High-throughput screening methodology to identify alpha-synuclein aggregation inhibitors

An increasing number of neurodegenerative diseases are being found to be associated with the abnormal accumulation of aggregated proteins in the brain. In Parkinson’s disease, this process involves the aggregation of alpha-synuclein (a-syn) into intraneuronal inclusions. Thus, compounds that inhibit a-syn aggregation represent a promising therapeutic strategy as disease-modifying agents for neurodegeneration. The formation of a-syn amyloid aggregates can be reproduced in vitro by incubation of the recombinant protein. However, the in vitro aggregation of a-syn is exceedingly slow and highly irreproducible, therefore precluding fast high throughput anti-aggregation drug screening. Here, we present a simple and easy-to-implement in-plate method for screening large chemical libraries in the search for a-syn aggregation modulators. It allows us to monitor aggregation kinetics with high reproducibility, while being faster and requiring lower protein amounts than conventional aggregation assays. We illustrate how the approach enables the identification of strong aggregation inhibitors in a library of more than 14, 000 compounds

Benzbromarone, quercetin, and folic acid inhibit amylin aggregation

Human Amylin, or islet amyloid polypeptide (hIAPP), is a small hormone secreted by pancreatic ß-cells that forms aggregates under insulin deficiency metabolic conditions, and it constitutes a pathological hallmark of type II diabetes mellitus. In type II diabetes patients, amylin is abnormally increased, self-assembled into amyloid aggregates, and ultimately contributes to the apoptotic death of ß-cells by mechanisms that are not completely understood. We have screened a library of approved drugs in order to identify inhibitors of amylin aggregation that could be used as tools to investigate the role of amylin aggregation in type II diabetes or as therapeutics in order to reduce ß-cell damage. Interestingly, three of the compounds analyzed—benzbromarone, quercetin, and folic acid—are able to slow down amylin fiber formation according to Thioflavin T binding, turbidimetry, and Transmission Electron Microscopy assays. In addition to the in vitro assays, we have tested the effect of these compounds in an amyloid toxicity cell culture model and we have found that one of them, quercetin, has the ability to partly protect cultured pancreatic insulinoma cells from the cytotoxic effect of amylin. Our data suggests that quercetin can contribute to reduce oxidative damage in pancreatic insulinoma ß cells by modulating the aggregation propensity of amylin

Design, synthesis and structure-activity evaluation of novel 2-pyridone-based inhibitors of a-synuclein aggregation with potentially improved BBB permeability

The treatment of Parkinson''s disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of a-synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with a-synuclein aggregation. Several of the derivatives obtained retain the parent''s compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD. © 2021 The Author

Inhibition of a-Synuclein Aggregation and Mature Fibril Disassembling With a Minimalistic Compound, ZPDm

Synucleinopathies are a group of disorders characterized by the accumulation of a-Synuclein amyloid inclusions in the brain. Preventing a-Synuclein aggregation is challenging because of the disordered nature of the protein and the stochastic nature of fibrillogenesis, but, at the same time, it is a promising approach for therapeutic intervention in these pathologies. A high-throughput screening initiative allowed us to discover ZPDm, the smallest active molecule in a library of more than 14.000 compounds. Although the ZPDm structure is highly related to that of the previously described ZPD-2 aggregation inhibitor, we show here that their mechanisms of action are entirely different. ZPDm inhibits the aggregation of wild-type, A30P, and H50Q a-Synuclein variants in vitro and interferes with a-Synuclein seeded aggregation in protein misfolding cyclic amplification assays. However, ZPDm distinctive feature is its strong potency to dismantle preformed a-Synuclein amyloid fibrils. Studies in a Caenorhabditis elegans model of Parkinson’s Disease, prove that these in vitro properties are translated into a significant reduction in the accumulation of a-Synuclein inclusions in ZPDm treated animals. Together with previous data, the present work illustrates how different chemical groups on top of a common molecular scaffold can result in divergent but complementary anti-amyloid activities

