The use of platelets microparticles and associated factors for improve the endometrium pontentiallity

Motivation: Despite, the high advances in assisted reproductive technology (ART), with technics like: i) intrauterine insemination (IUI); ii) in vitro fertilization (IVF) and; iii) intracytoplasmic sperm injection (ISCI); the rate of success still remains very low, about 30-33%. Implantation is a highly controlled process, involving a dialogue between the endometrium and the implanting embryo, which is crucial for the establishment and maintenance of pregnancy (Pafilis et al., 2007). When the endometrium is receptive and available to establish this dialogue is called the “window of implantation” (WOI). Nowadays, large amounts of resources are being invested are to find a way to improve or boost the potentiality of the endometrium. Objective: Characterize the effect of the microparticules and factors, derived from the activation of Platelets, to the endometrium with the goal of raise the ratio of embryo implantation.Methods: An in vitro model of embryo implantation, is used two different cell lines: Hec-1A in monolayer culture simulating the endometrium and JAR as trophoblast models (pseudoembryos). These lines are co-incubated using five different concentrations of MPPs and factors to quantify the ratio of adhesion. Finally, 192 genes that have been shown to be related to endometrial receptivity are studied using qPCR by BioMark HD Fluidigm.Conclusion: Through the use of MPPs and different factors (before and after embryo transfer) in this two cell lines and the 192 genes, it is intended to observe different changes in the transcriptomic endometrial profile, producing morphological and molecular changes that improves the endometrial receptivity during the WOI for a successful implantation raising the number of pregnancies

Importance of food sensitivities and personalized diet in patients with fibromyalgia

Fibromyalgia (FM) is a disease characterized by generalized chronic pain located in the locomotor system. Patients usually present obesity, metabolic syndrome, alterations in the microbiota and food sensitivities. The prevalence is 2.1% in the world population, affecting 2.3% of Europeans and 2.4% of Spaniards. By sex, the prevalence among men is estimated at 0.2%, compared to 4.2% in women. FM is one of the rheumatic diseases with more impact on the quality of life. Nutrition has been suggested as a relevant factor in the treatment and improvement of FM symptoms. The aim of this study was to measure mitochondrial homeostasis using several biomarkers for the diagnosis and monitoring of FM before and after the application of a personalized nutritional intervention with a rich antioxidant diet. Furthermore, we  evaluated the role of personalized nutrition as a potential treatment for patients with FM and identified new approaches for a better understanding of the disease.Motivation: Currently, a significant percentage of patients who suffer FM are not well diagnosed and face inadequate treatments. We proposed that personalized nutritional interventions can improve the symptomatology of patients and their quality of life.Methods: Fifty FM patients with clinical suspicion of food sensitivities were included in the study. Data were collected from nutritional questionnaries, the Fibromyalgia Impact Questionnaire (FIQ) and the ratio of mitochondrial mass and selective autophagy. Mitochondrial markers were measured pre and post nutritional interventions with the objective of evaluating if personalized diets improved clinical symptoms.Results: Rich antioxidant diets decreased radical oxygen species (ROS) levels in blood and improved significantly clinical symptoms. In addition, a balanced, personalized diet for each patient improved the ratio between mitochondrial mass and selective autophagy.Conclusions: FM patients presents several associated comorbidities such as food sensitivities which need more attention and research. The adequate consumption of antioxidants and other micronutrients is important in FM management. Therefore,  personalized antioxidant diets could be a promising approach for the improvement of clinical symptoms in FM

Producción de una hormona animal recombinante estable y con actividad in vivo.

