Serina 284 como regulador de la dimerización y de la localización celular de ERK2

ABSTRACT: Signals conveyed through ERK1/2 Mitogen-Activated Protein Kinases are well-known to play a critical role in cancer initiation, progression, and therapy resistance. It has been demonstrated that the balance between ERK monomers and dimers and cytoplasmic and nuclear sub-signals are critical for the biological outcomes resulting from ERK activation subcellular distribution. Studying the efficacy of DEL22379, a new compound that blocks ERK dimerization, we made the startling observation that ERK dimerization was restricted to mammalians. A comparison of the ERK2 sequence through the evolutionary scale unveiled that Serine284 (H.sapiens) was conserved in those species in which ERK2 dimerized, being suggestive of playing some role in ERK2 dimerization. Indeed, we have shown that Ser284 is necessary but not sufficient for ERK2 dimerization. Moreover, Ser284 is a phosphorylatable residue of ERK2, being a cytoplasmic marker of active ERK2. This phosphorylation, mediated by MEK1 and AKT1, enhances ERK affinity for the scaffold KSR1 and reduces ERK2 interaction with nuclear shuttles like IMP7; being critical for the regulation of ERK2 subcellular distribution. In addition, the sensitivity to BRAF mutant melanoma cells to vemurafenib treatment correlates with higher levels of phospho-Ser284. Thus, p-Ser284 levels could be used as a predictive biomarker for the response to vemurafenib in BRAF positive melanoma patients.RESUMEN: La señalización a través de la ruta de las MAP kinasas ERK1/2 está claramente implicada en el desarrollo y progresión tumoral, así como en la aparición de resistencias. Se ha demostrado que el equilibrio entre los monómeros y los dímeros de ERK y su señalización citoplasmática y nuclear son críticas para los efectos biológicos debidos a su activación en función de su distribución subcelular. Estudiando los efectos de DEL22379, un inhibidor de la dimerización de ERK, hicimos el sorprendente descubrimiento de que ERK2 dimeriza solo en mamíferos. A través de la comparación de la secuencia de ERK en distintas especies observamos que el residuo Ser284 (en humano) estaba conservado en aquellas especies donde ERK dimeriza, lo que sugiere que podría jugar un papel importante en la dimerización de ERK2. En efecto, hemos demostrado que la fosforilación de la Ser284 es necesaria pero no suficiente para la dimerización de ERK2. Dicha fosforilación, que es mediada por MEK1 y AKT1, aumenta la afinidad de ERK2 por el scaffold KSR1 y reduce la afinidad por proteínas transportadoras nucleares como la IMP7; siendo crítica para la regulación de la distribución subcelular de ERK2. Además, aquellas células tumorales de melanoma portadoras de la mutación en BRAF, sensibles al tratamiento con Vemurafenib, muestran una mayor fosforilación en Ser284. Por tanto, los niveles de p-Ser284 podrían servir como biomarcador predictivo de respuesta al tratamiento con Vemurafenib en pacientes con melanoma.La presente Tesis Doctoral titulada “Serine 284 as a regulator of ERK2 dimerization and cellular localization” ha sido realizada en el Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC) en el laboratorio de Regulación espacial de las señales RAS/ERK en cáncer gracias a la Ayuda para contratos predoctorales para la formación de doctores de la agencia estatal consejo superior de investigaciones científicas (BES-2016-077555) y a los proyectos financiados por: MINECO, Ciberonc Isciii, AECC. Durante el presente trabajo Vincenzo Cappitelli ha realizado una estancia predoctoral de 3 meses y medio en el laboratorio del Dr. Adam Hurlstone en la Faculty of Biology, Medicine and Health de la Universidad de Manchester, Reino Unido, gracias a la ayuda para la realización de estancias en otros centros de i+d del ministerio de economía, industria y competitividad (BES-2016-077555)

