Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients: Cellular model of pathology

The neurodegenerative disease Friedreich's ataxia (FRDA) is the most common autosomal-recessively inherited ataxia and is caused by a GAA triplet repeat expansion in the first intron of the frataxin gene. In this disease, transcription of frataxin, a mitochondrial protein involved in iron homeostasis, is impaired, resulting in a significant reduction in mRNA and protein levels. Global gene expression analysis was performed in peripheral blood samples from FRDA patients as compared to controls, which suggested altered expression patterns pertaining to genotoxic stress. We then confirmed the presence of genotoxic DNA damage by using a gene-specific quantitative PCR assay and discovered an increase in both mitochondrial and nuclear DNA damage in the blood of these patients (p<0.0001, respectively). Additionally, frataxin mRNA levels correlated with age of onset of disease and displayed unique sets of gene alterations involved in immune response, oxidative phosphorylation, and protein synthesis. Many of the key pathways observed by transcription profiling were downregulated, and we believe these data suggest that patients with prolonged frataxin deficiency undergo a systemic survival response to chronic genotoxic stress and consequent DNA damage detectable in blood. In conclusion, our results yield insight into the nature and progression of FRDA, as well as possible therapeutic approaches. Furthermore, the identification of potential biomarkers, including the DNA damage found in peripheral blood, may have predictive value in future clinical trials

EphA3 Expressed in the Chicken Tectum Stimulates Nasal Retinal Ganglion Cell Axon Growth and Is Required for Retinotectal Topographic Map Formation

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis

DMTs and Covid-19 severity in MS: a pooled analysis from Italy and France

We evaluated the effect of DMTs on Covid-19 severity in patients with MS, with a pooled-analysis of two large cohorts from Italy and France. The association of baseline characteristics and DMTs with Covid-19 severity was assessed by multivariate ordinal-logistic models and pooled by a fixed-effect meta-analysis. 1066 patients with MS from Italy and 721 from France were included. In the multivariate model, anti-CD20 therapies were significantly associated (OR&nbsp;=&nbsp;2.05, 95%CI&nbsp;=&nbsp;1.39–3.02, p&nbsp;&lt;&nbsp;0.001) with Covid-19 severity, whereas interferon indicated a decreased risk (OR&nbsp;=&nbsp;0.42, 95%CI&nbsp;=&nbsp;0.18–0.99, p&nbsp;=&nbsp;0.047). This pooled-analysis confirms an increased risk of severe Covid-19 in patients on anti-CD20 therapies and supports the protective role of interferon

Role of the Eph/ephrin system in the development of the central nervous system and its possible utility in strategies to regenerate the topographic orderer connections

