Development of antibody microarrays in beads format for the validation of osteoarthritis biomarkers

Traballo fin de mestrado (UDC.CIE). Biotecnoloxía avanzada. Curso 2017/2018[Resumen] La artrosis es la enfermedad reumática con mayor prevalencia en la población adulta. Los daños causados en la articulación por esta patología, silente en sus estadios iniciales, son irreversibles. A pesar de su elevado impacto en la sociedad, en la actualidad carece de tratamiento efectivo por lo que es muy importante su diagnóstico en estadios tempranos. Una buena estrategia para un diagnóstico precoz en la práctica clínica consistiría en el desarrollo de un panel de biomarcadores proteicos asociados a la artrosis, que idealmente permitiera la monitorización precisa de la enfermedad y la evaluación de la efectividad de nuevas estrategias terapéuticas. En este trabajo se pretende desarrollar un microarray analítico de anticuerpos en formato sándwich frente a proteínas con potencial biomarcador de artrosis que han sido identificadas en estudios previos del grupo de investigación. Dicho microarray permitirá el análisis múltiple del panel de proteínas en muestras de suero de pacientes artrósicos y controles. Para este desarrollo se trabajará con la tecnología xMAP de Luminex, basada en arrays en formato esfera con propiedades magnéticas

Dietary fatty acids and lipoproteins on progression of age-related macular degeneration

Age-related macular degeneration (AMD) is a medical condition of central loss vision and blindness. Numerous studies have revealed that changes on certain dietary fatty acids (FAs) could have useful for AMD management. This review summarizes the effects of dietary omega-3 long-chain PUFAs, MUFAs, and SFAs, and lipoproteins on AMD. Findings are consistent with the beneficial role of dietary omega-3 long-chain PUFAs, while the effects of dietary MUFAs and SFAs appeared to be ambiguous with respect to the possible protection from MUFAs and to the possible adverse impact from SFAs on AMD. Some of the pathological mechanisms associated with lipoproteins on AMD share those observed previously in cardiovascular diseases. It was also noticed that the effects of FAs in the diet and lipoprotein on AMD could be modulated by genetic variants. From a population health perspective, the findings of this review are in favour of omega-3 long-chain FAs recommendations in a preventive and therapeutic regimen to attain lower AMD occurrence and progression rates. Additional long-term and short-term nutrigenomic studies are required to clearly establish the role and the relevance of interaction of dietary FAs, lipoproteins, and genes in the genesis and progression of AMD

Mechanical harvesting at dawn in a super-high-density table olive orchard. Effect on the quality of fruits

BACKGROUND Mechanical harvesting with over-the-row harvesters in super-high-density (SHD) table olive orchards increases the effectiveness of fruit removal, although bruising can limit the fruit quality. Additionally, an early harvest in periods less favourable to quality production is increasingly frequent as a result of global warming. The present study explores the impact on olive quality of harvesting at dawn when the environmental temperature is low. The study was carried out for 2 years on two cultivars with different tolerance to bruising (‘Manzanilla de Sevilla’ and ‘Manzanilla Cacereña’), grown in SHD conditions and harvested at two timepoints: dawn and morning. RESULTS Fruit morphology was not modified by the moment of harvest in either of the cultivars. Fruit harvested at dawn produced less CO2 and ethylene and was less damaged externally and internally compared to fruit harvested in the morning. However, environmental conditions throughout development influenced the response because the highest values of bruising (incidence, area and volume of damaged area), total internal damage and the number of tissue ruptures increased in the year with the hottest summer, and the differences between harvest treatments were less evident