Novel Antibiofilm Non-Biocidal Strategies

Subaerial biofilm (SAB) formation on cultural heritage objects is often considered an undesirable process in which microorganisms and their by-products, e.g., enzymes and pigments, cause damage or alteration to a surface. Since biofilms are widespread phenomena, there has been a high demand for preventive and control strategies that resist their formation or reduce their negative effects once formed. Up to date, the main strategy to control biofilms has been the use of biocides. Because of their intrinsic properties, biocidal products can pose risks to humans, animals, and the environment. In this chapter, the authors call “green” only those alternative strategies to biocides able to prevent/control biofilms but that do not kill microorganisms, i.e., irrespective of the use of natural compounds. Here, we describe some of the methods that are most commonly used to test the effectiveness of antibiofilm compounds with multiple-species biofilm model systems. A unified terminology and well described protocols and guidelines are still required to compare and test the effectiveness of traditional or novel compounds against biofilms retrieved on heritage surfaces

The Ecology of Subaerial Biofilms in Dry and Inhospitable Terrestrial Environments

The ecological relationship between minerals and microorganisms arguably represents one of the most important associations in dry terrestrial environments, since it strongly influences major biochemical cycles and regulates the productivity and stability of the Earth's food webs. Despite being inhospitable ecosystems, mineral substrata exposed to air harbor form complex and self-sustaining communities called subaerial biofilms (SABs). Using life on air-exposed minerals as a model and taking inspiration from the mechanisms of some microorganisms that have adapted to inhospitable conditions, we illustrate the ecology of SABs inhabiting natural and built environments. Finally, we advocate the need for the convergence between the experimental and theoretical approaches that might be used to characterize and simulate the development of SABs on mineral substrates and SABs' broader impacts on the dry terrestrial environment

A simple and reliable methodology to detect egg white in art samples

A protocol for a simple and reliable dot-blot immunoassay was developed and optimized to test work of art samples for the presence of specific proteinaceus material (i.e. ovalbumin-based). The analytical protocol has been extensively set up with respect, among the other, to protein extraction conditions, to densitometric analysis and to the colorimetric reaction conditions. Feasibility evaluation demonstrated that a commercial scanner and a free image analysis software can be used for the data acquisition and elaboration, thus facilitating the application of the proposed protocol to commonly equipped laboratories and to laboratories of museums and conservation centres. The introduction of method of standard additions in the analysis of fresh and artificially aged laboratory-prepared samples, containing egg white and various pigments, allowed us to evaluate the matrix effect and the effect of sample aging and to generate threshold density values useful for the detection of ovalbumin in samples from ancient works of art. The efficacy of the developed dot-blot immunoassay was proved testing microsamples from 13th–16th century mural paintings of Saint Francesco Church in Lodi (Italy). Despite the aging, the altered conditions of conservation, the complex matrix, and the micro-size of samples, the presence of ovalbumin was detected in all those mural painting samples where mass-spectrometry-based proteomic analysis unambiguously detected ovalbumin peptides

Knowledge behind the scenes of a peer-reviewed journal helps in publishing your manuscript

This paper provides insights into the paper submission and review processes and the possible reasons behind paper acceptance or rejection. The main target of this paper is emerging researchers who are writing their first scientific papers in peer-reviewed journals. This manuscript gives clues on how to select a journal and what a preprint is, introduces authorship and the roles of editors, reviewers, and publishers, sheds light on the editorial process, and briefly introduces some ethical issues arising in scholarly publishing. The content is based on a course offered annually to doctoral students at the University of Milan and keeps into account frequently asked questions. The authors of this paper are an editor-in-chief and an associated editor of scientific journals, respectively

Interactions of microorganisms and synthetic polymers in cultural heritage conservation

Since the 1960s, synthetic polymers (at the time assumed to be resistant to microbial colonization) have been employed to mitigate the ongoing deterioration of cultural heritage. Since then, the use of synthetic materials in heritage conservation attracted much interest. This paper reviews the last two decades of advances in the relationship microorganisms - synthetic polymers in the cultural heritage conservation field. Three topics are considered: (1) biodeterioration of traditional synthetic polymers, (2) biosusceptibility of novel composites based on synthetic polymers, and (3) biocleaning to remove undesired synthetic polymers. It is know that, if the undesired polymer chemical structures are not fully known, they are particularly difficult to remove. Therefore, the future employment of synthetic polymers definitely requires a more critical holistic conservation assessment

