A haplotype-resolved draft genome of the European sardine (Sardina pilchardus)

The European sardine (Sardina pilchardus Walbaum, 1792) is culturally and economically important throughout its distribution. Monitoring studies of sardine populations report an alarming decrease in stocks due to overfishing and environmental change, which has resulted in historically low captures along the Iberian Atlantic coast. Important biological and ecological features such as population diversity, structure, and migratory patterns can be addressed with the development and use of genomics resources.Agência financiadora Portuguese national funds from FCT-Foundation for Science and Technology: UID/Multi/04326/2016; European Regional Development Fund (FEDER): 22153-01/SAICT/2016; ALG-01-0145-FEDER-022121; ALG-01-0145-FEDER-022231; MAR2020 operational programme of the European Maritime and Fisheries Fund (project SARDI-NOMICS): MAR-01.04.02-FEAMP-0024; European Union's Horizon 2020 research and innovation programme: 654008info:eu-repo/semantics/publishedVersio

Strategies to improve productivity of CHO-S cells expressing an anti-TNFα monoclonal antibody with biosimilar potential

Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFα drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn’s disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFα adalimumab. This project is based on the first development stage for an adalimumab biosimilar candidate with potential for national production through the generation of a productive and stable cell line and assessment of its functionality. The robotic system ClonePix was used for screening and isolation of colonies from transfected CHO-S stable pools plated in semisolid medium. Selected clones were expanded based on growth and productivity. Purified mAbs from different clones were tested for binding and functional activity. The binding affinity of the denominated adabut clones to TNFα and FcRs, tested by surface plasmon resonance, did not differ statistically when compared to reference adalimumab. One functional activity assay demonstrated the antibody neutralization capacity of the cytotoxicity induced by TNFα in L929 murine fibroblasts. A second assay confirmed adabut as an antagonist of the TNFα activity by the inhibition of the cell adhesion molecule expression in HUVEC cultures. The binding kinetics and functional analysis performed suggest a potential for further development of adabut as a biosimilar. The process of the cell line development for adabut generated data consisting of cell growth/viability, volumetric and specific productivity, antigen binding kinetics, functional assays and long-term stability, from which 3 clones were selected. One clone (123) was top ranked and 2 others (70 and 225) showed equivalent performance. To increase the basal productivity of clone 123, 0.6g/L, we run 3 fed-batch experiments to evaluate 6 basal media, 8 supplements and a temperature shift to 32 C on day 6 on some conditions. The experiments were conducted in 250 and 500mL-shaker flasks along 14 days or while cell viability was above 60%. The flasks were sampled daily for cell counting/viability by Vi-Cell XR cell counter and glucose monitoring. Samples were stored for additional metabolites and antibody concentration assessment. By testing 24 different fed-batch strategies we were able to increase up to 6 times the volumetric clone productivity, while maintaining product quality. No significant differences were observed between the mAbs purified from days 14, 16 or 17 fed-batch cultures and the reference commercial product by SDS-PAGE, SEC-HPLC, IEX-HPLC and IEF analyses

Radiolabeled nano-peptides show specificity for an animal model of human PC3 prostate cancer cells

OBJECTIVES: Cancer has been investigated using various pre-targeting techniques or models focusing on radiobombesin analogues; however, both are not offered together. In this study, nano-bombesin labeling by a pre-targeting system was undertaken to develop an alternative approach for prostate tumor treatment. METHODS: A two-step pre-targeting system utilizing a combination of streptavidin (SA), biotinylated morpholino (B-MORF), biotinylated BBN (B-BBN) with two different spacers (b-Ala and PEG), and a radiolabeled cMORF was evaluated in vitro and in vivo. RESULTS: Final conjugation conditions consisted of a 1:1:2 ratio of SA:B-MORF:B-BBN, followed by addition of 99mTc-cMORF to compensate for free MORF. In vitro binding experiments with prostate cancer cells (PC-3) revealed that total binding was time-dependent for the Ala spacer but not for the PEG spacer. The highest accumulation (5.06 ± 1.98 %) was achieved with 1 hour of incubation, decreasing as time progressed. Specific binding fell to 1.05 ± 0.35 %. The pre-targeting biodistribution in healthy Swiss mice was measured at different time points, with the best responses observed for 7-h and 15-h incubations. The effector, 99mTc-MAG3-cMORF, was administered 2 h later. Strong kidney excretion was always documented. The greatest tumor uptake was 2.58 ± 0.59 %ID/g at 7 h for B-bAla-BBN, with a region of interest (ROI) value of 3.9 % during imaging. The tumor/blood ratio was low due to the slow blood clearance; however, the tumor/muscle ratio was 5.95. CONCLUSIONS: The pre-targeting approach with a peptide was a viable concept. Further evaluation with modified sequences of MORF, including less cytosine, and additional test intervals could be worthwhile

