Rheologic and Physicochemical Characteristics of Hyaluronic Acid Fillers: Overview and Relationship to Product Performance.

Injections with hyaluronic acid (HA) fillers for facial rejuvenation and soft-tissue augmentation are among the most popular aesthetic procedures worldwide. Many HA fillers are available with unique manufacturing processes and distinct in vitro physicochemical and rheologic properties, which result in important differences in the fillers' clinical performance. The aim of this paper is to provide an overview of the properties most widely used to characterize HA fillers and to report their rheologic and physicochemical values obtained using standardized methodology to allow scientifically based comparisons. Understanding rheologic and physicochemical properties will guide clinicians in aligning HA characteristics to the facial area being treated for optimal clinical performance.

Structural and conformational requisites in DNA quadruplex groove binding: another piece to the puzzle.

The study of DNA G-quadruplex stabilizers has enjoyed a great momentum in the late years due to their application as anticancer agents. The recognition of the grooves of these structural motifs is expected to result in a higher degree of selectivity over other DNA structures. Therefore, to achieve an enhanced knowledge on the structural and conformational requisites for quadruplex groove recognition, distamycin A, the only compound for which a pure groove binding has been proven, has been chemically modified. Surprisingly, structural and thermodynamic studies revealed that the absence of Coulombic interactions results in an unprecedented binding position in which both the groove and the 3' end of the DNA are occupied. This further contribution adds another piece to the so far elusive puzzle of the recognition between ligands and DNA quadruplexes and will serve as a platform for a rational design of new groove binders.

Tandem application of virtual screening and NMR experiments in the discovery of brand new DNA quadruplex groove binders.

In the past decade, DNA G-quadruplexes have come into the limelight thanks to their biological implications and to their potential druggability in anticancer therapy. In particular, it has been found that small molecules that stabilize G-quadruplex structures are effective inhibitors of telomerase which plays a critical role in tumorigenesis. So far, the quadruplex groove recognition, which is expected to give a higher degree of selectivity over the other DNA structures, has been demonstrated for very few compounds. Thus with the aim of detecting new and structurally diverse groove binders, a structure-based virtual screening campaign has been performed using the X-ray structure of the [d(TGGGGT)](4) quadruplex. Remarkable results were achieved, and six brand new different molecular entities have been found to interact with the groove through NMR experiments. The reported results will certainly stimulate further studies aimed at the design and optimization of new quadruplex-specific groove binders to be applied as anticancer agents and for other diseases.

Competitive binding exchange between alkali metal ions (K+, Rb+, and Cs+) and Na+ ions bound to the dimeric quadruplex [d(G4T4G4)]2: a 23Na and 1H NMR study.

A comparative study of the competitive cation exchange between the alkali metal ions K+, Rb+, and Cs+ and the Na+ ions bound to the dimeric quadruplex [d(G4T4G4)]2 was performed in aqueous solution by a combined use of the 23Na and 1H NMR spectroscopy. The titration data confirm the different binding affinities of these ions for the G-quadruplex and, in particular, major differences in the behavior of Cs+ as compared to the other ions were found. Accordingly, Cs+ competes with Na+ only for the binding sites at the quadruplex surface (primarily phosphate groups), while K+ and Rb+ are also able to replace sodium ions located inside the quadruplex. Furthermore, the 1H NMR results relative to the CsCl titration evidence a close approach of Cs+ ions to the phosphate groups in the narrow groove of [d(G4T4G4)]2. Based on a three-site exchange model, the 23Na NMR relaxation data lead to an estimate of the relative binding affinity of Cs+ versus Na+ for the quadruplex surface of 0.5 at 298 K. Comparing this value to those reported in the literature for the surface of the G-quadruplex formed by 5'-guanosinemonophosphate and for the surface of double-helical DNA suggests that topology factors may have an important influence on the cation affinity for the phosphate groups on DNA.

A further contribution to the extreme variability of quadruplex structures from oligodeoxyribonucleotides containing inversion of polarity sites in the G-tract.

