2024 - X IDIES project

  The IDIES project began in 2014 at CEBAS-CSIC. The main objective of this project is the initiation of high school students (research modality) to scientific research participating and collaborating in projects led by researchers of recognized national and international prestige.

This year, under the supervision of Dr. Ávila-Gálvez, Dr. Giménez-Bastida, Dr. González-Sarrías and Prof. Juan Carlos Espín, students of the IES "Ramón y Cajal" (Murcia) will investigate how (poly)phenols are able to cross the blood brain barrier (BBB) and exert anti-inflammatory effects. 

The scientific activities will be mainly carried out at the University of Murcia (Cell culture service) and CEBAS-CSIC, where they will have the opportunity to get hands-on experience in cellular and molecular techniques (cell culture, cytotoxicity, ELISA assays and HPLC, among others). The data obtained and their analysis will be presented in the VIII IDIES Conference (poster and oral presentation).

FIRST SESSION: INTRODUCTION TO THE LABORATORY AND CELL CULTURE                                                               

The Cell Culture Service at the University of Murcia was the place where we performed all the activities planned for this day. In the first activity, we explained to the students the general laboratory safety rules. We also showed and explained the equipment they can found in a laboratory, including centrifuges, hoods, incubators, etc.

The first activity they worked on was the preparation of the compounds we want to investigate. This activity allowed them to learn important concepts related to the preparation of solutions such as molarity. Besides, the students also used the precision balance, micropipettes, multichannel pipettes and practiced the sterilization of compounds that will be used to treat cells under sterile conditions.

In a second set of experiments, the students learned about different types of culture medium, they used the microscope to observe cells, changed culture medium, participated in cell subculturing and counted them using the trypan blue dye exclusion technique.

Students of the IES Ramón y Cajal learning basic cell culture techniques

In this first session, although exhausting, our students demonstrated to have the making of a scientist.

SECOND SESSION: (POLY)PHENOL TRANSPORT ACROSS THE BBB (IN VITRO MODEL)                                             

Studying how a natural compounds such as (poly)phenols are able to cross the BBB is one of the cornerstones of this project. In this second session, our students will evaluate the transport through the BBB combining in vitro and HPLC techniques.

Specifically, the cultured cells will be treated with (poly)phenols mimicking conditions that can be detected in vivo (molecular form of the compounds, concentrations...). Next, after 2 hours, the culture medium will be collected, processed and further analyzed by HPLC. 

Will the (poly)phenols cross the BBB? Will they be metabolized? What will we find in our results?

Let´s figure it out in the third session.

THIRD SESSION: TREATMENT OF THE CELLS AND CULTURE MEDIUM COLLECTION                                                     

The use of natural compounds to modulate/ameliorate the inflammatory response is one of the cornerstones of this project. To investigate this topic it is essential to know how to treat the cells. This is what our student have learned in this session. The (poly)phenols under investigation were filter sterilized and added to the cells at (non-toxic) concetrations similar to those detected in vivo in the presence of pro-inflammatory molecules. After a few hours of treatment, the culture medium was collected and kept at -80 degrees. 

The analysis of this medium will provide information related to the anti-inflammatory activity of natural compounds. However, we have to wait until next week to figure it out.

FORTH SESSION: ELISA ANALYSIS OF MOLECULES RELATED TO INFLAMMATION                                                     

The enzyme-linked immunosorbent assay (ELISA) is a widely approached biochemistry technique described by Engvall and Perlmann in 1972 (doi: 10.4049/jimmunol.109.1.129). In this session we will used this assay to investigate how different natural compounds (phenolic compounds) are able to modulate key molecules involved in inflammation (i.e., cytokines). The different culture media corresponding to different treatments will be incubated in 96-well plates, pre-coated with antibodies that target a specific proinflammatory cytokine. The incubation is followed by different washing steps and incubations with detection antibodies, which are responsible of color development (see below). The measurement of the color obtained using an spectrophotometer will give and idea of the concentration of the cytokine and, therefore, the effect of the natural compounds tested on that molecule.

       

FIFTH SESSION: ANALYSIS OF METABOLITES Y CULTURE MEDIUM USING MASS SPECTROMETRY TECHNOLOGY

Our students not only learn what effects are exerted by the phenolic compounds investigated, but also mechanisms related to the transport of these compounds through the BBB model. This provides a more differentiatied view about the capacity of natural compounds to modulate the cellular response. The experiments carried out during sessions 2 and 3 were the basis to obtain the samples to be analyzed in this session.

The approach of this assay is intended to learn (i) basic concepts of mass spectrometry, (ii) sample processing (specifically, culture medium containing (poly)phenols) and (iii) the use of sophosticated softwares needed to design the analysis by mass spectrometry. A string of steps such as protein precipitation, centrifugation and filtration were performed prior to run the samples. The analysis of the chromatrograms generated is the final step of this experiment, but this is another exciting experience that we will live in the next (and last) session.