El objetivo central de este trabajo es estudiar los mecanismos moleculares que regulan la potencialidad evolutiva de las células madre/progenitoras de la retina, haciendo hincapié en el rol del sistema Eph/ephrinas. Se pretende que los hallazgos de este trabajo aporten: a) conocimientos básicos sobre los mecanismos celulares y moleculares del desarrollo del SNC y del sistema visual y b) conocimientos que sirvan de base para el diseño de estrategias de terapias regenerativas en la retina. Para tal efecto se emplea el sistema retinotectal del embrión de pollo como modelo experimental. El principal nicho de células madre/progenitoras de la retina reside en un anillo tisular que rodea a la retina llamado margen ciliar (MC). Éste contiene a las células madre/progenitoras del cuerpo ciliar (CC) y las células madre/progenitoras localizadas en la zona marginal ciliar (ZMC). Esta zona provee células retinianas durante la vida postnatal en peces y anfibios, permitiendo el crecimiento de la retina y su regeneración. Las aves y los mamíferos en cambio, pierden esta capacidad regenerativa. En aves, donde el crecimiento retiniano se produce durante el desarrollo, un pequeño MC persiste postnatalmente pero carece de la potencialidad evolutiva para aportar todos los tipos celulares de la retina. Las células madre/progenitoras del MC son capaces de 1) proliferar in vitro formando neuroesferas ó 2) de proliferar in vivo al ser estimuladas formando nueva retina neural. Sin embargo, se ha mostrado que no todas las células madre “son iguales” y que varían en su potencialidad evolutiva. En el presente trabajo: 1. Se evalúa si las células madre del MC se diferencian in vitro en células ganglionares de la retina (CGR) que posean información posicional y que sean competentes para responder a las moléculas del sistema Eph/ephrinas que guían el crecimiento axonal durante la formación del mapa retinotectal. 2. Se investiga si las retinas regeneradas a partir del MC y de la transdiferenciación expresan las moléculas del sistema Eph/ephrinas y presentan información posicional. 3. Se determina si la estimulación de la vía de señalización de Shh con su análogo activador SAG permite obtener retinas con regeneración completa a partir de las células madre/progenitoras del MC. En este trabajo se han establecido las condiciones in vitro que permiten obtener un alto grado de diferenciación de CGR a partir de células madre provenientes del MC. Estas CGR expresan moléculas del sistema Eph/ephrinas en forma topográficamente específica y son competentes para responder a moléculas del sistema Eph/ephrinas que guían a los axones durante la formación del mapa retinotectal. Se ha demostrado que la activación de la vía canónica de Shh con SAG induce la regeneración completa de la retina a partir de las células madre/progenitoras del MC in vivo y que las CGR de la misma tienen información posicional expresando el sistema Eph/ephrinas en forma de gradiente como las retinas en desarrollo. Nuestros resultados aumentan las probabilidades de obtener una eferencia que establezca conexiones topográficamente ordenadas en el tectum.The central aim of this work is to study the molecular mechanisms that regulate the evolutionary potential of the stem/progenitor cells of the retina, focusing on the role of the Eph/ephrin system. It is intended for the findings on this work to provide: a) basic knowledge on the cellular and molecular mechanisms of the CNS and visual system development, and b) knowledge to be used to design of regenerative therapy strategies for the retina. For this work we used the chick embryo retinotectal system is used as experimental model. The retina stem/progenitor cell niche resides in a ring around the retina called the ciliary margin (CM), which holds the stem/progenitor cells of the ciliary body (CB) and of the ciliary margin zone (CMZ). This zone provides retinal cells during the postnatal life in fish and amphibians, allowing for retinal growth and regeneration. However, avian and mammals have lost this regenerative capability. In avian, where retina growing takes place during development, a small CM persists postnatally, but it lacks of potenctial to generate all the cellular types of retina. The stem/progenitor cells of the CM are capable of 1) proliferating in vitro forming neurospheres, or 2) proliferating in vivo when stimulated forming new neural retina. However, it has been shown that not all the stem cells are ‘equal’ and that they vary in their evolutionary potentiality. In the present work: 1. Stem/progenitor cells of the CM are evaluated for their differentiation potential in vitro into retinal ganglion cells (RGC) which have positional information and are competent to respond to molecules of the Eph/ephrin system which guies axonal growth during the retinotectal map formation. 2. The expression patterns of Eph/ephrin and their positional information are studied during retina regeneration initiated from the CM and from retinal pigmented epithelium (RPE) transdifferentiation. 3. The sonic hedgehog (Shh) pathway and its analog activator SAG are evaluated for their potential to induce complete retina regeneration from the stem/progenitor cells of the CM. In this work we defined in vitro conditions that optimized for a high degree of RGC differentiation from the stem/progenitor cells present in the CM. These RGCs express molecules of the Eph/ephrin system in a topographic specific way and respond to the cues that guide the axons during the formation of the retinotectal map. We also show that activating the Shh canonical with SAG induces the complete retina regeneration from the CM stem/progenitor cells in vivo and that the RGCs from this regenerated retina have the same positional information expressing the Eph/ephrin system in a gradient form just as the retinas during development. Our results increase the probability of obtaining an efference that establishes a topographic order of connections in the tectum.Fil:Di Napoli, Jennifer. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Discovery of novel anti-HIV active G-quadruplex-forming oligonucleotides