Comparison of two peptide radiotracers for prostate carcinoma targeting

OBJECTIVES: Scintigraphy is generally not the first choice treatment for prostate cancer, although successful studies using bombesin analog radiopeptides have been performed. Recently, a novel peptide obtained using a phage display library demonstrated an affinity for prostate tumor cells. The aim of this study was to compare the use of a bombesin analog to that of a phage display library peptide (DUP-1) radiolabeled with technetium-99m for the treatment of prostate carcinoma. The peptides were first conjugated to S-acetyl-MAG3 with a 6-carbon spacer, namely aminohexanoic acid. METHODS: The technetium-99m labeling required a sodium tartrate buffer. Radiochemical evaluation was performed using ITLC and was confirmed by high-performance liquid chromatography. The coefficient partition was determined, and in vitro studies were performed using human prostate tumor cells. Biodistribution was evaluated in healthy animals at various time points and also in mice bearing tumors. RESULTS: The radiochemical purity of both radiotracers was greater than 95%. The DUP-1 tracer was more hydrophilic (log P = -2.41) than the bombesin tracer (log P = -0.39). The biodistribution evaluation confirmed this hydrophilicity by revealing the greater kidney uptake of DUP-1. The bombesin concentration in the pancreas was greater than that of DUP-1 due to specific gastrin-releasing peptide receptors. Bombesin internalization occurred for 78.32% of the total binding in tumor cells. The DUP-1 tracer showed very low binding to tumor cells during the in vitro evaluation, although tumor uptake for both tracers was similar. The tumors were primarily blocked by DUP1 and the bombesin radiotracer primarily targeted the pancreas. CONCLUSION: Further studies with the radiolabeled DUP-1 peptide are recommended. With further structural changes, this molecule could become an efficient alternative tracer for prostate tumor diagnosis

Eigenstructure of rank one updated matrices

[EN] The relationship among eigenvalues of a given square matrix A and the rank one updated matrix A+vkq⁎, where vk is an eigenvector of A associated with the eigenvalue λk and q is an arbitrary vector, was described by Brauer in 1952. In this work we study the relations between the Jordan structures of A and A+vkq⁎. More precisely, we analyze the generalized eigenvectors of the updated matrix in terms of the generalized eigenvectors of A, as well as the Jordan chains of the updated matrix. Further, we obtain similar results when we use a generalized eigenvector of A instead of the eigenvector vkSupported by the Spanish DGI grant MTM2013-43678-P.Bru García, R.; Cantó Colomina, R.; Urbano Salvador, AM. (2015). Eigenstructure of rank one updated matrices. Linear Algebra and its Applications. 485:372-391. https://doi.org/10.1016/j.laa.2015.07.036S37239148

An odorant-binding protein based electrical sensor to detect volatile organic compounds

