Neuronal plasticity: cell-based strategy for target identification and validation

While growing neurites are relatively plastic during development, their plasticity levels drop rapidly as neurons mature and become integrated into neuronal networks. As a consequence, the central nervous system ability to reorganize itself in response to injury or disease is insufficient. One of the main limitations for the design of therapeutic strategies to enhance neurite sprouting following neurological diseases is our poor understanding of the mechanisms underlying neurite structural plasticity. 

To overcome this limitation, we have implemented a strategy to identify, characterize and validate the most therapeutically relevant drug targets to modulate neuronal plasticity. This strategy is based on the hypothesis that the molecular regulation of the neurite shaft (controlling its ability to sprout) shares similarities with other cell systems. In an initial selection step (1), candidate targets are identified from the literature based on predetermined criteria (e.g. involvement in cell migration and growth cone collapse). (2) Compounds acting on those targets are tested in a neuronal cell-based assay for their effects on neurite sprouting as well as for early detection of undesired effects. (3) The selected targets are further analyzed during a verification step where their subcellular expression and activity is assessed. The regulation of the activity of the potential targets by branching factors is also determined at this point. (4) Once the biological relevance has been established, the signalling pathway in which the potential targets operates within neurons is mapped using activity reporters to uncover additional potential targets. 

This candidate approach, combined with the use of primary neurons, is designed to increase the probability of identifying suitable targets at the same time that it minimizes costs and time to validation. By using this strategy, we have been able to identify a set of proteins controlling neuronal sprouting together with an unsuspected mechanism of regulation of structural plasticity in neurons

Treatment Guidelines for Rare, Early-Onset, Treatment-Resistant Epileptic Conditions:A Literature Review on Dravet Syndrome, Lennox-Gastaut Syndrome and CDKL5 Deficiency Disorder

Background: Dravet syndrome (DS), Lennox-Gastaut syndrome (LGS) and CDKL5 deficiency disorder (CDD) are rare epileptic conditions, characterised by drug-resistant seizures. Seizure management in these patients requires careful therapy selection. This targeted literature review (TLR) aimed to collate and synthesise information from country-specific and international treatment guidelines for DS, LGS and CDD.Methods: A TLR was performed between 25th January and 11th March 2021. Online rare diseases and guideline databases were manually searched in addition to websites of national health technology assessment bodies for the following countries: Australia, Canada, France, Germany, Israel, Italy, Japan, Spain, Switzerland, UK and US, as defined by pre-specified eligibility criteria. Search terms, developed for each condition, were translated into local languages where appropriate. Descriptive analyses were performed to examine the geographical distribution of included guidelines; methodologies used to develop guidelines; cross-referencing of treatment recommendations made within other guidelines; patterns of treatment recommendations. An author map was created using R version 3.5.1, to visualise the extent of collaboration between authors.Results: Forty total guidelines were included, of which 29, 34 and 0 contained recommendations for DS, LGS and CDD, respectively (some provided recommendations for ≥1 condition). Most were country-specific, with guideline authors predominantly publishing in regional groups. Five guidelines were classified as “International” and displayed connections between author groups in the US, UK, France and Italy. Reported guideline development processes were lacking [43% (17 guidelines) had unclear/absent literature review methodologies] and those reported were variable, including both systematic and targeted literature reviews. Use of expert consultation was also variable. A high degree of heterogeneity was observed in the availability of treatment recommendations across disorders, with 271 and 190 recommendations for LGS and DS, respectively, and contradictory positive and negative treatment recommendations for several drugs in each indication [35% (11/31) and 22% (6/27) in LGS and DS, respectively].Conclusions: This review highlights the need for further high-quality international consensus-based treatment guidelines for LGS, DS, and particularly for CDD (for which no treatment guidelines were identified). Supra-national consensus guidance based on findings from a wider geographical range may improve resource allocation and establish an improved world-wide standard of care