A series of d((5')TGGGAG(3')) sequences, 5'-conjugated with a variety of aromatic groups through phosphodiester linkages, were synthesized, showing CD spectra diagnostic of parallel-stranded, tetramolecular G-quadruplex structures. When tested for anti-HIV-1 and HIV-2 activity, potent inhibition of HIV-1 infection in CEM cell cultures was found, associated with high selectivity index values. Surface Plasmon Resonance assays revealed specific binding to HIV-1 gp120 and gp41.status: publishe

Discovery of novel anti-HIV active G-quadruplex-forming oligonucleotides

A series of oligonucleotide sequences conjugated with a variety of aromatic groups through phosphodiester linkages were synthesized showing CD spectra diagnostic of parallel-stranded, tetramolecular G-quadruplex structures

Anaglyph of Retinal Stem Cells and Developing Axons: Selective Volume Enhancement in Microscopy Images

Retinal stem cell culture has become a powerful research tool, but it requires reliable methods to obtain high-quality images of living and fixed cells. This study describes a procedure for using phase contrast microscopy to obtain three-dimensional (3-D) images for the study of living cells by photographing a living cell in a culture dish from bottom to top, as well as a procedure to increase the quality of scanning electron micrographs and laser confocal images. The procedure may also be used to photograph clusters of neural stem cells, and retinal explants with vigorous axonal growth. In the case of scanning electron microscopy and laser confocal images, a Gaussian procedure is applied to the original images. The methodology allows for the creation of anaglyphs and video reconstructions, and provides high-quality images for characterizing living cells or tissues, fixed cells or tissues, or organs observed with scanning electron and laser confocal microscopy. Its greatest advantage is that it is easy to obtain good results without expensive equipment. The procedure is fast, precise, simple, and offers a strategic tool for obtaining 3-D reconstructions of cells and axons suitable for easily determining the orientation and polarity of a specimen. It also enables video reconstructions to be created, even of specimens parallel to the plastic base of a tissue culture dish, It is also helpful for studying the distribution and organization of living cells in a culture, as it provides the same powerful information as optical tomography, which most confocal microscopes cannot do on sterile living cells.Fil: Carri, Nestor Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Noo Bermúdez, Sebastián Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Fiore, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; ArgentinaFil: Di Napoli, Jennifer Irina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; ArgentinaFil: Scicolone, Gabriel Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia ; Argentin

5'-Modified G-quadruplex forming oligonucleotides endowed with anti-HIV activity: synthesis and biophysical properties

Oligodeoxyribonucleotides of sequence d(5'TGGGAG3') carrying bulky aromatic groups at the 5' end were found to exhibit potent anti-HIV activity [Hotoda, H., et al. (1998) J. Med. Chem. 41, 3655-3663 and references therein]. Structure-activity relationship investigations indicated that G-quadruplex formation, as well as the presence of large aromatic substituents at the 5'-end, were both essential for their antiviral activity. In this work, we synthesized some representative examples of the anti-HIV active Hotoda's 6-mers and analyzed the resulting G-quadruplexes by CD, DSC, and molecular modeling studies, in comparison with the unmodified oligonucleotide. In the case of the sequence carrying the 3,4-dibenzyloxybenzyl (DBB) group, identified as the best candidate for further drug optimization, we developed an alternative protocol to synthesize the 5'-DBB-thymidine phosphoramidite building block in higher yields. The thermodynamic and kinetic parameters for the association/dissociation processes of the 5'-conjugated quadruplexes, determined with respect to the unmodified one, were discussed in light of the molecular modeling studies. The aromatic groups at the 5' position of d(5'TGGGAG3') dramatically enhance both the equilibrium and the rate of formation of the quadruplex complexes. The overall stability of the investigated quadruplexes was found to correlate with the reported IC50 values, thus furnishing quantitative evidence for the hypothesis that the G-quadruplex structures are the ultimate active species, effectively responsible for the biological activity