Porphyrin Binding and Irradiation Promote G-Quadruplex DNA Dimeric Structure.

Nucleic acid sequences rich in guanines can organize into noncanonical DNA G-quadruplexes (G4s) of variable size. The design of small molecules stabilizing the structure of G4s is a rapidly growing area for the development of novel anticancer therapeutic strategies and bottom-up nanotechnologies. Among a multitude of binders, porphyrins are very attractive due to their light activation that can make them valuable conformational regulators of G4s. Here, a structure-based strategy, integrating complementary probes, is employed to study the interaction between TMPyP4 porphyrin and a 22-base human telomeric sequence (Tel22) before and after irradiation with blue light. Porphyrin binding is discovered to promote Tel22 dimerization, while light irradiation of the Tel22-TMPyP4 complex controls dimer fraction. Such a change in quaternary structure is found to be strictly correlated with modifications at the secondary structure level, thus providing an unprecedented link between the degree of dimerization and the underlying conformational changes in G4s.

Hypoxia-induced miR-210 modulates the inflammatory response and fibrosis upon acute ischemia.

Hypoxia-induced miR-210 is a crucial component of the tissue response to ischemia, stimulating angiogenesis and improving tissue regeneration. Previous analysis of miR-210 impact on the transcriptome in a mouse model of hindlimb ischemia showed that miR-210 regulated not only vascular regeneration functions, but also inflammation. To investigate this event, doxycycline-inducible miR-210 transgenic mice (Tg-210) and anti-miR-210 LNA-oligonucleotides were used. It was found that global miR-210 expression decreased inflammatory cells density and macrophages accumulation in the ischemic tissue. To dissect the underpinning cell mechanisms, Tg-210 mice were used in bone marrow (BM) transplantation experiments and chimeric mice underwent hindlimb ischemia. MiR-210 overexpression in the ischemic tissue was sufficient to increase capillary density and tissue repair, and to reduce inflammation in the presence of Wt-BM infiltrating cells. Conversely, when Tg-210-BM cells migrated in a Wt ischemic tissue, dysfunctional angiogenesis, inflammation, and impaired tissue repair, accompanied by fibrosis were observed. The fibrotic regions were positive for α-SMA, Vimentin, and Collagen V fibrotic markers and for phospho-Smad3, highlighting the activation of TGF-ß1 pathway. Identification of Tg-210 cells by in situ hybridization showed that BM-derived cells contributed directly to fibrotic areas, where macrophages co-expressing fibrotic markers were observed. Cell cultures of Tg-210 BM-derived macrophages exhibited a pro-fibrotic phenotype and were enriched with myofibroblast-like cells, which expressed canonical fibrosis markers. Interestingly, inhibitors of TGF-ß type-1-receptor completely abrogated this pro-fibrotic phenotype. In conclusion, a context-dependent regulation by miR-210 of the inflammatory response was identified. miR-210 expression in infiltrating macrophages is associated to improved angiogenesis and tissue repair when the ischemic recipient tissue also expresses high levels of miR-210. Conversely, when infiltrating an ischemic tissue with mismatched miR-210 levels, macrophages expressing high miR-210 levels display a pro-fibrotic phenotype, leading to impaired tissue repair, fibrosis, and dysfunctional angiogenesis.

Hypoxia-Induced miR-210 Is Necessary for Vascular Regeneration upon Acute Limb Ischemia.

Critical limb ischemia is the most serious form of peripheral artery disease, characterized by severe functional consequences, difficult clinical management and reduced life expectancy. The goal of this study was to investigate the miR-210 role in the neo-angiogenic response after acute limb ischemia. Complementary approaches were used in a mouse model of hindlimb ischemia: miR-210 loss-of-function was obtained by administration of LNA-oligonucleotides anti-miR-210; for miR-210 gain-of-function, a doxycycline-inducible miR-210 transgenic mouse was used. We tested miR-210 ability to stimulate vascular regeneration following ischemia. We found that miR-210 was necessary and sufficient to stimulate blood perfusion recovery, as well as arteriolar and capillary density increase, in the ischemic muscle. To clarify the molecular events underpinning miR-210 pro-angiogenic action, the transcriptomic changes in ischemic muscles upon miR-210 blocking were analyzed. We found that miR-210 impacted the transcriptome significantly, regulating pathways and functions linked to vascular regeneration. In agreement with a pro-angiogenic role, miR-210 also improved cardiac function and left ventricular remodeling after myocardial infarction. Moreover, miR-210 blocking decreased capillary density in a Matrigel plug assay, indicating that miR-210 is necessary for angiogenesis independently of ischemia. Collectively, these data indicate that miR-210 plays a pivotal role in promoting vascular regeneration.

Dysregulation of Circular RNAs in Myotonic Dystrophy Type 1.

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponges for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by "back-splicing", which is the linking covalently of 3'- and 5'-ends of exons. Thus, circRNA levels might be deregulated in conditions associated with altered RNA-splicing. Significantly, growing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG repeats in the DMPK gene which results in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of nine tested, four transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03, and ZNF609. Their circular fraction values correlated with skeletal muscle strength and with splicing biomarkers of disease severity, and displayed higher values in more severely affected patients. Moreover, Receiver-Operating-Characteristics curves of these four circRNAs discriminated DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and the plasma of DM1 patients, but they were not regulated significantly. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this pilot study identified circRNA dysregulation in DM1 patients.

Structure of human telomere G-quadruplex in the presence of a model drug along the thermal unfolding pathway.

A multi-technique approach, combining circular dichroism spectroscopy, ultraviolet resonance Raman spectroscopy and small angle scattering techniques, has been deployed to elucidate how the structural features of the human telomeric G-quadruplex d[A(GGGTTA)3GGG] (Tel22) change upon thermal unfolding. The system is studied both in the free form and when it is bound to Actinomycin D (ActD), an anticancer ligand with remarkable conformational flexibility. We find that at room temperature binding of Tel22 with ActD involves end-stacking upon the terminal G-tetrad. Structural evidence for drug-driven dimerization of a significant fraction of the G-quadruplexes is provided. When the temperature is raised, both free and bound Tel22 undergo melting through a multi-state process. We show that in the intermediate states of Tel22 the conformational equilibrium is shifted toward the (3+1) hybrid-type, while a parallel structure is promoted in the complex. The unfolded state of the free Tel22 is consistent with a self-avoiding random-coil conformation, whereas the high-temperature state of the complex is observed to assume a quite compact form. Such an unprecedented high-temperature arrangement is caused by the persistent interaction between Tel22 and ActD, which stabilizes compact conformations even in the presence of large thermal structural fluctuations.

Melting of DNA nonoriented fibers: a wide-angle X-ray diffraction study.

The melting transition of A- and B-DNA has been investigated by wide-angle X-ray diffraction. A significant crystalline phase is present in both the systems, even if the fibers have not been artificially aligned. The behavior of the intramolecular Bragg peaks of both A- and B-DNA as a function of the temperature clearly reveals the unfolding structural transition of the double helix. This transition occurs at the same temperature as the melting of the crystalline phase. The trends of the intramolecular correlations and the index of crystallinity are nicely described by the Peyrard-Bishop-Dauxois model for DNA melting. A description of the processes taking place at a microscopic level, i.e., double-helix deformation, crystalline dilation, and collapse, on approaching and during thermal melting is proposed.