Nogo, myelin and axonal regeneration

El sistema nerviós dels mamífers té una baixa capacitat de reparació axonal després d'una lesió. En els últims anys, diversos estudis han demostrat que els axons lesionats no poden recréixer a causa de la presència d'un gran nombre de molècules inhibitòries. Les molècules associades a la mielina limiten el creixement axonal i el seu bloqueig afavoreix la regeneració de diverses connexions. Tres d'aquestes proteïnes, Nogo, MAG i OMgp, comparteixen un mateix receptor: NgR. El clonatge recent de Nogo ha obert noves vies per estudiar la regeneració axonal. No obstant això, molts dels elements involucrats en la via inhibitòria de la mielina són desconeguts, i els primers estudis amb animals knockout són, a més, contradictoris. Per aquesta raó, Nogo i el seu receptor han de caracteritzar-se abans de desenvolupar noves tècniques per promoure regeneració axonal.Adult mammalian central nervous system (CNS) axons have very limited capacity of regrowth after injury. In recent years, advances in the field of axonal regeneration have proved that neurons do not regenerate, mainly because of the presence of inhibitory molecules. Myelin-associated proteins limit axonal outgrowth and their blockage improves the regeneration of damaged fiber tracts. Three of these proteins, Nogo, MAG and OMgp, share a common neuronal receptor (NgR), and together represent one of the main hindrances to neuronal regeneration. The recent molecular cloning of Nogo and its receptors opened a new door to the study of axon regeneration. However, many of the elements involved in the myelin inhibitory pathway are still unknown, and the preliminary experiments with knockout mice are rather contradictory. Because of this complexity, Nogo and NgR need to be characterized before precise strategies to promote axon regeneration in the CNS can be designed

Planning the teaching methodology adapted to EHEA: a proposal for Applied Economy subjects

Application of teaching methods adapted to the EHEA requires planning and teamwork among teachers of the same subject, but also between teachers of different subject, especially when we have to achieve a student with specific skills. This paper describes the experience of professors who teach Economic Policy and Financial System, and shows how these have coordinated the skills, methodologies and evaluation systems in two different subjects in order to develop the skills in a progressive form. Also, it analyzes the effort, in hours of work, of teachers when they apply a methodology as we have described

A cell viability study on an experimental model of human gingival fibroblasts cultured without foetal bovine serum

El objetivo del estudio es aportar más informaciones metodológicas y conceptuales a cerca del efecto del SBF 10% en ensayos de viabilidad celular sobre fibroblastos gingivales humanos, con la finalidad de optimizar las condiciones experimentales para la obtención de resultados más predictivos. Se utilizarán el ensayo colorimétrico WST-1 para la evaluación de proliferación celular, el ensayo de LDH para evaluación de la integridad de membrana celular, el ensayo de cuantificación de ADN mediante espectrometría y la microscopía óptica de contraste de fases para observar las alteraciones morfológicas. Entre 6 y 24 h, las células sufren poco con la ausencia del SBF 10% y no resulta imprescindible su utilización para evaluar proliferación celular y la integridad de membranas plasmática y nuclear. Sin embargo, la morfología de las células se altera, de forma intensa, a partir de 6 h de ausencia de SBF 10%, por lo que es conveniente utilizarlo para dichos estudios morfológicos.The aim of this study is to improve the methodological and conceptual knowledges about the effect of 10% foetal bovine serum in cell viability assays on human gingival fibroblasts, in order to optimize the experimental conditions to obtain more predictable results. In this study, different methods were used to determine cell proliferation, cell and nuclear membrane integrity and morphological study by using WST-1 assay, LDH assay, DNA quantification and phase contrast microscopy. Our results demonstrated that 10% foetal bovine serum deprivation for 6h up to 24h did not show changes in terms of cell proliferation, cell and nuclear membrane integrity. However, important morphological changes were observed under foetal bovine serum deprivation conditions after 6h. We conclude that, 10% foetal bovine serum does not result essential its utilization to evaluate cell proliferation and their deprivation do not compromise cell survival range and could be suppressed in cell proliferation, cell and nuclear membrane assays without affecting the assays results. In contrast, the utilization of 10% foetal bovine serum becomes crucial in morphological studies and we strongly recommend 10% foetal bovine serum supplement in morphological studies

Evaluation of the optical and biomechanical properties of bioengineered human skin generated with fibrin-agarose biomaterials