Analysis of the backward bending modes in damped rotating beams

[EN] This article presents a study of the backward bending mode of a simply supported rotating Rayleigh beam with internal damping. The study analyses the natural frequency behaviour of the backward mode according to the internal viscous damping ratio, the slenderness of the beam and its spin speed. To date, the behaviour of the natural frequency of the backward mode is known to be a monotonically decreasing function with spin speed due to gyroscopic effects. In this article, however, it is shown that this behaviour of the natural frequency may not hold for certain damping and slenderness conditions, and reaches a minimum value (concave function) from which it begins to increase. Accordingly, the analytical expression of the spin speed for which the natural frequency of the backward mode attains the minimum value has been obtained. In addition, the internal damping ratio and slenderness intervals associated with such behaviour have been also provided.The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors gratefully acknowledge the financial support of Ministerio de Ciencia, Innovacion y Universidades Agencia Estatal de Investigacion and the European Regional Development Fund (project TRA2017-84701-R), as well as Generalitat Valenciana (project Prometeo/2016/007) and European Commission through the project 'RUN2Rail - Innovative RUNning gear soluTiOns for new dependable, sustainable, intelligent and comfortable RAIL vehicles' (Horizon 2020 Shift2Rail JU call 2017, grant number 777564)Martínez Casas, J.; Denia Guzmán, FD.; Fayos Sancho, J.; Nadal, E.; Giner Navarro, J. (2019). Analysis of the backward bending modes in damped rotating beams. Advances in Mechanical Engineering. 11(4):1-13. https://doi.org/10.1177/1687814019840474S113114Zorzi, E. S., & Nelson, H. D. (1977). Finite Element Simulation of Rotor-Bearing Systems With Internal Damping. Journal of Engineering for Power, 99(1), 71-76. doi:10.1115/1.3446254Ku, D.-M. (1998). FINITE ELEMENT ANALYSIS OF WHIRL SPEEDS FOR ROTOR-BEARING SYSTEMS WITH INTERNAL DAMPING. Mechanical Systems and Signal Processing, 12(5), 599-610. doi:10.1006/mssp.1998.0159Dimentberg, M. F. (2005). Vibration of a rotating shaft with randomly varying internal damping. Journal of Sound and Vibration, 285(3), 759-765. doi:10.1016/j.jsv.2004.11.025Vatta, F., & Vigliani, A. (2008). Internal damping in rotating shafts. Mechanism and Machine Theory, 43(11), 1376-1384. doi:10.1016/j.mechmachtheory.2007.12.009Rosales, M. B., & Filipich, C. P. (1993). Dynamic Stability of a Spinning Beam Carrying an Axial Dead Load. Journal of Sound and Vibration, 163(2), 283-294. doi:10.1006/jsvi.1993.1165Mazzei, A. J., & Scott, R. A. (2003). Effects of internal viscous damping on the stability of a rotating shaft driven through a universal joint. Journal of Sound and Vibration, 265(4), 863-885. doi:10.1016/s0022-460x(02)01256-7Ehrich, F. F. (1964). Shaft Whirl Induced by Rotor Internal Damping. Journal of Applied Mechanics, 31(2), 279-282. doi:10.1115/1.3629598Vance, J. M., & Lee, J. (1974). Stability of High Speed Rotors With Internal Friction. Journal of Engineering for Industry, 96(3), 960-968. doi:10.1115/1.3438468Vila, P., Baeza, L., Martínez-Casas, J., & Carballeira, J. (2014). Rail corrugation growth accounting for the flexibility and rotation of the wheel set and the non-Hertzian and non-steady-state effects at contact patch. Vehicle System Dynamics, 52(sup1), 92-108. doi:10.1080/00423114.2014.881513Glocker, C., Cataldi-Spinola, E., & Leine, R. I. (2009). Curve squealing of trains: Measurement, modelling and simulation. Journal of Sound and Vibration, 324(1-2), 365-386. doi:10.1016/j.jsv.2009.01.048Bauer, H. F. (1980). Vibration of a rotating uniform beam, part I: Orientation in the axis of rotation. Journal of Sound and Vibration, 72(2), 177-189. doi:10.1016/0022-460x(80)90651-3Shiau, T. N., & Hwang, J. L. (1993). Generalized Polynomial Expansion Method for the Dynamic Analysis of Rotor-Bearing Systems. Journal of Engineering for Gas Turbines and Power, 115(2), 209-217. doi:10.1115/1.2906696Hili, M. A., Fakhfakh, T., & Haddar, M. (2006). Vibration analysis of a rotating flexible shaft–disk system. Journal of Engineering Mathematics, 57(4), 351-363. doi:10.1007/s10665-006-9060-3Young, T. H., Shiau, T. N., & Kuo, Z. H. (2007). Dynamic stability of rotor-bearing systems subjected to random axial forces. Journal of Sound and Vibration, 305(3), 467-480. doi:10.1016/j.jsv.2007.04.016Wang, J., Hurskainen, V.-V., Matikainen, M. K., Sopanen, J., & Mikkola, A. (2017). On the dynamic analysis of rotating shafts using nonlinear superelement and absolute nodal coordinate formulations. Advances in Mechanical Engineering, 9(11), 168781401773267. doi:10.1177/1687814017732672Lee, C.-W. (1993). Vibration Analysis of Rotors. Solid Mechanics and Its Applications. doi:10.1007/978-94-015-8173-8Genta, G. (1999). Vibration of Structures and Machines. doi:10.1007/978-1-4612-1450-2Cheng, C. C., & Lin, J. K. (2003). Modelling a rotating shaft subjected to a high-speed moving force. Journal of Sound and Vibration, 261(5), 955-965. doi:10.1016/s0022-460x(02)01374-

ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness

Atención primaria en España y Cataluña: una perspectiva desde la enfermería

Muchos años después de la Reforma de la Atención Primaria en España, se analizan los logros alcanzados en este nivel asistencial, así como los aspectos y expectativas aún pendientes, tanto para la disciplina médica como para la enfermería. También se analizan los planes de innovación recientemente puestos en marcha en diferentes comunidades autónomas españolas, especialmente el que se está desarrollando en Cataluña. Se aborda además en este texto, el actual desarrollo académico de la enfermería española, el desarrollo de las diferentes especialidades de enfermería y aspectos relativos a la prescripción de enfermería, el triaje y la atención de enfermería a la patología aguda, en Atención Primaria

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7–9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies

Recovery Signals of Rhodoliths Beds since Bottom Trawling Ban in the SCI Menorca Channel (Western Mediterranean)

One of the objectives of the LIFE IP INTEMARES project is to assess the impact of bottom trawling on the vulnerable benthic habitats of the circalittoral bottoms of the Menorca Channel (western Mediterranean), designated a Site of Community Importance (SCI) within the Natura 2000 network. The present study compares the epibenthic communities of four areas, subjected to different bottom trawl fishing intensity levels. The assignment of fishing effort levels was based on the fishing effort distribution in the area calculated from Vessel Monitoring System (VMS) data and the existence of two Fishing Protected Zones in the Menorca Channel. Biological samples were collected from 39 beam trawl stations, sampled during a scientific survey on April 2019. We compare the diversity, composition, and density of the epibenthic flora and fauna, together with the rhodoliths coverage and the morphology of the main species of rhodoliths of four areas subjected to different levels of bottom trawl fishing effort, including one that has never been impacted by trawling. Our results have shown negative impacts of bottom trawling on rhodoliths beds and the first signals of their recovery in areas recently closed to this fishery, which indicate that this is an effective measure for the conservation of this habitat of special interest and must be included in the management plan required to declare the Menorca Channel as a Special Area of Conservation.En prens