Genetic Diversity of Stone Fruit Cultivars Preserved On-Farm in Southern Spain

Old traditional cultivars are valuable genetic resources for crop improvement, but a great number of them have disappeared in the past century. This study aimed to characterize traditional cultivars of different Prunus species collected in small family orchards in southwestern Spain and to evaluate their genetic diversity and relationships. One hundred and twelve accessions belonging to 36 traditional cultivar denominations were analyzed using eight SSR loci transferable across the genus Prunus. The most useful loci to analyze different Prunus species were UDP96-005, BPPCT-002, UDP98-410 and ps02a12. A total of 152 alleles were observed, and 112 were unique to certain species. Sixty-eight different genotypes were found, revealing the possible existence of homonyms among traditional cultivar names. The clustering analysis was consistent with the taxonomic classification of the different species studied and with the geographical origins of the accessions within each species. The results showed wide genetic variability of traditional cultivars of stone fruits grown in small family orchards, which highlights the need to preserve them using both in-situ and ex-situ strategies. Twenty-eight of these accessions are currently conserved ex-situ at the University of Sevilla, Spain. The use of highly transferable SSRs has been proven as efficient in multi-species surveys performed on-farm

Suitability of Two Table Olive Cultivars (‘Manzanilla de Sevilla’ and ‘Manzanilla Cacereña’) for Mechanical Harvesting in Superhigh-density Hedgerows

At a time of increasing demand, the extremely high cost of manual labor required to harvest fruit in table olive groves is limiting the economic survival of the crop in many producing countries. New grove designs and management practices such as superhigh-density (SHD) groves now in use in oil olive production should be explored as an option to facilitate mechanical harvesting in table olives. The feasibility of two table olive cultivars, Manzanilla de Sevilla and Manzanilla Caceren˜a, to be harvested in a 5- year-old SHD grove (1975 trees/ha) was studied in 2012 when trees of both cultivars formed highly productive continuous hedgerows (’10,000 and 18,000 kg·haL1 , respectively). The differences between manual and mechanical harvesting using a grape straddle harvester were evaluated taking into consideration harvesting time, efficiency in fruit removal, and fruit quality both before and after processing as Spanish-style green olives. The average harvest time per hectare with a grape straddle harvester was less than 1.7 hours compared with 576 person/hour or more when done manually. Fruit removal efficiency was high in both cases, 98% for mechanical treatment and 100% for hand treatment. Mechanically harvested fruits had a high proportion of bruising damage (greater than 90%) and the severity of the damage was greater in ‘Manzanilla de Sevilla’ than in ‘Manzanilla Caceren˜a’. After Spanish-style green processing, however, the proportion of bruised fruits was below 3% in each cultivar. The fruit size in both cultivars was suitable for table olive processing and only 7% and 4% of ‘Manzanilla de Sevilla’ and ‘Manzanilla Caceren˜a’ fruits, respectively, were diverted to oil extraction as a result of insufficient size. Small differences were found between processed ‘Manzanilla Caceren˜a’ fruits that were manually or mechanically harvested. In contrast, mechanically harvested ‘Manzanilla de Sevilla’ fruits showed a significantly higher proportion of cutting (18%), a type of damage that may take place during harvesting, and lower firmness and texture than those harvested manually

A microRNA expression signature of the postprandial state in response to a high-saturated-fat challenge

The postprandial hypertriglyceridemia is an important and largely silent disturbance involved in the genesis of numerous pathological conditions. Exaggerated and prolonged states of postprandial hypertriglyceridemia are frequently related to the ingestion of meals enriched in saturated fatty acids (SFAs). MicroRNAs are noncoding RNAs that function as gene regulators and play significant roles in both health and disease. However, differential miRNA expression between fasting and postprandial states has never been elucidated. Here, we studied the impact of a high-saturated-fat meal, mainly rich in palmitic acid, on the miRNA signature in peripheral blood mononuclear cells (PBMCs) of nine male healthy individuals in the postprandial period by using a two-step analysis: miRNA array and validation through quantitative real-time polymerase chain reaction. Compared with miRNA expression signature in PBMCs at fasting, 36 miRNAs were down-regulated and 43 miRNAs were up-regulated in PBMCs at postprandial hypertriglyceridemic peak. Six chromosomes (3, 7, 8, 12, 14 and 19) had nearly half (48.1%) of dysregulated miRNA-gene-containing regions. Down-regulated miR-300 and miR-369-3p and up-regulated miR-495-3p, miR-129-5p and miR-7-2-3p had the highest number of target genes. The differentially expressed miRNAs and their predicted target genes involved pathways in cancer, MAPK signaling pathway, endocytosis and axon guidance. Only down-regulated miRNAs notably targeted PI3K-Akt signaling pathways, whereas only up-regulated miRNAs targeted focal adhesion, Wnt signaling pathway, transcriptional misregulation in cancer and ubiquitin-mediated proteolysis. This is the first study of miRNA expression analysis of human PBMCs during postprandial hypertriglyceridemia and offers insight into new potential mechanisms by which dietary SFAs influence health or disease.Ministerio de Economía y Competitividad AGL2011-29008, AGL2016-80852-

