures, their interaction is still poorly understood. Here, we employed a combination of several biophysical techniques to provide valuable insights into the interaction between a peptide containing an RGG motif shared by numerous human G-quadruplex-binding proteins (NIQI) and various biologically relevant G-quadruplex DNA structures with different topologies. We also shed light on the key amino acids involved in the binding process. Our findings contribute to lay the basis for the development of a new class of peptide-based G-quadruplex ligands as an alternative to small molecules. These ligands may serve as valid tools for interfering in DNA-protein interactions, with potential therapeutic applications.

Cytosine epigenetic modifications and conformational changes in G-quadruplex DNA: An ultraviolet resonance Raman spectroscopy study.

Epigenetic modifications of DNA are known to play important regulatory roles in biological systems, especially in regulation of gene expression, and are associated with many types of human diseases, including cancer. Alternative DNA secondary structures, such as G-quadruplexes, can also influence gene transcription, thus suggesting that such structures may represent a distinctive layer of epigenetic information. G-quadruplex structures and DNA epigenetic modifications often go side by side, and recent evidence reveals that cytosine modifications within loops of G-quadruplexes can play a role in modulating their stability and structural polymorphism. Therefore, the development and validation of experimental techniques that can easily and reliably analyse G-quadruplex structures are highly desirable. In the present study, we propose to exploit the advantages of UV resonance Raman (UVRR) spectroscopy to investigate cytosine epigenetic modifications along with conformational changes in G-quadruplex-forming DNA. Our findings show that clear and specific spectral changes occur when there is a change in a G-quadruplex structure. Moreover, UVRR spectral analysis can indirectly distinguish the spectral variations occurring because of modifications in the guanine glycosidic conformations, as well as detect changes in the loops induced by H-bond formation or hydration of nitrogenous bases. The results further underscore the utility of UVRR spectroscopy for G-quadruplex structure elucidation under biologically relevant solution conditions.

Bioengineered lipophilic Ru(III) complexes as potential anticancer agents.

Lipid-conjugated Ru(III) complexes - designed to obtain lipophilic analogues of the low molecular weight derivative AziRu, which is a NAMI-A-like anticancer agent - have been synthesized and fully characterized. A detailed biophysical investigation, including multiple, integrated techniques, allowed determining their molecular and self-assembling properties in aqueous solutions mimicking the extracellular environment, showing that our design produced a protective effect from hydrolysis of the Ru(III) complexes. In vitro biological experiments, carried out in comparison with AziRu, demonstrated that, among the novel lipophilic Ru(III) complexes synthesized, the compounds derivatized with palmitic and stearic acid, that we named PalmiPyRu and StePyRu respectively, showed attractive features and a promising antiproliferative activity, selective on specific breast cancer phenotypes. To get a deeper insight into their interactions with potential biomacromolecular targets, their ability to bind both bovine serum albumin (BSA), an abundant serum carrier protein, and some DNA model systems, including duplex and G-quadruplex structures, has been investigated by spectroscopic techniques. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis of the ruthenium amount incorporated in human MCF-7 and MDA-MB-231 breast cancer cells, after incubation in parallel experiments with PalmiPyRu and AziRu, showed a markedly higher cell uptake of the lipophilic Ru(III) complex with respect to AziRu. These data confirmed that the proper lipidic tail decorating the metal complex not only favoured the formation of aggregates in the extracellular media but also improved their cell membrane penetration, thus leading to higher antiproliferative activity selective on breast cancer cells.

The microstructure of Carbopol in water under static and flow conditions and its effect on the yield stress.

In this work, experimental observations of the microstructure of neutralized polyacrylic acid (Carbopol) in water by confocal microscopy under both static and flow conditions are presented. In the former case, a Carbopol-rich phase made by swollen particles dispersed in a water-rich continuous phase is found, so that the system will be henceforth referred to as a suspension, as long as particles are observed. The swollen particles form dendritic-like aggregates, which span the entire solution volume above a critical concentration. In such conditions, a percolated network can be formed, leading to the onset of a yield stress behavior. By separating the dispersed and continuous phase through centrifugation, we provide evidence of a miscibility gap in the phase behavior of Carbopol in water. When the Carbopol suspensions flow in a microfluidic capillary, a particle-concentrated plug core can be distinguished from a less concentrated layer corresponding to a steep velocity decrease. Confocal imaging also shows that the apparent slip found in Carbopol suspensions is due to a particle-concentrated near-wall region, where no flow is observed. Such flow-induced microstructure is responsible for the different nature of the yield stress values measured by classical rheometry and by flow velocimetry. While the yield stress measured by the former can be here related to the presence of a percolated network, the yield stress obtained from the velocity profile is due to the heterogeneous particle distribution along the capillary radius. These results provide a novel insight on the mechanisms governing yield stress in complex fluids.

Cross-sectional survey of canine leishmaniasis in Pantelleria island in Sicily.

Dogs are the major reservoir of Leishmania infantum, the causative agent of canine visceral and cutaneous human leishmaniasis in the Mediterranean basin. Canine and human leishmaniosis are endemic in Italy, particularly in central and southern regions, including islands. Here we show a preliminary, clinical, serological and molecular study carried out in Pantelleria island during 2017. In this study, we clinically examined 136 dogs for the presence of symptoms compatible with leishmaniasis, determined the titer of anti­Leishmania antibodies, and investigated Leishmania DNA by real time PCR in blood and/or lymph node of each dog. The prevalence of disease was equal to 27% with 95% CI [21%; 32%], lower than prevalence obtained in the other Sicily islands (Lampedusa, Lipari). We observed that enlarged lymph nodes was more positively associated with canine leishmaniasis (CanL)than other clinical signs. The results obtained showed that in an endemic area, such as Sicily, diagnosis of CanL needs to be carried out by including an immunological, molecular clinical approach.