Actualmente, en ganadería vacuna intensiva, la fertilización de las vacas se produce por inseminación artificial. Para obtener un mayor rendimiento, se extraen hormonas naturales de determinados animales para inyectarlas al ganado vacuno y conseguir la hiperovulación de éstos, pudiendo inseminar todas al mismo tiempo y con mayor probabilidad de éxito. El problema es que la extracción de estas hormonas es compleja y cara. Se ha intentado solventar este problema produciendo esta hormona en cultivos celulares de mamíferos o insectos mediante ingeniería genética. Aunque las enzimas recombinantes demuestran actividad in vitro, al inyectarlas a modelos animales o ganado no inducen la hiperovulación debido a una vida media en suero mucho más corta (minutos) que la versión natural (horas), ya que son eliminadas de la sangre rápidamente por la orina. Bionaturis ha propuesto la estabilización en suero de esta hormona recombinante mediante la fusión de secuencias estabilizadoras desarrolladas por la empresa a la secuencia hormonal.Bionaturis utiliza la plataforma FLYLIFE, que consiste en el uso del sistema baculovírico para la producción de proteínas recombinantes. A partir de secuencias estabilizadoras ya sintetizadas, se creó un vector de transferencia con las secuencias de interés mediante técnicas de clonación. Luego, mediante recombinación homóloga, se insertarán estas secuencias en baculovirus modificados genéticamente (Master Viral DNA de Bionaturis) que, en vez de sintetizar polihedrina, sintetizan la secuencia de interés, y así obtener baculovirus recombinantes de trabajo (Working Viral Banks). La obtención de los Working Virus Bank (WBK) se realiza mediante la co-transfección en células de insecto con el Master Viral DNA y el vector de transferencia. Hasta ahora, se ha llevado a cabo la construcción del vector de transferencia con éxito, los siguientes pasos a dar son la co-transfección para la obtención del WVB y su posterior caracterización mediante test de expresión de proteínas en cultivo celular. Posteriormente, se pretende la infección de larvas de lepidóptero con el virus recombinante obtenido que, al multiplicarse en su interior, producirá la proteína recombinante. Finalmente, se extraerá y se purificará la proteína de interés y se evaluará su actividad in vitro e in vivo

Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant β-Glucocerebrosidase Activity and Mitochondrial Function in Neuronopathic Forms of Gaucher Disease

Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes lysosomal β-glucocerebrosidase. Homozygosity for the L444P mutation in GBA1 is associated with high risk of neurological manifestations which are not improved by enzyme replacement therapy. Alternatively, pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the mutant enzyme represent promising alternative therapies.Here, we report on how the L444P mutation affects mitochondrial function in primary fibroblast derived from GD patients. Mitochondrial dysfunction was associated with reduced mitochondrial membrane potential, increased reactive oxygen species (ROS), mitophagy activation and impaired autophagic flux.Both abnormalities, mitochondrial dysfunction and deficient β-glucocerebrosidase activity, were partially restored by supplementation with coenzyme Q10 (CoQ) or a L-idonojirimycin derivative, N-[N’-(4-adamantan-1-ylcarboxamidobutyl)thiocarbamoyl]-1,6-anhydro-L-idonojirimycin (NAdBT-AIJ), and more markedly by the combination of both treatments. These data suggest that targeting both mitochondria function by CoQ and protein misfolding by PCs can be promising therapies in neurological forms of GD.España, Ministerio de Sanidad FIS PI13/00129España, Ministerio de Economía y Competitividad SAF2013-44021-R and CTQ2010-15848España, Junta de Andalucía CTS-5725 and FQM-146

Development of a tool for the study of the reproductive microbiome and its relationship with endometrial receptivity and functionality in infertility patients.

Motivation: The uterine cavity has been considered a sterile niche until few years ago; however, recent studies have shown a characteristic and functional microbiota resides over the endometrial tissue [1]. Thanks to the next generation sequencing (NGS) of the gene encoding the rRNA 16S of prokaryotic ribosomes, microorganisms present in the endometrium were identified; among those predominantly species of the genus Lactobacillus [2]. Bacteria of this genus are classified as acid-lactic bacteria, so they are able to secrete lactic acid, hydrogen peroxide and bacteriocins in order to generate a suitable environment with other microorganisms and avoid the proliferation of potential pathogens [3]. Despite the high percentage of lactobacilli in the endometrium of most women, the microbial composition of each one is highly individualized and the microbial profile can change due to different causes appearing episodes of dysbiosis, that can affect reproductive health, causing embryonic implantation failures, spontaneous miscarriage, premature deliveries and infectious diseases such as chronic endometritis.The main objective of this project is to develop a new tool to analyze the composition of endometrial microbiota of women with infertility problems using microfluidic techniques and studying its relationship with endometrial receptivity and functionality.Methods: In order to analyze the endometrial microbiota, a bibliographical research of the main microorganisms that reside in this tissue, as well as differential genes of these species was carried out. With these sequences, we designed specific primers for the genes of the microorganisms we identify, and they have will be associated with TaqMan probes. After a correct verification of primers and probes, we perform a DNA extraction from endometrial biopsies using a commercial extraction kit and the DNA obtained was pre-amplified by PCR. Finally, thanks to current microfluidic techniques, it was possible to make a single analysis with 96 samples of patients and the primers previously synthesized, to study the microbiological profile of each woman by qPCR

Study of technical validation of molecular biomarkers for the development of a diagnostic tool for Fibromyalgia.