LAST SESSION: DATA ANALYSIS AND CONCLUSIONS                                                                                                         

After several weeks of organizing experiments, working in the laboratory, and processing/analyzing samples, time is ripe to summarize, discuss and draw conclusions from the results obtained. Here, alongside our students, we engage in discussions regarding the information obtained from the ELISA assay, chromatograms as well as the transport of (poly)phenols using the BBB model. 

The main objective is to answer fundamental questions such as: are (poly)phenols able to exert anti-inflammatory effects? do these molecules have capacity to cross the BBB? are the differences observed significant from a statistic point of view? what statistical analysis should we use? The answers raised from this session will be the basis of the poster and the oral presentation that will be presented at the IDIES Conference.    

 

Dr. Mª Ángeles Ávila-Gálvez (Universidade Nova de Lisboa)

 

Dr. Ávila-Gálvez started her scientific career at CEBAS-CSIC (Murcia, Spain) to investigate the chemopreventive activity of (poly)phenols against breast cancer under the supervision of Prof. JC Espín and Dr. A. González-Sarrías. The broad scope of the project she worked on allowed her to get inmersed in a wide range of in vitro (celular and molecular biology techniques) and in vivo experiments following the approach "first in vivo and then in vitro". Her genuine interest in producing clean and reproducible data, her capacity of thinking in ways to solve problems and her ability to pay attention to detail are reflected in the Cum Laude mention received by her Thesis.

In 2021, Dr. Ávila-Gálvez took a new step in her research career and joined Dr. Claudia Santos group (Instituto de Biologia Experimental e Tecnológica -iBET-; Oreiras, Portugal). This step was a new challenge to deepen in the understanding of the anti-inflammatory potential of bioactive terpenes and the anti(neuro)inflammatory effects of (poly)phenols (including microbial and circulating metabolites) withing the ERC project led by Dr. Claudia Santos.

Her backgroung in nutrition and food science as well as her knowledge in metabolomics and molecular biology are the perfect match to accomplish the objectives of the PolyBiota and AngioPhenol projects. Let´s see if the recently published paper is the first of many.

4th International Conference on Food Bioactives & Health

The international "Food Bioactives and Health" conference has become one of the most important events to share the lastest research results in the field of food bioactives, including peptides, lipids and(or) (poly)phenols, among others. In 2022, the PolyBiota project was present in the 3rd international FBHC held in Parma where we presented a poster describing the capacity of curcuminoids to modulate processes related to angiogenesis. 

In 2023, Prague (Czech Republic) opened the door to hold the 4th international FBHC. This conference brought together inspirational keynote speakers, open dialogues, special oral presentations or interesting posters. This event was a nice chance to showcase further results of the PolyBiota project. Our oral presentation described "what" isoflavones were able to modulate angiogenesis and "how" these molecules exerted their effects. This oral presentation was the prelude of the later accepted manuscript entitled "(R,S)-Equol 7-beta-D-glucuronide, but not other circulating isoflavone metabolites, modulates migration and tubulogenesis in human aortic endothelial cells targeting the VEGF pathway".

From now on, it is time to work hard and get additional results allowing to bring the PolyBiota project to the 5th FBHC.

Dr. Alicia Martínez López

 

In July 2014, I completed my PhD in the Antiviral Strategies group led by Dr. María Amparo Estepa at the Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE) at Miguel Hernández University. During my PhD, I developed powerful tools in the aquaculture industry like the Recombinant vaccine against the Viral Hemorrhagic Septicemia Virus (VHSV) (WO 2015/025068 Al). We were also able to identify, for the first time, the two major determinants of the glycoprotein G of the VHSV implicated in triggering the host type I IFN antiviral response and its possible use as DNA molecular adjuvants. Moreover, I also studied the possible therapeutic applications of the Myticin-C antimicrobial peptide in the activation of the cellular regeneration and tissue reparation after an injury and its applications as a suitable template for the development of antimicrobial agents. In 2016 I got a postdoctoral position at the Albert Einstein College of Medicine in New York (2016-2020). Here I got involved in a project focused on understanding the role of SAMHD1 in the development of Aicardi-Goutières syndrome (AGS), a Type I Interferonopathy, and I characterized in detail the implication of the loss of function of SAMHD1 in these patients, results of high relevance to the biomedicine as we proposed the use of a small-molecule to treat patients with early-stage AGS. Additionally, I participated in a project studying the receptor interaction of Zika Virus and the design of drugs that restrict its infection. Currently I am a senior postdoctoral researcher in the group of Immunology, Inflammation and Cancer led by Dr. Victoriano Mulero at the Instituto Murciano de Investigación Biosanitaria (IMIB). Due to my extensive experience in the immunology field, my main project is now directed to the characterization of Type I Interferonopathies using the zebrafish animal model, and its use for the search of new treatments. All my investigations are now directed to elucidate pathological mechanisms and possible therapeutic targets. My role as a collaborator of the PolyBiota project is an opportunity to establish new collaborations as well as to branch out my knowledge to the beneficial effects of natural products.