Significance: Recent generation of bioengineered human skin allowed the efficient treatment of patients with severe skin defects. However, the optical and biomechanical properties of these models are not known. Aim: Three models of bioengineered human skin based on fibrin-agarose biomaterials (acellular, dermal skin substitutes, and complete dermoepidermal skin substitutes) were generated and analyzed. Approach: Optical and biomechanical properties of these artificial human skin substitutes were investigated using the inverse adding-doubling method and tensile tests, respectively. Results: The analysis of the optical properties revealed that the model that most resembled the optical behavior of the native human skin in terms of absorption and scattering properties was the dermoepidermal human skin substitutes after 7 to 14 days in culture. The time-course evaluation of the biomechanical parameters showed that the dermoepidermal substitutes displayed significant higher values than acellular and dermal skin substitutes for all parameters analyzed and did not differ from the control skin for traction deformation, stress, and strain at fracture break. Conclusions: We demonstrate the crucial role of the cells from a physical point of view, confirming that a bioengineered dermoepidermal human skin substitute based on fibrin-agarose biomaterials is able to fulfill the minimal requirements for skin transplants for future clinical use at early stages of in vitro development.Ministry of Science, Innovation and Universities of Spain PGC2018-101904-A-I0Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities, through AES 2017 AC17/00013Instituto de Salud Carlos III (ISCIII), Ministry of Science, Innovation and Universities within EuroNanoMed framework, EU AC17/00013University of Granada A.TEP.280.UGR18Junta de Andalucía PE-0395-2019Fundación Benéfica Anticancer San Francisco Javier y Santa Cándida, Granada, SpainOTRI.35A-0

In vivo time-course biocompatibility assessment of biomagnetic nanoparticles-based biomaterials for tissue engineering applications

Novel artificial tissues with potential usefulness in local-based therapies have been generated by tissue engineering using magnetic-responsive nanoparticles (MNPs). In this study, we performed a comprehensive in vivo characterization of bioengineered magnetic fibrin-agarose tissue-like biomaterials. First, in vitro analyses were performed and the cytocompatibility of MNPs was demonstrated. Then, bioartificial tissues were generated and subcutaneously implanted in Wistar rats and their biodistribution, biocompatibility and functionality were analysed at the morphological, histological, haematological and biochemical levels as compared to injected MNPs. Magnetic Resonance Image (MRI), histology and magnetometry confirmed the presence of MNPs restricted to the grafting area after 12 weeks. Histologically, we found a local initial inflammatory response that decreased with time. Structural, ultrastructural, haematological and biochemical analyses of vital organs showed absence of damage or failure. This study demonstrated that the novel magnetic tissue-like biomaterials with improved biomechanical properties fulfil the biosafety and biocompatibility requirements for future clinical use and support the use of these biomaterials as an alternative delivery route for magnetic nanoparticles.• Grants FIS-PI17/0391 and FIS-PI17/0393 from Instituto de Salud Carlos III - ISCIII (Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica I + D + i from the Spanish Ministerio de Ciencia e Innovación), co-financed by ERDF-FEDER, European Union. • Award number AC17/00013 (NanoGSkin) by ISCIII thorough AES 2017 and within the EuroNanoMed framework. • Grant FIS2017-85954-R funded by Ministerio de Economía, Industria y Competitividad, MINECO, and Agencia Estatal de Investigación, AEI, Spain, cofunded by Fondo Europeo de Desarrollo Regional, FEDER, European Union. • Grants CS PI-0257-2017 and CSyF PE-0395-2019 from Consejería de Salud y Familias, Junta de Andalucía, Spain. • Grant n° Res SECYT 411/18 from SECYT (Secretary of Science and Technology of National University of Córdoba, Argentina) • Project Future Investments UCA JEDI, No. ANR-15-IDEX-01, project “RheoGel” by the French “Agence Nationale de la Recherche”

Effects of Four Formulations of Prostaglandin Analogs on Eye Surface Cells. A Comparative Study