Fibromyalgia (FM) is a disease characterized by widespread pain and both physical and psychological symptoms. These influence the normal course of daily life on a personal and professional level, generating a considerable deterioration in the quality of life of patients. The prevalence of FM is estimated to be between 2% and 8% of the world population, with women being the most affected. A large proportion of patients with FM are currently not properly diagnosed and face sub-optimal treatment, whose efficacy is variable among patients. While the molecular pathomechanism of FM remains unclear, several factors that could be behind its pathogenesis have been suggested in recent years, including oxidative stress, inflammation, mitochondrial dysfunction and disturbances in the gut microbiome. Therefore, the aim of this study is to validate the use of various biomarkers for the molecular diagnosis of FM through the determination of mitochondrial metabolism balance, total antioxidant capacity and the presence of intestinal dysbiosis in patients with FM in order to develop a diagnostic tool. Methods: Peripheral blood and fecal samples were collected from patients diagnosed with FM and healthy controls. An isolation of peripheral blood mononuclear cells (PBMCs) and protein extraction was performed to measure the levels of certain proteins described to be altered in FM patients by Western blot. The plasma serum has also been isolated to evaluate several biomarkers that are related to intestinal permeability status. In addition, we have isolated the DNA from fecal samples for its subsequent sequencing. We are currently in the process of validating the total antioxidant capacity by spectrophotometry and intestinal dysbiosis by enzymeimmunoassay. Results: We have observed significative differences in the expression levels of VDAC and LC3B, which are proteins involved in autophagy and mitochondrial mass.  Conclusions: The results obtained so far confirm the validity of using molecular biomarkers related to mitochondrial metabolism balance levels to differentiate between FM patients and healthy people. Further studies will be performed to verify these results by a proteomic approach and sequencing of FM patients for the determination of the proteome and intestinal dysbiosis. A complete characterization of these biomarkers will contribute for a more accurate diagnosis of the disease, leading to a better course and allowing the monitorization of the treatment outcome

Study protocol for FIBROKIT: a new tool for fibromyalgia diagnosis and patient follow-up

Fibromyalgia (FM) is a complex disease that is characterized by chronic musculoskeletal pain and has great economic impact. FM prevalence is about 2% to 4% worldwide, affecting mainly middle-aged women, and its complex pathophysiology complicates diagnosis, treatment and the findings of solid biomarkers. Previous studies have suggested an association between the disease and oxidative stress, mitochondrial metabolism, intestinal microbiota and inflammation, providing sufficient data to support the multifactorial origin of FM. Hence, the objective of this randomized, prospective, low-interventional, double-blinded and placebo-controlled clinical trial is the development of a specific panel of FM biomarkers and the evaluation of their response to a six-month nutritional intervention based on the Mediterranean diet supplemented with extra virgin olive oil (EVOO). For this purpose, the experimental design implies the recruitment of a large cohort of female Spanish patients. Middle-aged women who meet the diagnostic criteria for FM according to the American College of Rheumatology (ACR) will be eligible, along with age-matched healthy women. Both groups will be randomly divided into placebo (olive oil, OO) and treatment groups (extra virgin olive oil, EVOO), and will provide samples at the beginning (T0), after 3 months of nutritional intervention (T1), at the end of the nutritional intervention in 6 months (T2), and 6 months after the end of nutritional intervention (TF), being enrolled for 1 year. Data will be collected through health questionnaires, and whole blood and stool samples will be taken and analyzed. Blood will be used for western-blotting and proteomic analysis of mitochondrial homeostasis and plasma proteome, while stool will undergo metagenomic analysis, respectively. This study represents the first low-interventional investigation with more than 200 participants focused on exploring the association of oxidative stress, mitochondrial metabolism, intestinal microbiota and related pathways with a nutritional intervention in the context of FM. As a result, the outcomes of this study will significantly contribute to the development of a comprehensive and robust panel of diagnostic biomarkers, and will shed some light on their modulation with non-pharmacological therapies such as nutrition.Clinical trial registration: https://clinicaltrials.gov/study/NCT05921409, identifier: NCT05921409