This work was funded by the European Research Council (ERC) under the EU Horizon 2020 research and innovation program [SCENTERC-2014-STG-639123, (2015–2022) and Grant Agreement No. 101069405-ENSURE-ERC-2022-POC1], and by national funds from FCT – Fundação para a Ciência e a Tecnologia, I.P., Portugal, for the project PROTEIOS (PTDC/CTM-CTM/3389/2021), for the Research Unit on Applied Molecular Biosciences the Associate Laboratory Institute for Health and Bioeconomy – i4HB (LA/P/0140/2020) and Associated Laboratory for Green Chemistry (LAQV) of the Network of Chemistry and Technology (REQUIMTE) – LAQV/REQUIMTE (UIDP/ 50006/2020). Stimulus, DOI: 10.54499/2022.07088.CEECIND/CP1725/CT0002 (CE).Artificial olfaction systems, such as electronic nose devices (ENs), can be used in various fields, from healthcare to food industry and the environmental sector. ENs consist of an array of sensors composed of different sensing materials. Incorporating Odorant Binding Proteins (OBPs) into gas-sensing materials can increase volatile organic compounds (VOCs) recognition and selectivity. OBPs are small soluble proteins found in vertebrates and insects, responsible for VOCs solubilization and transportation. In this work, OBP3 from Rattus norvegicus was selected to develop an OBP-based electrical VOC sensor, by optimizing protein production and immobilization on sensors surface. OBP3 was successfully produced in E. coli host cells (94 mg/L) and purified with high purity (88% purification yield, 96% purity). The protein folding and thermal stability were assessed by circular dichroism (Tm=71±1ºC) whereas ligand binding activity was verified in solution by fluorescence displacement against diisopropylphenol (Kd=0.24 µM). For the immobilization of OBP3 on gold interdigitated electrodes modified with reduced graphene oxide, we explored two strategies (covalent and non-covalent), establishing a reproducible and cost-effective methodology to develop OBP3-based electrical sensors. The non-covalent immobilization of a linker to the graphene-modified surface showed improved outcomes compared to the carbodiimide crosslinking chemistry. OBP3-sensors presented selectivity towards distinct model compounds in the gas phase (diisopropylphenol, dimethylpyrazine, menthone and decanol), in correlation with the dissociation constants measured by fluorescence displacement assays in solution. As a result, this study expands the practical applications of OBPs for gas-phase sensors, showcasing their potential for enhancing VOC detection.publishersversionpublishe

Saturated fatty acid-enriched small extracellular vesicles mediate a crosstalk inducing liver inflammation and hepatocyte insulin resistance

[Background & Aims]: Lipotoxicity triggers non-alcoholic fatty liver disease (NAFLD) progression owing to the accumulation of toxic lipids in hepatocytes including saturated fatty acids (SFAs), which activate pro-inflammatory pathways. We investigated the impact of hepatocyte- or circulating-derived small extracellular vesicles (sEV) secreted under NAFLD conditions on liver inflammation and hepatocyte insulin signalling. [Methods]: sEV released by primary mouse hepatocytes, characterised and analysed by lipidomics, were added to mouse macrophages/Kupffer cells (KC) to monitor internalisation and inflammatory responses. Insulin signalling was analysed in hepatocytes exposed to conditioned media from sEV-loaded macrophages/KC. Mice were i.v. injected sEV to study liver inflammation and insulin signalling. Circulating sEV from mice and humans with NAFLD were used to evaluate macrophage–hepatocyte crosstalk. [Results]: Numbers of sEV released by hepatocytes increased under NAFLD conditions. Lipotoxic sEV were internalised by macrophages through the endosomal pathway and induced pro-inflammatory responses that were ameliorated by pharmacological inhibition or deletion of Toll-like receptor-4 (TLR4). Hepatocyte insulin signalling was impaired upon treatment with conditioned media from macrophages/KC loaded with lipotoxic sEV. Both hepatocyte-released lipotoxic sEV and the recipient macrophages/KC were enriched in palmitic (C16:0) and stearic (C18:0) SFAs, well-known TLR4 activators. Upon injection, lipotoxic sEV rapidly reached KC, triggering a pro-inflammatory response in the liver monitored by Jun N-terminal kinase (JNK) phosphorylation, NF-κB nuclear translocation, pro-inflammatory cytokine expression, and infiltration of immune cells into the liver parenchyma. sEV-mediated liver inflammation was attenuated by pharmacological inhibition or deletion of TLR4 in myeloid cells. Macrophage inflammation and subsequent hepatocyte insulin resistance were also induced by circulating sEV from mice and humans with NAFLD. [Conclusions]: We identified hepatocyte-derived sEV as SFA transporters targeting macrophages/KC and activating a TLR4-mediated pro-inflammatory response enough to induce hepatocyte insulin resistance.This work was supported by grants PID2021-122766OB-I00 (AMV), PID2019-105989RB-I00 (JB), PID2020-113238RB-I00 (LB), PID2019-106581RB-I00 (MAM), PID2020-114148RB-I00 (MI), PID2019-107036RB-I00 (RF), and RD21/0006/0001 (ISCIII) (IL) funded by Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación/10.13039/501100011033 and ERDF ‘A way of making Europe’ by the European Union (MICINN/AEI/FEDER, EU), grant EFSD/Boehringer Ingelheim European Research Programme on ‘Multi-System Challenges in Diabetes’ from the European Foundation for the Study of Diabetes (AMV), P2022/BMD-7227 (Comunidad de Madrid, Spain) (AMV), Fundación Ramón Areces (Spain) (AMV), CIBERdem (AMV and JB), CIBERhed (RF), and CIBERcv (LB) (ISCIII, Spain). LB and AMV belong to the Spanish National Research Council’s (CSIC’s) Cancer Hub. We also acknowledge the Spanish Ministry of Economy and Competitiveness (MINECO) postdoctoral contract IJCI-2015-24758 to IGM and the Spanish Ministry of Education, Culture and Sport (MECD) FPU17/02786 grant to RA