We evaluated the cytotoxic effects of four prostaglandin analogs (PGAs) used to treat glaucoma. First we established primary cultures of conjunctival stromal cells from healthy donors. Then cell cultures were incubated with different concentrations (0, 0.1, 1, 5, 25, 50 and 100%) of commercial formulations of bimatoprost, tafluprost, travoprost and latanoprost for increasing periods (5 and 30 min, 1 h, 6 h and 24 h) and cell survival was assessed with three different methods: WST-1, MTT and calcein/AM-ethidium homodimer-1 assays. Our results showed that all PGAs were associated with a certain level of cell damage, which correlated significantly with the concentration of PGA used, and to a lesser extent with culture time. Tafluprost tended to be less toxic than bimatoprost, travoprost and latanoprost after all culture periods. The results for WST-1, MTT and calcein/AM-ethidium homodimer-1 correlated closely. When the average lethal dose 50 was calculated, we found that the most cytotoxic drug was latanoprost, whereas tafluprost was the most sparing of the ocular surface in vitro. These results indicate the need to design novel PGAs with high effectiveness but free from the cytotoxic effects that we found, or at least to obtain drugs that are functional at low dosages. The fact that the commercial formulation of tafluprost used in this work was preservative-free may support the current tendency to eliminate preservatives from eye drops for clinical use.This study was supported by the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I) from the Spanish Ministry of Economy and Competitiveness (Instituto de Salud Carlos III), grants FIS PI11/1582 and FIS PI14/0955 (co-financed by FEDER funds, European Union) and by Grant P10-CTS-6060 from the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucia, Spain (Proyectos de Excelencia)

Optical Behavior of Human Skin Substitutes: Absorbance in the 200–400 nm UV Range

The most recent generation of bioengineered human skin allows for the efficient treatment of patients with severe skin defects. Despite UV sunlight can seriously affect human skin, the optical behavior in the UV range of skin models is still unexplored. In the present study, absorbance and transmittance of the UGRSKIN bioartificial skin substitute generated with human skin cells combined with fibrin-agarose biomaterials were evaluated for: UV-C (200–280 nm), -B (280–315 nm), and -A (315–400 nm) spectral range after 7, 14, 21 and 28 days of ex vivo development. The epidermis of the bioartificial skin substitute was able to mature and differentiate in a time-dependent manner, expressing relevant molecules able to absorb most of the incoming UV radiation. Absorbance spectral behavior of the skin substitutes showed similar patterns to control native skin (VAF > 99.4%), with values 0.85–0.90 times lower than control values at 7 and 14- days and 1.05–1.10 times the control values at 21- and 28-days. UV absorbance increased, and UV transmission decreased with culture time, and comparable results to the control were found at 21 and 28 days. These findings support the use of samples corresponding to 21 or 28 days of development for clinical purposes due to their higher histological similarities with native skin, but also because of their absorbance of UV radiation.Junta de Andalucia PE-0395-2019University of Granada B-CTS-450-UGR20 A.TEP.280.UGR18Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades B-CTS-450-UGR20European Regional Development Fund (ERDF) through the "Una manera de hacer Europa" program Junta de Andalucia P20-00200Spanish Government PGC2018-101904-A-10

Histological assessment of nanostructured fibrin-agarose skin substitutes grafted in burnt patients. A time-course study

A previously developed fibrin-agarose skin model—UGRSKIN—showed promising clinical results in severely burnt patients. To determine the histological parameters associated to the biocompatibility and therapeutic effects of this model, we carried out a comprehensive structural and ultrastructural study of UGRSKIN grafted in severely burnt patients after 3 months of follow-up. The grafted epidermis was analogue to native human skin from day 30th onward, revealing well-structured strata with well-differentiated keratinocytes expressing CK5, CK8, CK10, claudin, plakoglobin, filaggrin, and involucrin in a similar way to controls, suggesting that the epidermis was able to mature and differentiate very early. Melanocytes and Langerhans cells were found from day 30th onward, together with a basement membrane, abundant hemidesmosomes and lack of rete ridges. At the dermal layer, we found an interface between the grafted skin and the host tissue at day 30th, which tended to disappear with time. The grafted superficial dermis showed a progressive increase in properly-oriented collagen fibers, elastic fibers and proteoglycans, including decorin, similarly to control dermis at day 60-90th of in vivo follow-up. Blood vessels determined by CD31 and SMA expression were more abundant in grafted skin than controls, whereas lymphatic vessels were more abundant at day 90th. These results contribute to shed light on the histological parameters associated to biocompatibility and therapeutic effect of the UGRSKIN model grafted in patients and demonstrate that the bioengineered skin grafted in patients is able to mature and differentiate very early at the epithelial level and after 60–90 days at the dermal level.Consejería de Salud y Familias, Junta de AndalucíaGrant/Award Numbers: PE- 0395-2019, PI-0458-2016Consejería de Transformación Económica, Industria,Conocimiento y Universidades, Grant/Award Number: B-CTS-450-UGR20Instituto de Salud Carlos III, Grant/Award Number: AC17/0001