Recent progress in bio-inspired biofilm-resistant polymeric surfaces

Any surface of human interest can serve as a substrate for biofilm growth, sometimes with detrimental effects. The social and economic consequences of biofilm-mediated damage to surfaces are significant, the financial impact being estimated to be billions of dollars every year. After describing traditional biocide-based approaches for the remediation of biofilm-affected surfaces, this review deals with more recent developments in material science, focusing on non-toxic, eco-sustainable nature-inspired biomaterials with anti-biofilm properties superior to the conventional biocide-based approaches in terms of addressing the biofilm problem

Sub-lethal Activity of Small Molecules from Natural Sources and their Synthetic Derivatives Against Biofilm Forming Nosocomial Pathogens

Nowadays, the patient safety is seriously jeopardized by the emergence and spread of nosocomial pathogens in the form of biofilm that is resistant to traditional and affordable antimicrobials. Although advances in organic synthesis have extended the lifetime of classic antibiotics through synthetic modifications, the search of innovative antibiofilm compounds from natural sources can provide new templates, novel targets and unique mechanisms that should have advantages over known antimicrobial agents. Testing sub-lethal concentrations of crude extracts and/or isolated compounds from plants and microorganisms is critical to acting on mechanisms subtler than the killing activity, e.g. those influencing the multicellular behavior, offering an elegant way to develop novel antimicrobial-free antibiofilm strategies. Herein we discussed the search and biological activity of small molecules from natural sources and their synthetic derivatives able to modulate biofilm genesis of nosocomial pathogens through non-microbicidal mechanisms (sub-lethal concentrations). The present work offers an overview about the approaches applied to the discovery of lead small molecules including a) conventional drug design methods like screening of chemical compounds obtained from nature and b) computer-aided drug design approaches. Finally, a classification (not exhaustive but representative) based on the natural origin of small molecules and their synthetic derivatives was reported. The information presented in this review should be of interest to a broad range of disciplines and represents an effort to summarize experimental research and advances in this field

Bioremoval of graffiti using novel commercial strains of bacteria

Previous studies have provided evidence that bioremediation deals a novel approach to graffiti removal, thereby overcoming well-known limitations of current cleaning methods. In the present study eight bacteria aerobic, mesophilic and culturable from the American ATCC and the German DSMZ collections of microorganisms, some isolated from car paint waste, coloured deposits in a pulp dryer and wastewater from dye works, were tested in the removal of silver and black graffiti spray paints using immersion strategies with glass slides. Absorbance at 600 nm and live/dead assays were performed to estimate bacterial density and activity in all samples. Also, pH and dissolved organic carbon (DOC) and inorganic carbon (DIC) measurements in the liquid media were made, as well as, thickness, colorimetric and infrared (FTIR) spectroscopy measurements in graffiti paint layers were used to evaluate the presence of the selected bacteria in the samples and the graffiti bioremoval capacity of bacteria. Data demonstrated that of the eight bacteria studied, Enterobacter aerogenes, Comamonas sp. and a mixture of Bacillussp., Delftia lacustris, Sphingobacterium caeni, and Ochrobactrum anthropiwere the most promising for bioremoval of graffiti. According to significant changes in FTIR spectra, indicating an alteration of the paint polymeric structure, coupled with the presence of a consistent quantity of live bacteria in the medium as well as a significant increase of DIC (a measure of metabolic activity) and a change in paint color

Methodological challenges for the investigation of the dual role of biofilms on outdoor heritage

Biofilm deterioration and biofilm protection should be considered as different aspects of the complex interactions between microbes and the surfaces of outdoor heritage (e.g. stones, bricks, mortar and plaster). Thus, it is urgent to verify and quantify to what extent the biofilm can protect from different weathering processes, to eventually determine the advisability of biofilm removal from the heritage surfaces. On one hand, it is necessary to more precisely describe the decaying processes caused by the microorganisms and to quantify the extent, severity, and rate at which the microorganisms are causing the decay. On the other hand, it is necessary to define methodologies to comprehensively study the bioprotection phenomena. So far, no decision-making tool is available to guide heritage professionals in deciding whether to remove or keep biofilms on heritage surfaces, and aesthetical alteration and discoloration is often the only criterion considered. In this work the different available approaches for the study of the dual role of biofilms on outdoor heritage have been critically reviewed. The open challenges and questions are also summarised