Inhibition of the Neuronal Calcium Sensor DREAM Modulates Presenilin-2 Endoproteolysis

Deregulated intracellular Ca2+ and protein homeostasis underlie synaptic dysfunction and are common features in neurodegenerative diseases. DREAM, also known as calsenilin or KChIP-3, is a multifunctional Ca2+ binding protein of the neuronal calcium sensor superfamily with specific functions through protein-DNA and protein-protein interactions. Small-molecules able to bind DREAM, like the anti-diabetic drug repaglinide, disrupt some of the interactions with other proteins and modulate DREAM activity on Kv4 channels or on the processing of activating transcription factor 6 (ATF6). Here, we show the interaction of endogenous DREAM and presenilin-2 (PS2) in mouse brain and, using DREAM deficient mice or transgenic mice overexpressing a dominant active DREAM (daDREAM) mutant in the brain, we provide genetic evidence of the role of DREAM in the endoproteolysis of endogenous PS2. We show that repaglinide disrupts the interaction between DREAM and the C-terminal PS2 fragment (Ct-PS2) by coimmunoprecipitation assays. Exposure to sub-micromolar concentrations of repaglinide reduces the levels of Ct-PS2 fragment in N2a neuroblastoma cells. These results suggest that the interaction between DREAM and PS2 may represent a new target for modulation of PS2 processing, which could have therapeutic potential in Alzheimer’s disease (AD) treatment

An atlas of the knee joint proteins and their role in osteoarthritis defined by literature mining

[Abstract] Osteoarthritis (OA) is the most prevalent rheumatic pathology. However, OA is not simply a process of wear and tear affecting articular cartilage but rather a disease of the entire joint. One of the most common locations of OA is the knee. Knee tissues have been studied using molecular strategies, generating a large amount of complex data. As one of the goals of the Rheumatic and Autoimmune Diseases initiative of the Human Proteome Project, we applied a text-mining strategy to publicly available literature to collect relevant information and generate a systematically organized overview of the proteins most closely related to the different knee components. To this end, the PubPular literature-mining software was employed to identify protein-topic relationships and extract the most frequently cited proteins associated with the different knee joint components and OA. The text-mining approach searched over eight million articles in PubMed up to November 2022. Proteins associated with the six most representative knee components (articular cartilage, subchondral bone, synovial membrane, synovial fluid, meniscus, and cruciate ligament) were retrieved and ranked by their relevance to the tissue and OA. Gene ontology analyses showed the biological functions of these proteins. This study provided a systematic and prioritized description of knee-component proteins most frequently cited as associated with OA. The study also explored the relationship of these proteins to OA and identified the processes most relevant to proper knee function and OA pathophysiology.PI19/01206; Instituto de Salud Carlos IIIPI20/00793; Instituto de Salud Carlos IIIPI20/01409; Instituto de Salud Carlos IIIPI22/01155; Instituto de Salud Carlos IIIRICORS-REIRD21/0002/0009; Instituto de Salud Carlos IIIED431E2018/03; Xunta de GaliciaIN607A2021/07; Xunta de GaliciaIN607D2020/10; Xunta de Galici

Eficiencia de la recolección mecánica y efecto sobre la calidad de la producción en plantaciones superintensivas de variedades de aceituna de mesa