Structural insight into DNA quadruplex structures formed by oligodeoxyribonucleotides 3'TG5'-5'GGGT3' (QS55) and 5'TG3'-3'GGGT5' (QS33) is presented. NMR analysis reveals that QS33 forms a parallel-like four-fold symmetric quadruplex, while QS55 possesses a two-fold symmetry and is characterized by a tetrameric antiparallel quadruplex embedded between two parallel tracts. The results reported here describe unprecedented quadruplex complexes provided by peculiar structural features never reported to date. These structures might inspire the design of new aptameric nucleic acids characterized by novel structural motifs hardly realizable with unmodified DNA/RNA.

Targeting DNA quadruplexes with distamycin A and its derivatives: an ITC and NMR study.

The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promising way to inhibit telomerase activity in tumor cells. In this paper, isothermal titration calorimetry (ITC) and 1H NMR studies have been conducted to examine the binding of distamycin A and its two carbamoyl derivatives (compounds 1 and 2) to the target [d(TGGGGT)]4 and d[AG3(T2AG3)3] quadruplexes from the Tetrahymena and human telomeres, respectively. The interactions were examined using two different buffered solutions containing either K+ or Na+ at a fixed ionic strength, to evaluate any influence of the ions present in solution on the binding behaviour. Experiments reveal that distamycin A and compound 1 bind the investigated quadruplexes in both solution conditions; conversely, compound 2 appears to have a poor affinity in any case. Moreover, these studies indicate that the presence of different cations in solution affects the stoichiometry and thermodynamics of the interactions.

Effect of rubidium and cesium ions on the dimeric quaduplex formed by the Oxytricha nova telomeric repeat oligonucleotide d(GGGGTTTTGGGG).

The DNA sequence d(GGGGTTTTGGGG) consists of 1.5 units of the repeat in telomeres of Oxytricha nova. It has been shown by NMR and x-ray crystallographic analysis that it is capable to form a dimeric quadruplex structure and that a variety of cations, namely K(+), Na(+), and NH(4)(+), are able to interact with this complex with different affinity, leading to complexes characterized by different local conformations. Thus, in order to improve the knowledge of this kind of molecule, and in particular to provide further insight into the role of monovalent cations in the G-quadruplex folding and conformation, we have investigated by (1)H-NMR the effect of the addition of Rb(+) and Cs(+) to the quadruplex formed by the oligonucleotide d(GGGGTTTTGGGG).

Molecular modelling studies of four stranded quadruplexes containing a 3'-3' or 5'-5' inversion of polarity site.

Recently we reported a preliminary study on the structure of two novel quadruplex structures, Q33 and Q55, formed by the oligodeoxynucleotides (5)'TGG(3)'-(3)'GGT(5)' and (3)'TGG(5)'-(5)'GGT(3)', respectively. Here we report their solution structures at the atomic level. The obtained structures reveal that Q55 and Q33 possess a different stacking among G-quartets and different twist angle (and therefore different helical winding) at the inversion of polarity level.

Structural insight into the hTERT intron 6 sequence d(GGGGTGAAAGGGG) from 1H-NMR study.

The interest in DNA quadruplex structures has been fueled by the recognition that telomeres, the 3' single stranded guanine-rich overhangs found at the termini of chromosomes, are likely to form G-tetrads type structures important in cell senescence and cancer. In addition to their presence in telomeres, where they may play a role in maintaining the stability and integrity of chromosomes, guanine-rich regions are found in other region of the genome, amongst these is intron 6 of hTERT a gene codifying for the enzyme telomerase. Interestingly, the formation of G-quadruplexes in this region is involved in the down-regulation of telomerase activity caused by an alteration of the hTERT splicing pattern. Therefore, we have analyzed several sequences of that intron by (1)H-NMR and CD spectroscopy, and we have found that the sequence d(GGGGTGAAAGGGG) is able to fold in a single well-defined antiparallel quadruplex structure consisting of four G-tetrads, possessing a twofold symmetry, and containing four Gs in a syn glycosidic conformation.

Biophysical properties of quadruplexes containing two or three 8-bromodeoxyguanosine residues.