Retinal Thickness Changes Over Time in a Murine AD Model APP NL-F/NL-F.

Background: Alzheimer's disease (AD) may present retinal changes before brain pathology, suggesting the retina as an accessible biomarker of AD. The present work is a diachronic study using spectral domain optical coherence tomography (SD-OCT) to determine the total retinal thickness and retinal nerve fiber layer (RNFL) thickness in an APPNL-F/NL-F mouse model of AD at 6, 9, 12, 15, 17, and 20 months old compared to wild type (WT) animals. Methods: Total retinal thickness and RNFL thickness were determined. The mean total retinal thickness was analyzed following the Early Treatment Diabetic Retinopathy Study sectors. RNFL was measured in six sectors of axonal ring scans around the optic nerve. Results: In the APPNL-F/NL-F group compared to WT animals, the total retinal thickness changes observed were the following: (i) At 6-months-old, a significant thinning in the outer temporal sector was observed; (ii) at 15-months-old a significant thinning in the inner temporal and in the inner and outer inferior retinal sectors was noticed; (iii) at 17-months-old, a significant thickening in the inferior and nasal sectors was found in both inner and outer rings; and (iv) at 20-months-old, a significant thinning in the inner ring of nasal, temporal, and inferior retina and in the outer ring of superior and temporal retina was seen. In RNFL thickness, there was significant thinning in the global analysis and in nasal and inner-temporal sectors at 6 months old. Thinning was also found in the supero-temporal and nasal sectors and global value at 20 months old. Conclusions: In the APPNL-F/NL-F AD model, the retinal thickness showed thinning, possibly produced by neurodegeneration alternating with thickening caused by deposits and neuroinflammation in some areas of the retina. These changes over time are similar to those observed in the human retina and could be a biomarker for AD. The APPNL-F/NL-F AD model may help us better understand the different retinal changes during the progression of AD.This research was funded by the Ophthalmological Network OFTARED (RD16/0008/0005) of the Institute of Health of Carlos III of the Spanish Ministry of Science and Innovation; and the Research Network RETIBRAIN (RED2018-102499-T) and Grant PID2019-106581RB-I00 of the Spanish Ministry of Science and Innovation; and Leducq Foundation for Cardiovascular Research TNE-19CVD01. IL-C was currently supported by a Pre-doctoral Fellowship (CT42/18-CT43/18) from the Complutense University of Madrid. JF-A was currently supported by a Pre-doctoral Fellowship (FPU17/01023) from the Spanish Ministry of Science, Innovation, and Universities.S