Coenzyme Q10 partially restores pathological alterations in a macrophage model of Gaucher disease

Background Gaucher disease (GD) is caused by mutations in the GBA1 gene which encodes lysosomal β-glucocerebrosidase (GCase). In GD, partial or complete loss of GCase activity causes the accumulation of the glycolipids glucosylceramide (GlcCer) and glucosylsphingosine in the lysosomes of macrophages. In this manuscript, we investigated the effects of glycolipids accumulation on lysosomal and mitochondrial function, inflammasome activation and efferocytosis capacity in a THP-1 macrophage model of Gaucher disease. In addition, the beneficial effects of coenzyme Q10 (CoQ) supplementation on cellular alterations were evaluated. Chemically-induced Gaucher macrophages were developed by differentiateing THP-1 monocytes to macrophages by treatment with phorbol 12-myristate 13-acetate (PMA) and then inhibiting intracellular GCase with conduritol B-epoxide (CBE), a specific irreversible inhibitor of GCase activity, and supplementing the medium with exogenous GlcCer. This cell model accumulated up to 16-fold more GlcCer compared with control THP-1 cells. Results Chemically-induced Gaucher macrophages showed impaired autophagy flux associated with mitochondrial dysfunction and increased oxidative stress, inflammasome activation and impaired efferocytosis. All abnormalities were partially restored by supplementation with CoQ. Conclusion These data suggest that targeting mitochondria function and oxidative stress by CoQ can ameliorate the pathological phenotype of Gaucher cells. Chemically-induced Gaucher macrophages provide cellular models that can be used to investigate disease pathogenesis and explore new therapeutics for GD.info:eu-repo/semantics/publishedVersio

Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease

This is an open access article distributed under the terms of the Creative Commons Attribution License.[Introduction]: Fibromyalgia is a chronic pain syndrome with unknown etiology. Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of fibromyalgia. However, it is still not clear whether oxidative stress is the cause or the effect of the abnormalities documented in fibromyalgia. Furthermore, the role of mitochondria in the redox imbalance reported in fibromyalgia also is controversial. We undertook this study to investigate the role of mitochondrial dysfunction, oxidative stress, and mitophagy in fibromyalgia. [Methods]: We studied 20 patients (2 male, 18 female patients) from the database of the Sevillian Fibromyalgia Association and 10 healthy controls. We evaluated mitochondrial function in blood mononuclear cells from fibromyalgia patients measuring, coenzyme Q10 levels with high-performance liquid chromatography (HPLC), and mitochondrial membrane potential with flow cytometry. Oxidative stress was determined by measuring mitochondrial superoxide production with MitoSOX™ and lipid peroxidation in blood mononuclear cells and plasma from fibromyalgia patients. Autophagy activation was evaluated by quantifying the fluorescence intensity of LysoTracker™ Red staining of blood mononuclear cells. Mitophagy was confirmed by measuring citrate synthase activity and electron microscopy examination of blood mononuclear cells. [Results]: We found reduced levels of coenzyme Q10, decreased mitochondrial membrane potential, increased levels of mitochondrial superoxide in blood mononuclear cells, and increased levels of lipid peroxidation in both blood mononuclear cells and plasma from fibromyalgia patients. Mitochondrial dysfunction was also associated with increased expression of autophagic genes and the elimination of dysfunctional mitochondria with mitophagy. [Conclusions]: These findings may support the role of oxidative stress and mitophagy in the pathophysiology of fibromyalgia.This work was supported by grants FIS PI080500 and FIS EC08/00076, Ministerio de Sanidad, Spain. The authors dedicate this manuscript to FM patients and AFIBROSE (Asociación de Fibromialgia de Sevilla) for their unconditional help.Peer Reviewe