Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington’s disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer

Rare Variants in Autophagy and Non-Autophagy Genes in Late-Onset Pompe Disease: Suggestions of Their Disease-Modifying Role in Two Italian Families

Pompe disease is an autosomal recessive disorder caused by a deficiency in the enzyme acid alpha-glucosidase. The late-onset form of Pompe disease (LOPD) is characterized by a slowly progressing proximal muscle weakness, often involving respiratory muscles. In LOPD, the levels of GAA enzyme activity and the severity of the clinical pictures may be highly variable among individuals, even in those who harbour the same combination of GAA mutations. The result is an unpredictable genotype–phenotype correlation. The purpose of this study was to identify the genetic factors responsible for the progression, severity and drug response in LOPD. We report here on a detailed clinical, morphological and genetic study, including a whole exome sequencing (WES) analysis of 11 adult LOPD siblings belonging to two Italian families carrying compound heterozygous GAA mutations. We disclosed a heterogeneous pattern of myopathic impairment, associated, among others, with cardiac defects, intracranial vessels abnormality, osteoporosis, vitamin D deficiency, obesity and adverse response to enzyme replacement therapy (ERT). We identified deleterious variants in the genes involved in autophagy, immunity and bone metabolism, which contributed to the severity of the clinical symptoms observed in the LOPD patients. This study emphasizes the multisystem nature of LOPD and highlights the polygenic nature of the complex phenotype disclosed in these patients

Next Generation Molecular Diagnosis of Hereditary Spastic Paraplegias: An Italian Cross-Sectional Study

Hereditary spastic paraplegia (HSP) refers to a group of genetically heterogeneous neurodegenerative motor neuron disorders characterized by progressive age-dependent loss of corticospinal motor tract function, lower limb spasticity, and weakness. Recent clinical use of next generation sequencing (NGS) methodologies suggests that they facilitate the diagnostic approach to HSP, but the power of NGS as a first-tier diagnostic procedure is unclear. The larger-than-expected genetic heterogeneity-there are over 80 potential disease-associated genes-and frequent overlap with other clinical conditions affecting the motor system make a molecular diagnosis in HSP cumbersome and time consuming. In a single-center, cross-sectional study, spanning 4 years, 239 subjects with a clinical diagnosis of HSP underwent molecular screening of a large set of genes, using two different customized NGS panels. The latest version of our targeted sequencing panel (SpastiSure3.0) comprises 118 genes known to be associated with HSP. Using an in-house validated bioinformatics pipeline and several in silico tools to predict mutation pathogenicity, we obtained a positive diagnostic yield of 29% (70/239), whereas variants of unknown significance (VUS) were found in 86 patients (36%), and 83 cases remained unsolved. This study is among the largest screenings of consecutive HSP index cases enrolled in real-life clinical-diagnostic settings. Its results corroborate NGS as a modern, first-step procedure for molecular diagnosis of HSP. It also disclosed a significant number of new mutations in ultra-rare genes, expanding the clinical spectrum, and genetic landscape of HSP, at least in Italy

Driven Assembly of Lignin into Microcapsules for Storage and Delivery of Hydrophobic Molecules

Oil-filled microcapsules of kraft lignin were synthe- sized by first creating an oil in water emulsion followed by a high- intensity, ultrasound-assisted cross-linking of lignin at the water/oil interface. The rationale behind our approach is based on promoting documented lignin hydrophobic interactions within the oil phase, followed by locking the resulting spherical microsystems by covalent cross-linking using a high intensity ultrasound treatment. As further evidence in support of our rationale, confocal and optical microscopies demonstrated the uniformly spherical morphology of the created lignin microparticles. The detailed elucidation of the cross-linking processes was carried out using gel permeation chromatography (GPC) and quantitative 31P NMR analyses. The ability of lignin microcapsules to incorporate and release Coumarin-6 was evaluated in detail. In vitro studies and confocal laser scanning microscopy analysis were carried out to assess the internalization of capsules into Chinese hamster ovary (CHO) cells. This part of our work demonstrated that the lignin microcapsules are not cytotoxic and readily incorporated in the CHO cells