Las plantaciones superintensivas de olivar (Olea europaea L.), con densidades superiores a los 1.000 árboles por hectárea, se han convertido en una interesante alternativa al cultivo de olivar de almazara en la que el coste de recolección disminuye por la drástica reducción de la mano de obra y del tiempo requerido por el empleo de cosechadoras cabalgantes. No hay, sin embargo, experiencia alguna en relación al olivar de mesa. El objetivo de este trabajo ha sido evaluar la aptitud de dos variedades tradicionalmente destinadas al aderezo, como son la ‘Manzanilla de Sevilla’ y ‘Manzanilla Cacereña’, a la recolección con cosechadora cabalgante cuando se cultivan en superintensivo. El trabajo se realizó en septiembre de 2012 y 2014 en una plantación adulta. Antes de la recolección se caracterizaron los setos. Posteriormente se analizó la eficiencia de la recolección mecánica, en términos de porcentaje de frutos derribados y tiempo de recolección, así como las proporciones de frutos con daños. En 2012 también se analizaron los daños en frutos aderezados en verde al estilo sevillano. El tamaño de los setos fue adecuado para la recolección mediante cosechadora cabalgante. La cosechadora derribó prácticamente la totalidad de los frutos en un tiempo no superior a las dos horas por hectárea. La producción media fue mayor en la ‘Manzanilla Cacereña’ que en la ‘Manzanilla de Sevilla’ y la calidad del fruto apta para la comercialización, si bien los frutos de esta última variedad presentaban una mayor susceptibilidad al daño.Junta de Andalucía SGAEX/C

Association of the serological status of rheumatoid arthritis patients with two circulating protein biomarkers: a useful tool for precision medicine strategies

[Abstract] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints and presence of systemic autoantibodies, with a great clinical and molecular heterogeneity. Rheumatoid Factor (RF) and anti-citrullinated protein antibodies (ACPA) are routinely used for the diagnosis of RA. However, additional serological markers are needed to improve the clinical management of this disease, allowing for better patient stratification and the desirable application of precision medicine strategies. In the present study, we investigated those systemic molecular changes that are associated with the RF and ACPA status of RA patients. To achieve this objective, we followed a proteomic biomarker pipeline from the discovery phase to validation. First, we performed an iTRAQ-based quantitative proteomic experiment on serum samples from the RA cohort of the Hospital of Santiago de Compostela (CHUS). In this discovery phase, serum samples from the CHUS cohort were pooled according to their RF/ACPA status. Shotgun analysis revealed that, in comparison with the double negative group (RF-/ACPA-), the abundance of 12 proteins was altered in the RF+/ACPA+ pool, 16 in the RF+/ACPA- pool and 10 in the RF-/ACPA+ pool. Vitamin D binding protein and haptoglobin were the unique proteins increased in all the comparisons. For the verification phase, 80 samples from the same cohort were analyzed individually. To this end, we developed a Multiple Reaction Monitoring (MRM) method that was employed in a comprehensive targeted analysis with the aim of verifying the results obtained in the discovery phase. Thirty-one peptides belonging to 12 proteins associated with RF and/or ACPA status were quantified by MRM. In a final validation phase, the serum levels of alpha-1-acid glycoprotein 1 (A1AG1), haptoglobin (HPT) and retinol-binding protein 4 (RET4) were measured by immunoassays in the RA cohort of the Hospital of A Coruña (HUAC). The increase of two of these putative biomarkers in the double seropositive group was validated in 260 patients from this cohort (p = 0.009 A1AG1; p = 0.003 HPT). The increased level of A1AG1 showed association with RF rather than ACPA (p = 0.023), whereas HPT showed association with ACPA rather than RF (p = 0.013). Altogether, this study has allowed a further classification of the RA seropositive patients into two novel clusters: RF+A1AG+ and ACPA+HPT+. The determination of A1AG1 and HPT in serum would provide novel information useful for RA patient stratification, which could facilitate the effective implementation of personalized medicine in routine clinical practice.Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI17/00404Instituto de Salud Carlos III; PI19/01206Instituto de Salud Carlos III; PI20/00793Instituto de Salud Carlos III; RD16/0012/0002Instituto de Salud Carlos III; RD21/0002/0009Xunta de Galicia; IN607A2021/07Xunta de Galicia; IN607D2020/1