A physico-chemical characterization, based on NMR and CD spectroscopy, of quadruplexes formed by the oligonucleotide d(TGGGT), where two or three Gs are substituted by 8-bromo-2'-deoxyguanosine residues (dGBr), is reported. The oligonucleotidic sequences d(TGBr GBr GT), d(TGBr GGBr T), d(TGGBr GBr T), and d(TGBr GBr GBr T) have been synthesized. Only sequences d(TGBr GGBr T) and d(TGBr GBr GT) were able to fold into a well defined quadruplex structure, and their CD profiles and thermal stabilities turned out to be very different from those observed for the natural counterpart, indicating that the 8-Br-dG residues dramatically affect the structure of the quadruplex.

Effect of the incorporation of 2'-deoxy-8-(hydroxyl)adenosine on the stability of quadruplexes formed by modified human telomeric DNA.

Differential scanning calorimetry (DSC) and circular dichroism (CD) techniques were used to investigate the physico-chemical properties of the quadruplexes formed by the two different truncations of human telomeric sequence d(TAGGGT) and d(AGGGT), where the adenines were substituted by 2'-deoxy-8-(hydroxyl)adenosine (A --> A OH). CD spectra show that the modified sequences are able to form parallel-stranded quadruplex structure. Analysis of the thermodynamic parameters reveals that the introduction of the modified adenine affects in different way the thermal stability of the [d(TAGGGT)]4 and [d(AGGGT)]4 quadruplexes.

Thermodynamic analysis of quadruplex DNA-drug interaction.

This work studies the binding properties of distamycin and its carbamoyl analog, containing four pyrrole units, with the [d(TGGGGT)](4) quadruplex by means of isothermal titration calorimetry (ITC). Analysis of the ITC data reveals that drug/quadruplex binding stoichiometry is 1:1 for both interactions and that distamycin analog gives approximately a 10-fold increase in the quadruplex affinity.

Structural and thermodynamic studies of the interaction of distamycin A with the parallel quadruplex structure [d(TGGGGT)]4.

The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by 1H NMR spectroscopy and isothermal titration calorimetry (ITC). To unambiguously assert that distamycin A interacts with the grooves of the quadruplex [d(TGGGGT)]4, we have analyzed the NMR titration profile of a modified quadruplex, namely [d(TGGMeGGT)]4, and we have applied the recently developed differential frequency-saturation transfer difference (DF-STD) method, for assessing the ligand-DNA binding mode. The three-dimensional structure of the 4:1 distamycin A/[d(TGGGGT)]4 complex has been determined by an in-depth NMR study followed by dynamics and mechanics calculations. All results unequivocally indicate that distamycin molecules interact with [d(TGGGGT)]4 in a 4:1 binding mode, with two antiparallel distamycin dimers that bind simultaneously two opposite grooves of the quadruplex. The affinity between distamycin A and [d(TGGGGT)]4 enhances ( approximately 10-fold) when the ratio of distamycin A to the quadruplex is increased. In this paper we report the first three-dimensional structure of a groove-binder molecule complexed to a DNA quadruplex structure.

A novel thrombin binding aptamer containing a G-LNA residue.

In this work, we report the solution structure, thermodynamic studies, and the pharmacological properties of a new modified thrombin binding aptamer (TBA) containing a G-LNA residue, namely d(5'-GGTTGGTGTGGTTGg-3'), where upper case and lower case letters represent DNA and LNA residues, respectively. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the three-dimensional structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT, and TT loops. d(5'-GGTTGGTGTGGTTGg-3') is characterized by the same folding of TBA, being two strands parallel to each other and two strands oriented in opposite manner. This led to a syn-anti-syn-anti and anti-syn-anti-syn arrangements of the Gs in the two tetrads. d(5'-GGTTGGTGTGGTTGg-3') possesses an anticoagulant activity, even if decreased with respect to the TBA.

A new modified thrombin binding aptamer containing a 5'-5' inversion of polarity site.

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5'-5' inversion of polarity site, namely d(3'GGT5'-5'TGGTGTGGTTGG3'), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(3'GGT5'-5'TGGTGTGGTTGG3') is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4 degrees C higher than the corresponding unmodified TBA. d(3'GGT5'-5'TGGTGTGGTTGG3') continues to display an anticoagulant activity, even if decreased with respect to the TBA.