Prevalence of hyponatraemia in patients over the age of 65 who have an in-hospital fall

Background and aim: Hyponatraemia is the most common electrolyte disorder. Some studies have found that it increases morbidity and mortality. There are new lines of research that are investigating the link between hyponatraemia and patient falls. Aim: To determine if hyponatraemia is associated with falls in elderly hospitalised patients. Methods: Design observational, analytical, case-control study. Study population: Patients older than 65 years who had fallen during their hospitalisation at Gregorio Marañón Hospital (Madrid) were considered cases. Patients who did not fall were considered to be controls, paired according to the following variables: hospital ward, age, length of hospital stay, gender and Downton fall risk index. The sample size was 206 subjects. Data collection: Socio-demographic factors, variables included in the falls record sheet, Downton fall risk index and sodium levels were studied (hyponatraemia was considered Na+&lt; 135 mmol/l). Analysis: A descriptive analysis was performed to determine the sample homogeneity. The OR was calculated, and an analytical analysis using Chi-square test and a multivariate logistic regression analysis were also performed. Results: Of 103 cases recruited, 61 were men (50.4%) and 42 were women (49.4%). Hyponatraemia was detected in 29 cases with an association with falls of P: 0.002. The adjusted OR was 3.708 (1.6-8.3), 95% CI. Risk factors for falls were identified as hyponatraemia and limb sensory deficits. Conclusions: Given that hyponatraemia could be considered a risk factor for falls, the inclusion of the determination of sodium level would be important for fall prevention strategies in the elderly.Fundamento y objetivo: La hiponatremia es el trastorno electrolítico más frecuente. Algunos estudios afirman que aumenta la morbimortalidad. Existen nuevas líneas de investigación que buscan la relación entre hiponatremia y caídas. Objetivo: Determinar si la hiponatremia es un factor relacionado con las caídas en ancianos hospitalizados. Método: Disen˜ o observacional analítico de casos y controles. Población de estudio: Se consideraron casos los pacientes mayores de 65 an˜ os que experimentaron una caída durante su ingreso en unidades de hospitalización del Hospital General Universitario Gregorio Maran˜ ón de Madrid. Los controles fueron pacientes que no wxperimentaron caída, pareados según las variables: unidad, edad, periodo de ingreso, género y Downton. El taman˜ o fue de 206 sujetos. Recogida de datos: Se estudiaron factores sociodemográficos, las variables incluidas en la ficha de registro de caídas y escala de Downton, y el sodio sérico. Se consideró hiponatremia Na+ < 135 mmol/l. Análisis: Se realizó un análisis descriptivo para valorar la homogeneidad de la muestra, un análisis analítico utilizando el test chi cuadrado, calculando la OR y un análisis multivariante con regresión logística. Resultados: De 103 casos, 61 eran hombres (50,4%) y 42 mujeres (49,4%). En 29 se detectó hiponatremia; la relación con las caídas fue p: 0,002. La OR ajustada fue de 3,708 (1,6-8,3), IC 95%. Se identificaron como factores de riesgo para las caídas: hiponatremia y déficits sensoriales en extremidade

15N Natural Abundance Evidences a Better Use of N Sources by Late Nitrogen Application in Bread Wheat

This work explores whether the natural abundance of N isotopes technique could be used to understand the movement of N within the plant during vegetative and grain filling phases in wheat crop (Triticum aestivum L.) under different fertilizer management strategies. We focus on the effect of splitting the same N dose through a third late amendment at flag leaf stage (GS37) under humid Mediterranean conditions, where high spring precipitations can guarantee the incorporation of the lately applied N to the soil-plant system in an efficient way. The results are discussed in the context of agronomic parameters as N content, grain yield and quality, and show that further splitting the same N dose improves the wheat quality and induces a better nitrogen use efficiency. The nitrogen isotopic natural abundance technique shows that N remobilization is a discriminating process that leads to an impoverishment in 15N of senescent leaves and grain itself. This technique also reflects the more efficient use of N resources (fertilizer and native soil-N) when plants receive a late N amendment